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Defining intrarenal sexual dimorphisms to obesity and hypertension: understanding the female advantage

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Defining intrarenal sexual dimorphisms to obesity and hypertension: understanding the female advantage

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Defining Intrarenal Sexual Dimorphisms to Obesity and Hypertension:
Understanding the Female Advantage

by

Brandon Eugene McFarlin

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MEDICAL BIOLOGY)

May 2022

Copyright 2022 Brandon Eugene McFarlin
i
Epigraph

“Throw out your conceited opinions, for it is impossible for a person to begin
to learn what he thinks he already knows”
-Epictetus, Discourses, 2.17.1

ii
Acknowledgements
I would like to sincerely thank Dr. Alicia McDonough and Donna Ralph for their unwavering
guidance and support throughout this endeavor. The lessons I’ve learned from you both expand
far beyond science. I would also like to express deep gratitude to my dissertation committee Dr.
János Peti-Peterdi (Chair), Dr. Laura Perin, Dr. Jang-Hyun Youn, and Dr. Zhongwei Li for their
mentorship and for keeping me on track through the years. To my family, I owe a debt of
gratitude for your unconditional love and support.
iii
Table of Contents
Epigraph ........................................................................................................................................... i
Acknowledgements ......................................................................................................................... ii
Table of Contents ........................................................................................................................... iii
List of Tables ................................................................................................................................. iv
List of Figures ................................................................................................................................. v
Abbreviations ................................................................................................................................ vii
Abstract ........................................................................................................................................... x
Introduction ..................................................................................................................................... 1
The role of the kidneys in regulating effective circulating volume .................................... 1
Obesity, diabetes, and chronic kidney disease .................................................................... 3
Dissertation aims and key findings ..................................................................................... 4
Chapter 1. Sexual Dimorphisms in Acute Pressure Natriuresis in Female and Male Sprague
Dawley Rats .................................................................................................................................... 7
Abstract ............................................................................................................................... 7
Introduction ......................................................................................................................... 9
Methods............................................................................................................................. 12
Results ............................................................................................................................... 18
Discussion ......................................................................................................................... 36
Supplemental Information ................................................................................................ 42
Chapter 2. Sex-Specific Adaptations to Obesity Preserve Kidney Function in Female ZSF1 Rats
....................................................................................................................................................... 46
Abstract ............................................................................................................................. 46
Introduction ....................................................................................................................... 49
Methods............................................................................................................................. 51
Results ............................................................................................................................... 58
Discussion ......................................................................................................................... 80
Supplemental Information ................................................................................................ 88
General conclusions ...................................................................................................................... 95
Bibliography ................................................................................................................................. 97
Appendix ..................................................................................................................................... 112

iv
List of Tables

Table 1-1. Physiological parameters in male and female SD rats with acute hypertension……..23
Table 1-2. Impact of ABT on physiological parameters in female and male SD rats with acute
hypertension……………………………………………………………………………...29
Supplemental Table 1-1. Antibody and immunoblot protocol details…………………………...43
Table 2-1. Physiological parameters of ZSF1 rats at 18 weeks of age…………………………..59
Table 2-2. Organ weights and tibia lengths of ZSF1 rats at 18 weeks of age…………………...60
Table 2-3. Physiological parameters after 10 weeks of 0.3% Na
+
or 0.1% Na
+
diet…………….78
Supplemental Table 2-1. Antibody and immunoblot protocol details………………………..89-92

v
List of Figures
Figure 1-1. Experimental protocol schematic for acute hypertension surgeries…………………13
Figure 1-2. Similar hemodynamic response to vascular constriction in male and female SD
rats………………………………………………………………………………………..19
Figure 1-3. More robust natriuresis response in females with acute hypertension………………21
Figure 1-4. Impact of acute hypertension on transporters along the nephron in male and female
SD rats……………………………………………………………………………………25
Figure 1-5. Transporter profile summary in male and female SD rats with acute hypertension...27
Figure 1-6. ABT shift renal function curves leftward in both sexes but only increases sodium
excretion in males..............................................................................................................31
Figure 1-7. ABT treatment has little effect on transporter abundance and covalent modifications
in female SD rats................................................................................................................32
Figure 1-8. ABT treatment lowers transporter abundance and covalent modifications in male SD
rats......................................................................................................................................34
Supplemental Figure 1-1. GFR, UV vs MAP, UKV, and UKV vs MAP in male and female SD
rats with acute hypertension...............................................................................................44
Supplemental Figure 1-2. Impact of ABT on UV vs MAP, UKV, and UKV vs MAP in male and
female SD rats with acute hypertension.............................................................................45
Figure 2-1. Experimental protocol schematic for ZSF1 rats..........................................................53
Figure 2-2. OM, not OF, develop hyperglycemia despite hyperinsulinemia and greater SGLT1
abundance in both sexes.....................................................................................................62
Figure 2-3. Obese ZSF1 rats exhibit progressively worsening proteinuria, tissue albumin, and
glomerular fibrosis.............................................................................................................64
vi
Figure 2-4. Altered renal function with time in OM but not OF…………………....................67
Figure 2-5. Impact of hyperglycemia and obesity on transporters, channels, claudins, and
regulators along the proximal tubule and medullary thick ascending limb……………...70
Figure 2-6. Impact of hyperglycemia and obesity on transporters, channels, claudins, and
regulators along the distal tubule and collecting duct…………………............................74
Figure 2-7. Summary of key transporters, channels, claudins, and regulators along the nephron in
ZSF1 rats............................................................................................................................76
Figure 2-8. 10 weeks 0.1% Na
+
intake versus 0.3% Na
+
tends to blunt proteinuria.....................79
Supplemental Figure 2-1. Assessment of equal loading by protein staining of loading gel and
linearity of the detection system........................................................................................93
Supplemental Figure 2-2. Pronounced perirenal fat, lipidemia, and hyperleptinemia in obese
rats......................................................................................................................................94
Appendix Figure 2-1. Summary of transporters, channels, claudins, and regulators along the
nephron in SD females and lean ZSF1 females normalized to respective males............113
Appendix Figure 2-2. Summary of full transporters, channels, claudins, and regulators along the
nephron in ZSF1 rats........................................................................................................114
Appendix Figure 2-2. Full longitudinal analysis of physiological parameters 1.........................115
Appendix Figure 2-3. Full longitudinal analysis of physiological parameters 2.........................117

vii
Abbreviations
AA: Arachidonic acid
ABT: 1-aminobenzotriazole
ACE: angiotensin converting enzyme
Alix: ALG-2-interacting protein X
AngII: Angiotensin II
AngII-HTN: Angiotensin II hypertension
A’ogen: Angiotensinogen
AQP: aquaporin
AT1: angiotensin type I receptor
AT2: angiotensin type II receptor
BCA: Bicinchonic acid
BP: Blood pressure
C: Clearance (i.e. CLi+, CNa+)
CD: Collecting duct
CKD: Chronic kidney disease
Cldn: claudin
CVD: Cardiovascular disease
CYP: Cytrochrome P450
DCT: Distal convoluted tubule
DKD: Diabetic kidney disease
DPP-IV: Dipeptidyl Peptidase IV
ECV: effective circulating volume
viii
EETs: epoxyeicosatrienoic acids
ENaC: epithelial Na channel
ESRD: End stage renal disease
F: Female
MABT: Female treated with 1-aminobenzotriazole
MAP: mean arterial pressure
GFR: Glomerular filtration rate
HBP: High blood pressure
HO-1: heme oxygenase -1
HTN: Hypertension
IHC: Immunohistochemistry
Kir4.1: inwardly rectifying K
+
channel 4.1
K-pNPPase: K
+
-Dependent p-nitrophenyl phosphatase
LM, LF: Lean male, female
M: Male
m-: medullary
MABT: Male treated with 1-aminobenzotriazole
Na
+
: Na
+

NaPi2: Na+, Phosphate cotransporter 2
NBCe-1A: sodium bicarbonate cotransporter 1A
NCC: Na+, Cl+ cotransporter
NCCpS71,pT53: -P at S71 or T53
NHERF1: Na
+
/H
+
Exchanger Regulatory Factor
ix
NHE3: Na+,H+ exchanger isoform 3
NHE3pS552: -P associated with inhibition
NKA: Na+,K+-ATPase
NKCC(2): apical Na+,K+, 2Cl+ cotransporter
NKCCp: -P at T96 , T101
OM,OF: obese male, female
-P: Phosphorylation
PT: Proximal Tubule
RAAS: Renin angiotensin aldosterone system
RBF: Renal blood flow
ROMK: renal outer medullary K
+
channel
SD: Sprague Dawley
SPAK: Ste/SPS-1 related proline-alanine rich kinase
Transporters: transporters, channels, claudins, and regulators
T2D: Type II diabetes mellitus
TAL(H): Thick ascending limb of Henle’s loop
UV: Urine volume
UKV: U K+ excretion
UNaV: U Na+ excretion
ZSF1: Zucker Diabetic Fatty Spontaneously Hypertensive Heart Failure F1 hybrid rat
20-HETE: 20-hydroxyeicosatetraenoic acid
x
Abstract
In the United States, it’s estimated that 74% of American adults are overweight or obese which is
associated with a range of cardiometabolic disorders including the two leading causes of chronic
kidney disease – hypertension and diabetes – and is now considered an independent risk factor
for kidney disease. Hypertension and type 2 diabetes mellitus together account for three-fourths
of all chronic kidney disease cases. Furthermore, these two risk factors have high comorbidity
that exacerbates disease progression and increases the risk of cardiovascular-related mortality. It
is established that pre-menopausal women exhibit lower blood pressure, the prevalence of
diabetes, and the risk of cardiovascular and kidney disease compared to age-matched men. Our
lab has previously highlighted sexual dimorphic patterns of kidney transporters and electrolyte
handling that may confer a “head-start” in kidney adaptations to disease. However, mechanisms
accounting for this “female advantage” remain an important gap in renal physiology. This
dissertation aimed to define intrarenal sexual dimorphisms to hypertension and obesity with the
goal to provide a foundation for optimizing sex-specific therapies and provide disease-specific
targets for future therapeutics.
Pressure natriuresis is a powerful mechanism used by the kidneys for acute and chronic
maintenance of effective circulating volume. Our lab previously showed that redistribution of
proximal tubule Na
+
,H
+
exchanger isoform 3 (NHE3) from within the microvilli to the
microvillar base (inactive) plays an important role in pressure natriuresis response to
vasoconstriction. Additional studies show NHE3 is localized to the base of the microvilli in
females as baseline. In Chapter 1, we aimed to define the physiological and molecular responses
contributing to pressure natriuresis in female SD rats. We determined that acutely increasing
vascular resistance via vasoconstriction leads to similar levels of hypertension between sexes but
xi
females exhibit more robust pressure natriuresis mediated by baseline sex-specific transporter
patterns and complemented by further inhibition of proximal tubule to medullary thick ascending
limb transporters. In males, distinct patterns of lower proximal tubule and distal tubule
transporters contribute to pressure natriuresis. We additionally show that inhibition of
arachidonic acid metabolites with 1-aminobenzotraizole (ABT) similarly lowers blood pressure
and shifts renal function curves to the left in both sexes. In males, not females, ABT treatment
also lowered sodium transporter abundance contributing to the leftward shift in renal function
curves. Together these findings suggest sex-specific changes in pool size of Na
+
transporters that
contribute to pressure natriuresis.
In Chapter 2, we aimed to define sexual dimorphisms in renal tubular response to obesity
and early diabetic kidney disease and to determine the physiological consequences of the
differences on disease progression. To accomplish this aim, experimental protocols were
performed using the highly translatable ZSF1 rat model. Our findings show both obese males
and females develop metabolic complications including hypertrophy, lipidemia,
hyperinsulinemia, and glomerular fibrosis; only obese males develop hyperglycemia and
glycosuria. We provide evidence that hyperglycemia may exacerbate kidney pathology, but
obesity, independent of hyperglycemia, may induce initial kidney pathology. Both obese males
and females similarly exhibit patterns of lower pool sizes of key Na
+
transporters. In both
chapters, we show the female advantage to slower disease progression is partially mediated by
baseline sex-specific differences along the nephron that give females a head-start in kidney
adaptations to hypertension and metabolic challenges.
1
Introduction

The role of the kidneys in regulating the effective circulating volume
The kidney system has many physiological roles including regulation of acid-base
balance, red blood cell production, and regulation of vitamin D production. However, the two
major functions of the kidneys are excretion of metabolic waste and regulation of volume and
electrolyte homeostasis. To accomplish this, the kidneys receive the largest proportion of cardiac
output relative to organ weight and filter 180 L of blood a day on average in a healthy adult at
rest [1]. As the heart pumps blood, the force in which blood pushes against arterial walls is
defined as blood pressure, and the kidneys are tasked with maintaining the volume within the
cardiovascular system that effectively perfuses all tissues, known as effective circulating volume
(ECV). These functions are accomplished by filtration at the renal corpuscle, reabsorption,
secretion along the nephron (the functional unit within the kidney), and ultimately excretion via
urination. Chronic kidney disease (CKD) is defined as a gradual loss of these functions which
increases the risk of end-stage kidney disease necessitating transplantation and increases the risk
of cardiovascular disease and mortality [2]. The two main causes of chronic kidney disease are
hypertension and diabetes which together account for nearly three-fourths of all cases [3].
Chronically, high blood pressure or hypertension acts as a homeostatic signal to correct ECV. In
2017, the American Heart Association redefined hypertension as blood pressure above 130
(systolic) over 80 mmHg (diastolic), and in 2021, KDIGO updated its guidelines to recommend
adults maintain systolic blood pressure under 120 mmHg [4, 5]. These shifts to lower target
blood pressures expand the estimated prevalence of hypertension to nearly 50% of the adult
population in the United States [6]. Despite advances in treatment options, hypertension remains
2
the leading global burden of disease risk factor and heightens the demand for research to better
understand molecular mechanisms and develop more effective therapies [7].
Building on the seminal work of Ernest H. Starling, Guyton and colleagues [8]
conceptualized the kidney-body fluid feedback mechanism which outlines how pressure
natriuresis is a powerful mechanism used by the kidneys for acute and chronic maintenance of
ECV. To maintain homeostasis, intake must be equal to output and any imbalances in this
equilibrium such as abnormal increases in sodium (Na
+
) reabsorption or failure of the kidneys to
excrete ingested sodium leads to parallel increases in water reabsorption and movement of water
from tissues into the blood to correct circulating sodium concentration, which together ultimately
increases ECV. Increases in ECV elevate arterial pressure, which serves as a homeostatic error
signal to correct ECV via pressure natriuresis. The proximal tubule is responsible for two-thirds
of all reabsorption along the nephron and is layered with microvilli to increase transport surface
area. As defined in males, a key mechanism of pressure natriuresis is the inhibition of proximal
tubule (PT) sodium transport [9]. The PT Na
+
/H
+
exchanger isoform 3 (NHE3) and Na
+
/Pi
exchanger isoform 2 (NaPi2) redistribute from within the microvilli (active transport) to the base
of microvilli (associated with phosphorylation and inactivation) or internalized into
the microvillar cleft, respectively [10-14]. In female rats, however, NHE3 is localized at
the base of the microvilli under basal conditions, further contributing to the ability to excrete a
Na
+
load more rapidly than in males [15]. While mechanisms regulating ECV are complex and
multifaceted, mediators of pressure natriuresis and regulation of fluid volume are ill-defined in
females which motivates the questions asked in the current dissertation.

3
Obesity, diabetes, and chronic kidney disease
Global levels of obesity have reached epidemic proportions with approximately 39% of
adults overweight and 13% obese [16]. The World Health Organization defines overweight (BMI
≥25 kg/m
2
) and obesity (BMI ≥30 kg/m
2
) as abnormal or excess accumulation of adipose tissue.
Body Mass Index (BMI) is a measure of body fat estimated by body mass divided by the square
of body height. In the United States, it is estimated that 74% of American adults are overweight
or obese [17]. Obesity is associated with a range of cardiometabolic disorders including cardiac
hypertrophy [18], maladaptive alteration of gut microbiota [19], and the two leading causes of
CKD – hypertension and diabetes – and obesity is now considered an independent risk factor for
kidney disease [20, 21]. Diabetes mellitus is clinically defined as defects in pancreatic beta-cell
function causing defects in insulin production (type I) or insufficient insulin production and
insulin resistance (type 2) that both causes elevated blood glucose (hyperglycemia) [22]. The
increasing prevalence of diabetes is primarily driven by increases in type 2 diabetes mellitus
(T2DM) which parallels the increase in global obesity [23]. With nearly half of all diabetic
patients developing diabetic kidney disease [24], the burden of kidney disease is expected to rise
over the next decade. This heightens the demand for researchers to better understand the
pathogenesis of these high incidence, chronic, metabolic diseases with the goal to develop more
effective therapies.
Historically, females are underrepresented in both clinical trials and biomedical research
although it is clear that significant sex disparities are evident in prevalence, disease pathogenesis,
and treatment outcomes for obesity and diabetic kidney disease [25-27]. While BMI, the most
common measure of obesity, is higher in women, the prevalence of diabetes is higher in men
[28]. Studies suggest the “female advantage” may include differences in fat distribution
4
(subcutaneous versus visceral and ectopic fat) and estrogens which improve insulin sensitivity
and impair gluconeogenesis [29, 30]. However, this female advantage seems to be limited to
disease onset whereas following disease prognosis women may be at greater risk of disease-
related mortality [29]. Many useful animal models have contributed to our current understanding
of pathogenesis but a major limitation to advancement in obesity and diabetic kidney disease
research is the lack of animal models that truly mimic human disease. A majority of these
models do not meet all the criteria put forth by the Animal Models of Diabetic Complications
Consortium (AMDCC), often lacking glomerular histology and progression to end-stage kidney
disease (ESKD) as seen in humans [31, 32]. However, a recent analysis of disease progression in
Zucker Diabetic Spontaneously Hypertensive Heart Failure F1 hybrid (ZSF1) rats suggests this
model does meet all criteria put forth by the AMDCC, mimics disease progression seen in
humans, and offers the best currently available model to study kidney pathology related to
T2DM [33, 34].

Dissertation aims and key findings
The central objective of the current dissertation aims to define sexual dimorphisms in
acute and chronic intrarenal mechanisms contributing to sex disparities in chronic kidney
disease. First, the current dissertation aimed to define the physiological and molecular responses
contributing to pressure natriuresis in female Sprague Dawley (SD) rats. We tested the
hypothesis that females (versus males) would have more robust pressure natriuresis mediated by
baseline lower fractional Na
+
reabsorption in the proximal tubule and suppression of medullary
sodium reabsorption. To address the important physiological gap in understanding fluid and
electrolyte handling in females, a classic pressure natriuresis surgical protocol [35] was adapted
5
for our lab to acutely increase total peripheral vascular resistance in both female and male SD
rats. In addition, the generation of cytochrome P450 metabolites of arachidonic acid were
inhibited using 1-aminobenzotriazole to determine the role of these metabolites in pressure
natriuresis in females. Results defining acute physiological responses and determining
subcellular distribution, abundance, and covalent modification of key Na
+
transporters in
response to acute hypertension are described in Chapter 1.
Second, the current dissertation aimed to define sexual dimorphisms in renal tubular
response to obesity and early diabetic kidney disease and determine the physiological
consequences these differences have in disease progression. To accomplish this aim,
experimental protocols were performed using the highly translatable ZSF1 rat model. Results
defining sex-specific differences in the progression of pathophysiology, fluid and electrolyte
handling, and abundance and covalent modifications of transporters, channels, claudins, and
regulators are described in Chapter 2. In both chapters, we show the female advantage is partially
mediated by baseline sex-specific differences along the nephron that give females a head-start in
kidney adaptations to hypertension and metabolic challenges. Together these aims contribute to
our understanding of the female advantage to chronic kidney disease and provide a foundation
for optimizing sex-specific therapeutics and providing disease-specific targets for future
therapies.
Key Findings:
Chapter 1: Sexual dimorphisms in acute pressure natriuresis in female and male SD Rats
• Females (versus males) have a more robust acute pressure natriuretic response that
evokes a leftward shift in renal function curves (i.e., greater capacity to excrete sodium at
lower blood pressure) and greater sum-total UNaV (Figure 1-3).
6
• Distinct patterns of kidney transporter abundance and covalent modifications suggest
natriuresis stems from PT-mTAL in females and PT and DCT in males (Figure 1-4).
• CYP metabolite inhibition with 1-aminobenzotriazole lowered transporter abundances
and evoked greater sodium excretion in males (not females) but shifted renal function
curves leftward in both sexes (Figures 1-6, 1-7, and 1-8).

Chapter 2: Define sexual dimorphisms in renal tubular response to obesity and early diabetic
kidney disease
• Obesity provokes a lower abundance of key Na
+
transporters along the nephron
independent of hyperglycemia or baseline sexual dimorphic patterns. However, obese
females exhibit distinct proximal tubule abundance patterns that may suggest remodeling
and potentially influence disease progression (Figures 2-5, 2-6, and 2-7).
• Despite resistance to hyperglycemia, obese females develop hyperinsulinemia (Figure 2-
3), glomerular fibrosis, progressively worsening proteinuria, kidney tissue albumin
accumulation (Figure 2-2), lipidemia, and perirenal fat (Supp Figure 2-2) seen in obese
diabetic males.
• Lowering dietary sodium blunts proteinuria progression in obese males (Figure 2-8).
• Despite similar genetic background (leptin receptor deficiency), obese females do not
develop hyperleptinemia to the same extent as obese males which may contribute to
more severe pathology in males (Supp Figure 2-2).

7
Chapter 1. Sexual Dimorphisms in Acute Pressure Natriuresis in Female and
Male Sprague Dawley Rats

Abstract
Background. Increases in ECV or vasoconstriction can increase blood pressure. Elevated blood
pressure triggers pressure natriuresis and diuresis which are fundamental for the restoration of
ECV homeostasis. Previous studies in males show acute increases in blood pressure by
vasoconstriction lead to rapid redistribution of transporters to the base of the microvilli (NHE3)
or sub-apical pools (NaPi2, NCC) which facilitates natriuresis. Our lab has previously shown
normotensive females excrete a saline load more rapidly than normotensive males. This is partly
facilitated by the localization of NHE3 to the base of the microvilli as NHE3p (inactive) at
baseline in females which resembles males with hypertension. However, mechanisms
contributing to pressure natriuresis in females remain a poorly understood gap in renal
physiology. The current study aimed to define the physiological and molecular responses
contributing to pressure natriuresis in female Sprague Dawley (SD) rats.

Methods. Female and male SD rats (8-10 weeks of age; 55 rats total) underwent surgery to
acutely raise arterial pressure by constriction of celiac artery, superior mesenteric artery, and
abdominal aorta for 25-35 minutes or Sham (no vasoconstriction) surgery. A subset of rats
received 1-aminobenzotraizole (ABT; 50 mg/kg/day for 5 days) or untreated (saline vehicle or
no injection) before undergoing surgery to determine the role of cytochrome P450 metabolites of
arachidonic acid on pressure natriuresis in females. Mean arterial pressure was measured via
carotid artery cannulation, urine collected via bladder cannulation, electrolytes assessed by flame
8
photometry, and kidneys were excised and either dissected for semi-quantitative immunoblotting
or surface fixed for immunohistochemistry.

Results. At baseline, females (versus males) exhibit lower blood pressure and greater sodium
excretion, urinary volume excretion, and clearance (CLi and CNa). Vascular constriction acutely
increased blood pressure by 25-34 mmHg in both sexes. Acute hypertension provokes a 21- and
19-fold increase in UNaV in females and males, respectively. Despite similar increases in UNaV
with acute hypertension, females exhibit a leftward shift in renal function curves and excrete
more urinary sodium compared to males. Contributing to increased natriuresis, both females and
males exhibit 21-22% greater proximal tubule NHE3p. Additionally, females exhibit 21% lower
medullary (m-) NHE3 and 17% lower mNKCC2 abundance whereas males exhibit 16% lower
NCC, 29% lower NCCpS71, and 30% greater full-length gENaC (no change in cleavage). ABT
treatment lowered blood pressure by 9-12% and shifted renal function curves leftward in both
females and males. ABT treatment in males, not females, lowered Na
+
transporter abundances
along the nephron and facilitated greater urinary sodium excretion compared to untreated rats.

Conclusion. Females exhibit more robust pressure natriuresis than males in response to acute
hypertension likely mediated by baseline lower blood pressure set-point, baseline sexual
dimorphisms in transporters, and distinct alterations in transporters along the nephron with acute
hypertension. While CYP metabolites may have similar effects within the vasculature, we
provide evidence of sexual dimorphism in the kidney adaptations. Together, these results suggest
mediators of acute pressure natriuresis and physiological responses to acute hypertension are
sex-specific.
9
Introduction
Hypertension is the leading global burden of disease risk factor and a leading contributor
to chronic kidney disease [3, 7]. Pressure natriuresis is a powerful mechanism used by the
kidneys for acute and chronic maintenance of ECV. Early studies in male rats demonstrate that
acute increases in blood pressure by vasoconstriction rapidly inhibit fluid reabsorption in the
proximal tubule and increase flow rate and urinary excretion with minimal effect on glomerular
filtration rate (GFR) or renal blood flow [35-37]. Our lab previously expanded these findings by
showing vasoconstriction reduced Na
+
,K
+
-ATPase activity [11], provoked redistribution of
NHE3 to the base of the microvilli [11, 13], and redistribution NaPi2 and distal tubule Na
+
,Cl
-
cotransporter (NCC) to subapical endosomes [13, 38, 39]. Together, these adaptations facilitate
the increases in urinary excretion and volume leaving the proximal tubule in response to acute
hypertension. The same molecular responses mediating acute pressure natriuresis also occur in
models of chronic hypertension [40-42]. Previous studies implicate cytochrome P450 (CYP)
metabolites of arachidonic acid, epoxyeicosatrienoic acids (EET) and 20-
hydroxyeicosatetraenoic acid (20-HETE; most abundant CYP metabolite), as potential mediators
of pressure natriuresis. In the kidney, 20-HETE inhibits Na
+
,K
+
-ATPase activity and reduces
sodium reabsorption from the proximal tubule to the medullary thick ascending limb. Studies
inhibiting 20-HETE formation describe attenuated pressure natriuresis in response to increases in
renal perfusion pressure [43-47]. However, overexpression of 20-HETE in the proximal tubule
promotes salt-sensitive hypertension, greater NCC phosphorylation, and a rightward shift in
renal function curves [48]. In the vasculature, 20-HETE promotes increases in vascular tone,
endothelial dysfunction, and renin-angiotensinogen-aldosterone system (RAAS) activation [44,
49]. Mice overexpressing 20-HETE in vascular smooth muscle cells exhibit hypertension and
10
reduced vasodilatory capacity which was reversed with 20-HETE inhibition [50]. These studies
emphasize the complex actions of 20-HETE as both antihypertensive and prohypertensive.
While great progress has contributed to our understanding of mechanisms involved in the
maintenance of fluid and electrolyte homeostasis, these studies are primarily performed in males
[51]. The notion that inclusion of females increases variability has been discredited and recent
notices from the NIH (NOT-OD-15-102) emphasize the need to consider sex as a significant
biological variable [52]. It is now well established that sex hormones and sex-specific differences
in kidney physiological function contribute to lower blood pressure and the lower prevalence of
kidney disease in premenopausal females compared to males [27]. These differences are also
evident in experimental rodent models where studies show male testosterone is associated with
greater expression of pro-hypertensive angiotensin II (AngII) type I (AT1) receptor, sodium
retention, greater oxidative stress, and a shift in the renin-angiotensin-aldosterone system
(RAAS) towards the pressor pathway in males [27, 53, 54]. Whereas in females, estrogens
reduce AT1 receptor expression, enhance anti-hypertensive angiotensin II type 2 (AT2) receptor
expression, and enhance angiotensin-converting enzyme 2 (ACE2) expression which breaks
down the main effector of RAAS, AngII [55-57]. Our lab has recently shown that sex-specific
differences exist in abundance and covalent modifications of kidney Na
+
transporters, channels,
claudins, and regulatory factors (collectively called transporters) [15]. Together with
mathematical models, these studies indicate females have lower fractional reabsorption in the
proximal tubule and greater reabsorption in the distal tubule which mediate a more rapid
natriuresis in females versus males when challenged with a saline load [58, 59]. Interestingly, in
normotensive females, NHE3 is localized to the base of the microvilli (mimicking the
distribution pattern seen in males with hypertension) which facilitates the greater capacity to
11
excrete a saline load. These observations underlie the aims of the current study to address this
important physiological gap in our understanding of pressure natriuresis in females. This study
aimed to define the physiological and molecular responses contributing to pressure natriuresis in
female Sprague Dawley rats.
12
Methods
Animal protocol
All animal procedures were approved by the Institutional Animal Care and Use Committee of the
Keck School of Medicine of the University of Southern California and were conducted in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Rats.
Experiments were performed using 22 male and 33 female Sprague Dawley (SD) rats, 8-
10 weeks of age, purchased from Envigo (Indianapolis, IN). Rats were housed under 12-hour
light-dark cycles with ab libitum access to food (Rodent diet 5001, LabDiet) and water. Rats
were anesthetized with a small dose of intramuscular ketamine (100 µl) followed by
intraperitoneal thiobutabarbital (Inactin, 100-125 mg/kg). Body temperature was maintained
thermostatically at 37ºC. Polyethylene (PE-50) catheters filled with heparinized saline (20 U
heparin/ml), connected to the transducer, signal amplifier, and flatbed recorder were inserted into
the carotid artery for in vivo blood pressure monitoring. PE-50 catheters were inserted in the
jugular vein for constant infusion (50-100 ul/min) of 4% Bovine Serum Albumin in 0.9% saline
to maintain euvolemia. Urine was collected and measured via bladder cannulation directly into
pre-weighed microcentrifuge tubes. Vaginal smears were obtained by lavage (0.3 ml sterile
saline – 0.9% NaCl w/v) and cytology via 10X objective (Nikon TMS, Japan) used to determine
stage in estrus cycle in females. Acute increases in mean arterial pressure were accomplished by
increases total peripheral vascular resistance as first introduced by Roman and Cowley [35] and
described in Figure 1-1. In set 1 (n=5/group), mean arterial pressure was acutely raised by step-
wise constriction of the celiac artery and superior mesenteric artery (15 min) followed by
abdominal aorta (20 min) below the renal arteries with black braided surgical ligatures as
13
described by Ivy, J.R. and Bailey, M.A. [60]. SHAM surgery underwent identical procedure with
ligatures placed around arteries but did not undergo vasoconstriction.

Figure 1-1. Experimental protocol schematic for acute hypertension surgeries. Euvolemia was
maintained by constant infusion of 4% Bovine Serum Albumin in 0.9% saline via jugular vein
catheter (50-100 µl/min). For a subset of rats (n=10 total), 0.1% FITC-sinistrin (MediBeacon, St.
Louis, MO) was added to the jugular vein infusate. Polyethylene catheters filled with heparinized
saline (20 U heparin/ml), connected to a transducer, signal amplifier, and flatbed recorder were
inserted into the carotid artery for in vivo blood pressure monitoring. Tracheotomy was
performed to facilitate breathing. Surgical ligatures were places around celiac artery, superior
mesenteric artery, and abdominal aorta. Acute hypertension was evoked by vasoconstriction and
SHAM surgery underwent identical surgery without vasoconstriction. Urine was collected and
measured via bladder cannulation directly into pre-weighed microcentrifuge tubes. Kidneys were
rapidly excised following experiment and processed as described below.
Cannulated
Jugular Vein
Cannulated Carotid
Artery
In vivo BP Measure
Suture around Celiac &
Mesenteric Arteries
Suture around
Abdominal Aorta
Bladder cannula
Urine collection
BSA+
0.9% saline
Tracheotomy
BSA
+
Saline
+
*FITC-
Sinistrin
14
In set 2 (n=2-7/group), rats were treated with 1-aminobenzotrizaole (1-ABT, 50
mg/kg/day) or untreated (vehicle: 0.9% saline or no injection) via intraperitoneal injection under
brief anesthesia (2% inhaled isoflurane) for 5 days before surgery. Mean arterial pressure was
acutely raised by simultaneous constriction of the celiac artery, superior mesenteric artery, and
abdominal aorta arteries for 25 minutes or SHAM surgery. In both sets, blood samples were
taken through carotid artery cannula during pre- and post-vasoconstriction periods for collection
of plasma. Renal arteries were clamped and kidneys excised. One kidney was decapsulated,
dissected into cortex and medulla, and immediately homogenized (Ultra-Turrax T25) in isolation
buffer [5% sorbitol, 0.5 mM disodium EDTA, and 5 mM histidine-imidazole buffer, pH = 7.5,
with the addition of 0.2 mM phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 µl/ml of
phosphatase inhibitor cocktail (sigma P0044)]. Homogenates were quick-frozen in liquid
nitrogen and stored at -80°C as single-use aliquots. The other kidney was cut along the coronal
plane and bathed in ice-cold periodate-lysine-paraformaldehyde fixative (2% paraformaldehyde,
75 mM lysine, and 10 mM Na-periodate, pH 7.4) for 4 hours before being cryoprotected
overnight in 30% sucrose-PBS, embedded in Tissue-Tek OCT Compound, and frozen at -80ºC.

Glomerular Filtration Rate (GFR) determination
For a subset of rats (n=10 total), 0.1% FITC-sinistrin (MediBeacon, St. Louis, MO) was added to
the jugular vein infusate (4% BSA + 0.9% saline) for measurement of glomerular filtration rate.
Plasma and urine samples were diluted 1:10 and 1:200, respectively, in 0.5 M HEPES (pH 7.4)
and 100 µl loaded onto a 96 well plate. Fluorescence was measured using SpectraMax iD3
Multi-Mode Microplate Reader (excitation wavelength 480 nm and emission wavelength
530nm). Concentration of FITC-sinistrin was calculated by: [FITC-sinistrin]=((mean RFU ± y-
15
intercept)/slope) X dilution factor. GFR was calculated by: GFR (ml.min)= GFR = (UFITC-sinistrin x
UV)/PFITC-sinistrin. Where P is the plasma sinistrin concentration (mg/ml) calculated as the mean of
the serum samples collected before and after vasoconstriction; V is the urine flow rate (ml/min);
and U is the urine sinistrin concentration (mg/ml).

Electrolyte analysis
Urine volumes were measured gravimetrically. [Na
+
], [K
+
], and [Li
+
] in plasma and urine were
measured by flame photometry (Cole-Parmer, model 02655-10. IL, USA). Endogenous lithium
clearance (CLi), an estimate of volume flow leaving the proximal tubule and medullary thick
ascending limb, was calculated classically as (urinary [Li
+
] X urine volume)/plasma [Li
+
].
Sodium clearance (CNa), an estimate of overall sodium reabsorption along nephron, was
calculated as (urinary [Na
+
] X urine volume)/Plasma [Na
+
].

K
+
-Dependent p-nitrophenyl phosphatase (K-pNPPase) enzymatic assay
K
+
-pNPPase activity, a measure of K
+
dependent ATPase activity [11, 47], was measured in
kidney cortical and medullary homogenates. K
+
-pNPPase reaction product, p-nitrophenyl, is can
be determined colorimetrically. A sodium solution [100 mM TRIS, 20 mM MgCl2, 10 mM
EDTA, and 200 mM NaCl], potassium solution [100 mM TRIS, 20 mM MgCl2, 10 mM EDTA,
and 200 mM KCl], and quench solution [1 N NaOH, 50 mM EDTA, and 0.1% Triton] were
made fresh prior to assay. In 15-second intervals, 10 µl of cortical or medullary homogenates
were added to 0.5 ml of sodium or potassium solution in triplicate. After 30 minutes, the reaction
was stopped by addition of 2 ml of quench solution in 15 second intervals. Sample absorbance
was read using a 96-well plate and plate reader at 410 nm wavelength. µmole Pi was calculated
16
by [(potassium absorbancesample - sodium absorbancesample) – (potassium absorbancebackground –
sodium absorbancebackground] X [(1000 µmole/L)/extinction coefficient)] X final reaction volume
(L). Total K
+
-pNPPase activity was calculated by µmole Pi/[(protein concentrationsample (mg/ul))
X Time (hr) X sample volume assayed (ul)].

Semiquantitative immunoblotting and immunofluorescence
Protein concentration in each homogenate was determined using a bicinchoninic acid (BCA)
assay. Homogenates with equal protein concentration were denatured in Laemmli sample buffer
for 20 min at 60°C. Uniform sample preparation was confirmed by densitometry of Coomassie-
stained gels loaded with equal amounts of protein as previously described [61]. To validate the
linearity of the detection system, 1X and ½X amounts of samples were loaded in parallel and
quantitated. Normalized values of 1X and ½X runs were averaged for statistical analysis.
Supplemental Table A-1 provides loading amounts, antibody information, dilutions, and
incubation times. Signals were detected and quantified with the Odyssey Infrared Imaging
System and the accompanying software (LI-COR, Lincoln, NE). For immunofluorescence,
kidneys were excised, bisected, and fixed in PLP fixative for 4 hours. The kidneys were then
cryoprotected overnight in 30% sucrose-PBS, embedded in Tissue-Tek OCT compound, and
frozen at -80ºC. Cryosections (5µm) were cut and transferred to charged glass slides, dried then
rehydrated in PBS for 10 minutes, washed in 50 mM NH4Cl in PBS then antigen retrieval with
1% SDS in PBS for 5 minutes. Sections were blocked using 1% BSA in PBS to reduce
background before double-labeling with polyclonal ani-NHE3 and monoclonal anti-villin (both
1:100) in 1% BSA in PBS for 2 hours. Following the same protocol, additional sections were
double labeled with polyclonal anti-NaPi2 (1:50) and monoclonal anti-a adaptin (1:50). The
17
sections were incubated with a mixture of Alexa 488-conjugated goat anti-rabbit and Alexa 568-
conjugated goat anti-mouse secondary antibodies diluted 1:500 in 1% BSA in PBS for 1 hour
before mounting in Prolong Antifade and dried overnight. Sections were examined using the
Leica TCS SP8 DIVE (Leica Microsystems, Wetzlar, Germany) confocal/multiphoton laser
scanning microscope system.

Statistical analysis
Statistical comparisons were performed using GraphPad Prism 9.2.0 (San Diego, CA) two-way
ANOVA followed by Dunnett multiple comparisons post hoc test or mixed-effects analysis
followed by Sidaks multiple comparisons post hoc test. Comparisons for renal function curves
were performed using correlation tests and linear regression analysis. P values provided.
18
Results
Effect of vasoconstriction on mean arterial pressure in female versus male rats
To define acute pressure natriuretic response in females, both female (F) and male (M) SD rats
underwent surgical procedures to acutely raise vascular resistance via constriction of the celiac
artery, superior mesenteric artery, and abdominal aorta as previously reported [10, 12, 13, 40]. In
protocol 1, rats underwent step-wise vasoconstriction to determine the influence of different
arterial beds on blood pressure response (Figure 1-2A). At baseline, females (91 ± 5 mmHg)
exhibit lower mean arterial pressure (MAP) versus males (105 ± 3, P=0.03). Vasoconstriction of
the celiac artery and mesenteric artery raised blood pressure from 91 ± 5 to 126 ± 5 mmHg in F
and 105 ± 3 to 130 ± 3 mmHg in M, P<0.001 for both (Figure 2-2A). The change in MAP with
vasoconstriction tended to be greater in F but did not reach statistical significance (D34 ± 4 in F
and D25 ± 2 mmHg in M, P=0.07). Constriction of the abdominal aorta raised MAP to similar
levels as celiac and mesenteric artery. In protocol 2, simultaneous constriction of the three
arterial beds raised blood pressure from 90 ± 2 to 123 ± 3 in F and 93 ± 2 to 132 ± 3 mmHg in
M, P<0.01 for both (Figure 1-2B). Given the similar response between step-wise constriction and
simultaneous constriction, excretion data from pre- to 25 minutes post-vasoconstriction from
protocol 1 (n=5/group) and protocol 2 (n=4-7/group) have been combined as one group moving
forward.

19

Figure 1-2. Similar hemodynamic response to vascular constriction in male and female SD rats.
Comparisons of mean arterial pressure (MAP) following (A) step-wise constriction of the celiac
and mesenteric arteries before constriction of the abdominal aorta in male (blue circle; n=5) and
female (red triangles; n=5) or (B) simultaneous constriction of the celiac, mesenteric, and
abdominal aorta arteries in male (blue circles; n=4) and female (red triangles; n=7) rats. Mean
arterial pressure was measured throughout the experiment via carotid artery cannulation
connected to the transducer, signal amplifier, and flatbed recorder. Data expressed as individual
values with error bars denoting mean ± SEM. Statistical comparisons were performed using
GraphPad Prism (9.2.0) one-way ANOVA followed by Sidaks multiple comparisons post hoc
test. *P<0.05 versus pre-constriction mean arterial pressure.
A
B
Pre- 5 10 15 20 25 30 35
Pre vs Post:
Male P=0.009
Female P<0.001
Pre- 5 10 15 20 25 30 35
60
80
100
120
140
160
MAP (mmHg)
Pre- 5 10 15 20 25
60
80
100
120
140
160
MAP (mmHg)
Pre- 5 10 15 20 25
Pre vs Post:
Male P=0.004
Female P=0.004
Celiac &
Mesenteric
Abdominal Aorta
Celiac &
Mesenteric
Abdominal Aorta
Celiac, Mesenteric, & abdominal aorta Celiac, Mesenteric, & abdominal aorta
Female Male
20
Impact of acute hypertension on renal function and kidney transporters
Physiological responses to acute hypertension are summarized in Figure 1-3, Table 1-1, and
Supplemental Figure 1-1. At baseline, females (5 ± 1 µmol/min) exhibit 2.5-fold greater sodium
excretion (UNaV) compared to males (2 ± 1µmol/min). Urine volume (UV) was 75% greater in
females versus males with no difference in urinary potassium excretion (UKV) (Supplemental
Figure 1-1B-D). UNaV increased 21-fold in females and 19-fold in males in response to acute
hypertension. UV increased 16- and 13-fold and UKV increased 8- and 9-fold in females and
males, respectively. Pressure natriuresis response was sustained over 25 minutes following
vasoconstriction. Females exhibit 45% greater sum-total UNaV over 25 minutes compared to
males however it did not reach statistical significance (P=0.09) (Figure 1-3C-D). Renal function
curves depict the relationship between excretion and MAP. UNaV was strongly correlated with
MAP in F (P<0.001) and moderately correlated in M (P=0.004) (Figure 1-3B). Linear regression
analysis exhibits a leftward shift in renal function curves in females compared to males, evident
by a significant difference in elevation or y-int, P<0.001. This leftward shift is also evident in
UV vs MAP but not UKV vs MAP (Supplemental Figure 1-1B,D).

21
Figure 1-3. More robust natriuresis response in females with acute hypertension. Urine was
collected and measured via bladder cannulation directly into pre-weighed microcentrifuge tubes
over 5-minute intervals in male (blue circles; n=9) and females (red triangles; n=12) SD rats. Na
was measured by flame photometry and displayed as (A) Urinary sodium excretion (UNaV), (B)
UNaV plotted against mean arterial pressure (MAP), and (C-D) sum-total UNaV. Data expressed
as individual measurements (A-C) or mean ± SEM. Statistical comparisons were performed
using GraphPad Prism (9.2.0) two-way ANOVA followed by Dunnetts multiple comparisons
post hoc test (A,C-D) or correlation test followed by linear regression analysis (B).

Pre- 5 10 15 20 25
0
20
40
60
80
100
UNaV (µmol/min)
Time post-constriction (min)
5 10 15 20 25
0
500
1000
1500
2000
Sum-Total UNaV

(µmol)
Pre- 5 10 15 20 25
Time post-constriction (min)
Two-Way ANOVA
P
HTN
<0.001
P
Sex
=0.10
P
Interaction
=0.047
5 10 15 20 25
Two-Way ANOVA
P
HTN
<0.001
P
Sex
=0.09
P
Interaction
=0.13
80 100 120 140
0
20
40
60
80
MAP (mmHg)
UNaV (µmol/min)
M
F
r=0.35; P=0.004
r=0.66; P<0.001
Linear regression:
Slope P=0.12
Elevation P<0.001
5 10 15 20 25
0
500
1000
1500
2000
Time (min)
Sum-Total UNaV

(µmol)
P=0.09
Males
Females
Time post-constriction (min)
A B
C D
22
Glomerular filtration rate (GFR) was assessed in a subset of rats using FITC-sinistrin
infused through jugular vein cannulation. Average GFR increased 16- to 20-fold in both sexes in
response to acute hypertension but rapidly autoregulated by 10 minutes and sustained 2-fold
higher than baseline levels for the duration of the experiment (Table 1-1 and Supplemental
Figure 1-1A). This pattern was mirrored by potassium clearance (CK) which increased 6.3- fold
in males and 4.6-fold in females with acute hypertension but rapidly reduced by 10 minutes post-
vasoconstriction and sustained for the duration of the experiment (Table 1-1 and Supplemental
Figure 1-1C). Endogenous lithium clearance (CLi), a measure of volume flow from the proximal
tubule and medullary thick ascending limb, and sodium clearance (CNa), a measure of whole
nephron volume flow was determined (Table 1-1). At baseline, females (versus males) exhibit
1.2- and 2.5-fold greater CLi and CNa, respectively, although neither reached statistical
significance. Following vasoconstriction, CLi increased 7-fold in both sexes while CNa
increased 9-fold in females and 11-fold in males (all P<0.02). Elevated clearance of lithium and
sodium was sustained over 20-25 minutes (Table 1-1).

23
Table 1-1. Physiological parameters in male and female SD rats with acute hypertension

Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism
(9.2.0) mixed-effects analysis followed by Tukeys multiple comparisons post hoc test. P<0.05
considered significant *versus male basal value or ^versus female basal value.
#
For GFR, n=3 for
male and n=7 for female.

Parameter
Male (n=9) Female (n=12)
Basal
Pre-
5 min
High BP
20-25 min
High BP
Basal
Pre-
5 min
High BP
20-25 min
High BP
Glomerular Filtration Rate
#
(GFR; ml/min)
1.3 ± 0.3 16 ± 8* 2.7 ± 0.3 1.1 ± 0.2 20 ± 7^ 2.5 ± 0.3
Urine Volume (μl/min) 12 ± 3 123 ± 30* 71 ± 16* 21 ± 4 208 ± 19^ 102 ± 12
Lithium Clearance (μl/min) 82 ± 23 540 ± 99* 261 ± 48* 94 ± 13 625 ± 99^ 344 ± 68^
Sodium Clearance (μl/min) 15 ± 7 169 ± 48* 135 ± 22* 38 ± 8 324 ± 34^ 239 ± 20^
Potassium Clearance (μl/min) 0.8 ± 0.2 5 ± 1* 1.4 ± 0.2 0.8 ± 0.1 3.7 ± 0.4^ 1.32 ± 0.09
24
After 25-35 minutes of acute hypertension (vs respective SHAM), females exhibit 21%
lower medullary (m-) NHE3 and 17% lower mNKCC2 abundance whereas males exhibit 16%
lower NCC, 29% lower NCCpS71, and 30% greater full-length gENaC (no change in cleavage)
(Figure 1-4 A-B). Both females and males exhibit 21-22% greater proximal tubule NHE3p
although it did not reach statistical significance. Changes in abundance and covalent
modifications are summarized in Figure 1-5. To determine K
+
dependent ATPase activity, K
+
-
Dependent p-nitrophenyl phosphatase (K-pNPPase activity) enzymatic assay was performed in
kidney cortex and medulla homogenates (Figure 1-4C). Females (vs males) exhibit 18% and
31% greater K-pNPPase activity in the cortex and medulla, respectively. However, acute
hypertension did not change K-pNPPase activity in either cortex or medulla in either sex. Kidney
sections from female rats after acute hypertension (or SHAM) were stained and visualized on the
same slide (Figure 1-4D). Villin (red, left), the microvillar actin bunding protein, was used to
indicate PT microvilli whereas AP-2 (red, right), a clathrin adaptor protein, was used to indicate
clathrin-coated pits. NHE3 in females (green, left) was distributed at the base of the microvilli in
SHAM rats consistent with our previous reports [15], with no evidence of further redistribution
with hypertension. NaPi2 (green, right) staining shows strong signal within the microvilli and
loss of signal with vasoconstriction suggesting internalization or degradation.

25
Figure 1-4. Impact of acute hypertension on transporters along the nephron in male and female
SD rats. Effect of vascular constriction on transporters, channels, and regulatory kinases along
the nephron. 1X and 1/2X amounts were assayed to confirm linearity of detection system (one
amount shown); assay and immunoblot details are provided in Supplemental Table 1. (A)
Immunoblots for proximal tubule to collecting duct proteins (m prefix denotes medulla). Relative
abundances (arbitrary density values) were normalized to SHAM = 1 for both male and females;
data expressed as mean ± SEM; n=7-12/group were assayed and used for statistical comparisons
(n=5/group shown for immunoblots). kD indicated apparent molecular weight of the stained
markers and indicated lane loaded with prestained protein ladders (BioRad). (B) Graphs
expressing individual values of immunoblots plotted as relative abundance normalized to SHAM
A
B C
D
- 75
- 75
- 100
1.00 ± 0.07 1.10 ± 0.09 1.0 ± 0.1 1.06 ± 0.06
- 75
- 75
- 100
NHE3
Male
SHAM HTN
Female
SHAM HTN kD
NHE3p
1.00 ± 0.05 1.21 ± 0.08 1.0 ± 0.1 1.22 ± 0.06
1.00 ± 0.08 0.97 ± 0.09 1.00 ± 0.09 0.79 ± 0.05^
1.00 ± 0.09 0.90 ± 0.08 1.0 ± 0.1 0.79 ± 0.07
- 75
- 100
1.0 ± 0.1 0.99 ± 0.05 1.00 ± 0.05 0.92 ± 0.04
Villin
- 75
1.00 ± 0.04 1.09 ± 0.06 1.00 ± 0.05 1.11 ± 0.06
NaPi2
mNHE3
mNHE3p
- 150
- 250
- 150
- 250
1.00 ± 0.08 0.82 ± 0.08 1.00 ± 0.07 0.83 ± 0.04^
1.0 ± 0.1 0.84 ± 0.07 1.0 ± 0.1 0.91 ± 0.05
mNKCC2
mNKCC2p
- 75
- 75
1.00 ± 0.07 0.99 ± 0.09 1.0 ± 0.1 0.85 ± 0.08
1.0 ± 0.1 0.7 ± 0.1 1.0 ± 0.2 0.6 ± 0.2
mSPAK
mSPAKp
- 75
- 100
Male
SHAM HTN
Female
SHAM HTN kD
1.00 ± 0.03 1.29 ± 0.05* 1.00 ± 0.04 1.02 ± 0.04
!ENaC
FLà
Clà
- 150
- 150
- 250
- 150
- 250
- 150
1.00 ± 0.06 1.11 ± 0.1 1.00 ± 0.08 1.01 ± 0.06
- 250
NKCC2
NKCC2p
1.00 ± 0.04 1.02 ± 0.08 1.00 ± 0.05 0.97 ± 0.05
1.00 ± 0.03 0.84 ± 0.05* 1.00 ± 0.06 1.04 ± 0.03
1.00 ± 0.04 0.85 ± 0.08 1.00 ± 0.07 1.02 ± 0.08
1.00 ± 0.03 0.71 ± 0.08* 1.00 ± 0.1 0.96 ± 0.07
NCC
NCCpT53
NCCpS71
- 75
- 75
1.0 ± 0.09 1.7 ± 0.3 1.0 ± 0.2 1.11 ± 0.1
1.00 ± 0.1 0.95 ± 0.09 1.00 ± 0.1 0.9 ± 0.1
SPAKà
SPAKpà
1.00 ± 0.05 1.02 ± 0.06 1.00 ± 0.03 1.10 ± 0.06
- 250
- 250
- 150
Male Female
0.0
0.5
1.0
1.5
2.0
NHE3
Male Female
0.0
0.5
1.0
1.5
2.0
mNHE3
P=0.04
Male Female
0.0
0.5
1.0
1.5
2.0
mNKCC2
P=0.03
Male Female
0.0
0.5
1.0
1.5
2.0
NKCC2
Male Female
0.0
0.5
1.0
1.5
2.0
NCCpS71
P=0.01
Male Female
0.0
0.5
1.0
1.5
2.0
NHE3p
P=0.06 P=0.050
Male Female
0.0
0.5
1.0
1.5
2.0
mNHE3p
Male Female
0.0
0.5
1.0
1.5
2.0
mNKCC2p
Male Female
0.0
0.5
1.0
1.5
2.0
NKCC2p
Male Female
0.0
0.5
1.0
1.5
2.0
Villin
Male Female
0
1
2
3
4
SPAK
P=0.052
Male Female
0.0
0.5
1.0
1.5
2.0
γ ENaC - Fl
P<0.001
Male Female
0.0
0.5
1.0
1.5
2.0
NaPi2
Male Female
0.0
0.5
1.0
1.5
2.0
mSPAK
P=0.09
Male Female
0.0
0.5
1.0
1.5
2.0
SPAKp
Male Female
0.0
0.5
1.0
1.5
2.0
γ ENaC - Cl
Male Female
0.0
0.5
1.0
1.5
2.0
mSPAKp
P=0.09
Male Female
0.0
0.5
1.0
1.5
2.0
NCC
P=0.02
Relative Abundance (SHAM=1)
SHAM
HTN
NHE3
Villin
NHE3
Villin
NaPi2
AP-2
NaPi2
AP-2
Female SHAM
Female HTN
M F
0.4
0.5
0.6
0.7
0.8
0.9
Cortex
K-pNPPase activity
(umole Pi/mg*hr)
SHAM
HTN
P=0.005
P=0.005
M F
0
1
2
3
Medulla
K-pNPPase activity
(umole Pi/mg*hr)
P=0.01
P=0.009
26
= 1 and error bars denoting mean ± SEM. (C) K+-dependent p-nitrophenyl phosphatase (K-
pNPPase) activity, estimate of K
+
dependent ATPase activity, was measured in kidney cortex
and medullary homogenates (see methods for details). (D) Immunofluorescence microscopy of
NHE3 (green, left; polyclonal anti-NHE3) and NaPi2 (green, right; polyclonal anti-NaPi2 1:50).
Villin (red, left monoclonal anti-villin 1:100), the microvillar actin bunding protein, was used to
indicate PT microvilli whereas AP-2 (red, right; monoclonal anti-AP2 1:50), a clathrin adaptor
protein, was used to indicate clathrin-coated pits. Statistical comparisons were performed using
GraphPad Prism (9.2.0) unpaired students t test. P<0.05 *versus SHAM male and ^versus
SHAM female (A) and P values provided (B-C). Abbreviations: ENaC: epithelial Na
+
channel γ
full length (Fl) or cleaved (Cl); NaPi2: Na
+
-phosphate cotransporter 2A; NHE3: Na
+
/H
+

exchanger isoform 3; NCC: Na
+
-Cl
-
cotransporter; NCCp -S71 –T53: NCC phosphorylated at
S71 and T53; NHE3p: NHE3 phosphorylated at S552; NKCC2: Na
+
-K
+
-2Cl
-
cotransporter
isoform 2; NKCC2p -T39T101: NKCC2 phosphorylated at T39, T101; SPAK: Ste/SPS-1 related
proline-alanine rich kinase; SPAKp: SPAK phosphorylated at S373.

27

Figure 1-5. Transporter profile summary in male and female SD rats with acute hypertension
(n=7-11/group). Summary of data shown in Figure 1-4. Data expressed as mean ± SEM for each
protein normalized to respective SHAM = 1.

NHE3
NHE3p
NaPi2
Villin
NHE3
NHE3p
SPAK
SPAKp
NKCC2
NKCC2p
SPAK
SPAKp
NKCC2
NKCC2p
NCC
NCCpT53
NCCpTS71
yENaC - Fl
yENaC - Cl
0.0
0.5
1.0
1.5
2.0
2.5
Relative Abundance
(SHAMs = 1)
Male HTN Female HTN
* *
*
* *
Cortex Medulla Cortex
28
Physiological and transporter responses to ABT
To investigate the influence of cytochrome P450 (CYP) metabolites of arachidonic acid (AA) on
pressure natriuresis in females, both female and male rats were treated with 1-
aminobenzotriazole (ABT; 50 mg/kg/day) for 5 days in order to block formation of
epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) formation.
Rats received 5 days of treatment with ABT, vehicle (0.9% saline), or were untreated (no
injections) prior to undergoing identical surgeries as previously outlined in this study. No
differences were detected between vehicle treated and untreated rats as outlined in Figure 1 and
Table 1-1. These rats were combined and collectively termed untreated. Both ABT treated
females (FABT) and males (MABT) exhibit 9-12% lower baseline MAP compared to untreated
rats but only reached statistical significance in females (Table 1-2). There was no difference in
the rise of MAP in response to vasoconstriction in either sex with ABT treatment. ABT treatment
did not affect UNaV (Figure 1-6A,E) or UV (Table 1-2) at baseline in either sex. However, CLi
was 64% and 51% lower in FABT and MABT versus untreated rats, respectively, but only
reached statistical significance in females. CNa tended to be lower in FABT and greater in
MABT versus untreated rats but did not reach statistical significance either (Table 1-2).

29
Table 1-2. Impact of ABT on physiological parameters in female and male rats with acute HTN
Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism
(9.2.0) mixed-effects analysis followed by Tukeys multiple comparisons post hoc test. P<0.05
considered significant *versus untreated basal value, ^versus ABT basal value.

Female Parameters
Untreated (F; n=12) ABT (FABT; n=8)
Basal
Pre-
5 min
High BP
20-25 min
High BP
Basal
Pre-
5 min
High BP
20-25 min
High BP
Mean Arterial Pressure
(MAP; mmHg)
90 ± 3 124 ± 3* 117 ± 4* 80 ± 5* 112 ± 9^ 100 ± 7
Urine Volume (μl/min) 21 ± 4 208 ± 19* 102 ± 12* 14 ± 5 164 ± 30^ 60 ± 7^
Lithium Clearance (μl/min) 94 ± 13 625 ± 99* 344 ± 68* 34 ± 7* 408 ± 66^ 124 ± 34^
Sodium Clearance (μl/min) 38 ± 8 324 ± 34* 239 ± 20* 26 ± 6 405 ± 61^ 161 ± 28^
Potassium Clearance (μl/min) 0.8 ± 0.1 3.7 ± 0.4* 1.32 ± 0.09 1.2 ± 0.3 9 ± 2^ 2.2 ± 0.05
Male Parameters
Untreated (M; n=9) ABT (MABT; n=4)
Basal
Pre-
5 min
High BP
20-25 min
High BP
Basal
Pre-
5 min
High BP
20-25 min
High BP
Mean Arterial Pressure
(MAP; mmHg)
100 ± 3 131 ± 2* 121 ± 4* 91 ± 6 137 ± 1^ 119 ± 1^
Urine Volume (μl/min) 12 ± 3 123 ± 30* 71 ± 16* 11 ± 4 262 ± 63 140 ± 40
Lithium Clearance (μl/min) 82 ± 23 540 ± 99* 261 ± 48* 40 ± 19 457 ± 61^ 181 ± 38
Sodium Clearance (μl/min) 15 ± 7 169 ± 48* 135 ± 22* 25 ± 14 471 ± 83^ 281 ± 64
Potassium Clearance (μl/min) 0.8 ± 0.2 5 ± 1* 1.4 ± 0.2 0.5 ± 0.2 9 ± 2 5 ± 1^
30
ABT treated females (versus untreated females) exhibit similar increases in UNaV and
sum-total UNaV in response to acute hypertension (Figure 1-6A,C, and D). However, ABT
treated males (versus untreated males) exhibit greater UNaV (PABT=0.01) and sum-total UNaV
(P=0.01) (Figure 1-6A,G, and H). Renal function curves plotting UNaV vs MAP, exhibit a
leftward shift with ABT treatment in both females (elevation P<0.001) and males (slope
P=0.002) (Figure 1-6B,F) that is also evident in UV (Supplemental Figure 1-2 A-B). ABT
treatment had no effect on UKV (Supplemental Figure 1-2 C-F) in either sex. ABT treatment did
not affect abundance or covalent modification of kidney transporters in female SHAM rats
(Figure 1-7 A-B). Acute hypertension in ABT treated females (vs ABT treated SHAM) exhibit
similar transporter patterns previously described in this study for females, greater cortical (c-)
NHE3p, lower mNHE3 and mNKCC2 but only reaching significance for mNKCC2 (Figure 1-7
A-C). Additionally, acute hypertension in ABT treated females exhibited greater full-length
cgENaC (no change in cleavage) not previously seen in untreated females (Figure 1-7 A-C).
ABT treatment in male SHAM rats versus untreated SHAM rats, exhibit lower cNHE3, cNaPi2,
mNHE3, cSPAK, cSPAKp, cNKCC2, cNKCC2p, c cNCCpT53 and -S71 (Figure 1-8 A-B).
However, only reaching statistical significance in NCCpT53 and NCCpS71, respectively. Acute
hypertension in untreated males exhibit similar responses previously described in this study
whereas ABT treated males had no statistically significant difference in any transporter
compared to respective SHAM rats (Figure 1-8 A-C).

31
Figure 1-6. ABT shift renal function curves leftward in both sexes but only increases sodium
excretion in males. Female (F and FABT; n=8-12/group) and males (M and MABT; n=4-
9/group) were either treated with ABT (50 mg/kg/day for 5 days; FABT and MABT) or
untreated (receiving either vehicle injection of 0.9% saline or no injection; F and M) prior to
acute hypertension surgery. Urine was collected and measured via bladder cannulation directly
into pre-weighed microcentrifuge tubes over 5-minute intervals Na was measured by flame
photometry and displayed as (A,E) Urinary sodium excretion (UNaV), (B,F) UNaV plotted
against mean arterial pressure (MAP), and (C-D, G-H) sum-total UNaV. Data expressed as
individual measurements (A-C, E-G) or mean ± SEM (D,H). Statistical comparisons were
performed using GraphPad Prism (9.2.0) Correlation test and linear regression analysis (B, F) or
mixed effects analysis followed by Sidaks multiple comparisons post hoc test (D,H).
Pre- 5 10 15 20 25
0
20
40
60
80
100
UNaV (µmol/min)
Time post-constriction (min)
5 10 15 20 25
0
500
1000
1500
2000
Sum-Total UNaV

(µmol)
Pre- 5 10 15 20 25
Time post-constriction (min)
Two-Way ANOVA
P
HTN
<0.001
P
ABT
=0.67
P
Interaction
=0.03
5 10 15 20 25
Two-Way ANOVA
P
HTN
<0.001
P
ABT
=0.43
P
Interaction
=0.89
60 80 100 120 140
0
20
40
60
80
100
MAP (mmHg)
UNaV (µmol/min)
F
FABT
r=0.66; P<0.001
r=0.68; P<0.001
Linear regression:
Slope P=0.30
Elevation P<0.001
5 10 15 20 25
0
500
1000
1500
2000
Time (min)
Sum-Total UNaV

(µmol)
Pre- 5 10 15 20 25
0
20
40
60
80
100
UNaV (µmol/min)
Time post-constriction (min)
5 10 15 20 25
0
500
1000
1500
2000
Sum-Total UNaV

(µmol)
Pre- 5 10 15 20 25
Time post-constriction (min)
Two-Way ANOVA
P
HTN
<0.001
P
ABT
=0.01
P
Interaction
<0.001
5 10 15 20 25
Two-Way ANOVA
P
HTN
<0.001
P
ABT
=0.01
P
Interaction
<0.001
60 80 100 120 140
0
20
40
60
80
100
MAP (mmHg)
UNaV (µmol/min)
M
MABT
r=0.35; P=0.004
r=0.73; P<0.0001
Linear regression:
Slope P=0.002
5 10 15 20 25
0
500
1000
1500
2000
Time (min)
Sum-Total UNaV

(µmol)
P=0.01
F
FABT
Time post-constriction (min)
M
MABT
Time post-constriction (min)
A
B
C D
E
F
G H
32

NHE3
NHE3p
NaPi2
Villin
NHE3
NHE3p
SPAK
SPAKp
NKCC2
NKCC2p
NKA α1
NKA α1
SPAK
SPAKp
NKCC2
NKCC2p
NCC
NCCpT53
NCCpTS71
yENaC - Fl
yENaC - Cl
0.0
0.5
1.0
1.5
2.0
2.5
Relative Abundance
(Untreated SHAM = 1)
Untreated SHAM ABT SHAM
NHE3
NHE3p
NaPi2
Villin
NHE3
NHE3p
SPAK
SPAKp
NKCC2
NKCC2p
NKA α1
NKA α1
SPAK
SPAKp
NKCC2
NKCC2p
NCC
NCCpT53
NCCpTS71
yENaC - Fl
yENaC - Cl
0.0
0.5
1.0
1.5
2.0
2.5
Relative Abundance
(SHAMs = 1)
Untreated HTN ABT HTN
*
*
Cortex Medulla Cortex
- 75
- 75
- 75
- 100
1.00 ± 0.06 0.97 ± 0.05 0.91 ± 0.06
- 75
- 75
- 75
- 150
- 250
- 100
NHE3
ABT
SHAM
Untreated
SHAM
kD
NHE3p
NaPi2
Villin
mNHE3
mNHE3 à
mSPAKp
mNKCC2
mNKCC2p
mNKA⍺1
- 75
mSPAKà
ABT
HTN
Untreated
HTN
- 75
- 150
- 250
- 75
- 75
0.91 ± 0.09
1.00 ± 0.07 1.17 ± 0.05 1.06 ± 0.08 0.90 ± 0.08
1.00 ± 0.08 1.1 ± 0.1 0.94 ± 0.02 0.82 ± 0.04
1.00 ± 0.07 0.85 ± 0.05 1.19 ± 0.07 1.1 ± 0.1
1.00 ± 0.05 0.80 ± 0.06 0.73 ± 0.07 0.85 ± 0.6
1.0 ± 0.2 0.90 ± 0.07 0.8 ± 0.1 1.1 ± 0.2
1.00 ± 0.04 0.8 ± 0.2 0.9 ± 0.1 0.9 ± 0.1
1.0 ± 0.3 0.7 ± 0.2 0.8 ± 0.2 0.9 ± 0.3
1.00 ± 0.01 0.90 ± 0.06 0.81 ± 0.07^ 1.10 ± 0.07
1.0 ± 0.2 1.02 ± 0.05 1.0 ± 0.1 1.2 ± 0.1
1.00 ± 0.07 0.95 ± 0.06 0.76 ± 0.07 0.96 ± 0.07
"ENaC
- 75
- 50
- 75
- 150
- 250
- 150
- 250
- 75
SPAKà
SPAKpà
NKCC2
NKCC2p
NCC
NCCpT53
NCCpS71
- 75
NKA ⍺1
1.00 ± 0.04 0.99 ± 0.03 0.97 ± 0.05 1.06 ± 0.06
1.0 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 1.0 ± 0.1
1.0 ± 0.2 1.1 ± 0.2 1.0 ± 0.1 1.2 ± 0.2
1.00 ± 0.08 0.92 ± 0.08 0.86 ± 0.05 0.93 ± 0.05
1.0 ± 0.1 0.94 ± 0.07 0.96 ± 0.05 1.04 ± 0.06
1.0 ± 0.1 1.04 ± 0.04 1.22 ± 0.08 1.13 ± 0.09
1.0 ± 0.1 1.0 ± 0.1 1.23 ± 0.08 1.2 ± 0.1
1.0 ± 0.1 1.0 ± 0.1 1.1 ± 0.1 1.2 ± 0.2
1.00 ± 0.06 1.10 ± 0.06 1.16 ± 0.04^ 0.9 ± 0.1
1.00 ± 0.06 1.23 ± 0.05 1.2 ± 0.1 1.24 ± 0.07
FLà
Clà
- 100
- 150
- 250
- 150
- 250
- 150
- 250
Females
33
Figure 1-7. ABT treatment has little effect on transporter abundance and covalent modifications
in female SD rats. 1X and 1/2X amounts were assayed to confirm linearity of detection system
(one amount shown); assay and immunoblot details are provided in Supplemental Table 1. (A)
Immunoblots for proximal tubule to collecting duct proteins (m prefix denotes medulla). Relative
abundances (arbitrary density values) were normalized to untreated SHAM surgery = 1; data
expressed as mean ± SEM; n=3-6/group were assayed and used for statistical comparisons. First
three lanes of vehicle SHAM are repeats from protocol 1 rats in Figure 4 to assess reproducibility
but excluded from statistical comparisons here. kD indicated apparent molecular weight of the
stained markers and indicated lane loaded with prestained protein ladders (BioRad). (B)
Transporter profile summarizing impact of ABT treatment without hypertension through
comparisons of ABT treated SHAM surgery rats. (C) Transporter profile summarizing impact of
ABT treatment with acute hypertension through comparisons of untreated rats and ABT treated
rats normalized to their respective SHAM = 1. Data expressed as mean ± SEM. Statistical
comparisons were performed using GraphPad Prism (9.2.0) Two-way ANOVA followed by
Sidak’s multiple comparisons post hoc test. P<0.05 ^versus ABT SHAM female in (A) and
*versus ABT SHAM female in (B). Abbreviations: ENaC: epithelial Na
+
channel γ full length
(Fl) or cleaved (Cl); NaPi2: Na
+
-phosphate cotransporter 2A; NHE3: Na
+
/H
+
exchanger isoform
3; NCC: Na
+
-Cl
-
cotransporter; NCCp -S71 –T53: NCC phosphorylated at S71 and T53; NHE3p:
NHE3 phosphorylated at S552; NKA: Na
+
,K
+
-ATPase alpha 1 subunit; Na NKCC2: Na
+
-K
+
-2Cl
-

cotransporter isoform 2; NKCC2p -T39T101: NKCC2 phosphorylated at T39, T101; SPAK:
Ste/SPS-1 related proline-alanine rich kinase; SPAKp: SPAK phosphorylated at S373.

34

Untreated
HTN
ABT
SHAM
Untreated
SHAM
-75
-75
-75
-100
0.6 ± 0.1 1.00 ± 0.07 1.1 ± 0.2 0.9 ± 0.2
-75
-75
-75
-150
-250
-100
NHE3
kD
NHE3p
0.9 ± 0.2 1.00 ± 0.03 1.3 ± 0.2 1.2 ± 0.2
0.59 ± 0.01 1.00 ± 0.04 0.98 ± 0.06 0.7 ± 0.1
0.99 ± 0.02 1.0 ± 0.1 1.08 ± 0.06 1.11 ± 0.04
0.77 ± 0.04 1.00 ± 0.06 0.83 ± 0.07 0.7 ± 0.1
0.95 ± 0.05 1.0 ± 0.2 0.80 ± 0.09 0.7 ± 0.1
0.7 ± 0.2 1.0 ± 0.4 0.8 ± 0.2 1.2 ± 0.5
1.05 ± 0.02 1.0 ± 0.4 0.84 ± 0.05 1.0 ± 0.2
1.4 ± 0.1 1.0 ± 0.3 0.8 ± 0.1 0.9 ± 0.1
1.10 ± 0.04 1.0 ± 0.1 1.1 ± 0.1 1.1 ± 0.1
NaPi2
Villin
mNHE3
mNHE3pà
mSPAKp
mNKCC2
mNKCC2p
mNKA⍺1
-75
1.16 ± 0.02 1.0 ± 0.2 1.1 ± 0.1 1.2 ± 0.4
mSPAKà
ABT
HTN
-75
-150
-250
-75
-75
-75
-150
-250
0.4 ± 0.1 1.00 ± 0.01 1.0 ± 0.2 0.73 ± 0.2
-150
-250
SPAKà
SPAKpà
0.5 ± 0.2 1.06 ± 0.05 0.83 ± 0.06 0.7 ± 0.1
0.50 ± 0.04 1.0 ± 0.1 0.84 ± 0.1 0.7 ± 0.2
0.64 ± 0.03 1.0 ± 0.1 0.81 ± 0.05 0.80 ± 0.09
0.61 ± 0.02 1.00 ± 0.03 0.74 ± 0.05 0.8 ± 0.1
0.52 ± 0.07* 1.0 ± 0.2 0.68 ± 0.04* 0.63 ± 0.06
0.43 ± 0.03* 1.00 ± 0.03 0.62 ± 0.05* 0.48 ± 0.08
1.1 ± 0.1 1.00 ± 0.02 1.20 ± 0.08 1.3 ± 0.1
NKCC2
NKCC2p
NCC
NCCpT53
NCCpS71
FLà
Clà
-100
NKA ⍺1
0.78 ± 0.1 1.0 ± 0.1 0.84 ± 0.05 0.81 ± 0.05
0.90 ± 0.04 1.00 ± 0.08 1.3 ± 0.2 0.87 ± 0.07
-75
-50
-150
-250
-150
-250
-150
-250
-75 "ENaC
Males
NHE3
NHE3p
NaPi2
Villin
NHE3
NHE3p
SPAK
SPAKp
NKCC2
NKCC2p
NKA α1
NKA α1
SPAK
SPAKp
NKCC2
NKCC2p
NCC
NCCpT53
NCCpTS71
yENaC - Fl
yENaC - Cl
0.0
0.5
1.0
1.5
2.0
2.5
Relative Abundance
(Untreated SHAM = 1)
Untreated SHAM ABT SHAM
*
*
NHE3
NHE3p
NaPi2
Villin
NHE3
NHE3p
SPAK
SPAKp
NKCC2
NKCC2p
NKA α1
NKA α1
SPAK
SPAKp
NKCC2
NKCC2p
NCC
NCCpT53
NCCpTS71
yENaC - Fl
yENaC - Cl
0.0
0.5
1.0
1.5
2.0
2.5
Relative Abundance
(SHAMs= 1)
Untreated HTN ABT HTN
* *
Cortex Medulla Cortex
35
Figure 1-8. ABT treatment lowers transporter abundance and covalent modifications in male SD
rats. 1X and 1/2X amounts were assayed to confirm linearity of detection system (one amount
shown); assay and immunoblot details are provided in Supplemental Table 1. (A) Immunoblots
for proximal tubule to collecting duct proteins (m prefix denotes medulla). Relative abundances
(arbitrary density values) were normalized to vehicle treated SHAM surgery = 1; data expressed
as mean ± SEM; n=2-5/group were assayed and used for statistical comparisons. First two lanes
of untreated SHAM and first lane of untreated HTN are repeats from protocol 1 rats in Figure 4
to assess reproducibility but excluded from statistical comparisons here. kD indicated apparent
molecular weight of the stained markers and indicated lane loaded with prestained protein
ladders (BioRad). (B) Transporter profile summarizing impact of ABT treatment without
hypertension through comparisons of ABT treated SHAM surgery rats. (C) Transporter profile
summarizing impact of ABT treatment with acute hypertension through comparisons of
untreated rats and ABT treated rats normalized to their respective SHAM = 1. Data expressed as
mean ± SEM. Statistical comparisons were performed using GraphPad Prism (9.2.0) Two-way
ANOVA followed by Sidak’s multiple comparisons post hoc test. *P<0.05 versus untreated
SHAM male. Abbreviations: ENaC: epithelial Na
+
channel γ full length (Fl) or cleaved (Cl);
NaPi2: Na
+
-phosphate cotransporter 2A; NHE3: Na
+
/H
+
exchanger isoform 3; NCC: Na
+
-Cl
-

cotransporter; NCCp -S71 –T53: NCC phosphorylated at S71 and T53; NHE3p: NHE3
phosphorylated at S552; NKA: Na
+
,K
+
-ATPase alpha 1 subunit; Na NKCC2: Na
+
-K
+
-2Cl
-

cotransporter isoform 2; NKCC2p -T39T101: NKCC2 phosphorylated at T39, T101; SPAK:
Ste/SPS-1 related proline-alanine rich kinase; SPAKp: SPAK phosphorylated at S373.

36
Discussion
This study aimed to determine the physiological and molecular responses contributing to
pressure natriuresis in female Sprague Dawley (SD) rats. We demonstrate females (versus males)
have a more robust acute pressure natriuretic response to acute hypertension that evokes a
leftward shift in renal function curves (i.e., greater capacity to excrete sodium at lower blood
pressure) and greater sum-total UNaV. The pressure natriuresis response is mediated by distinct
patterns in abundance and covalent modifications which suggests natriuresis derives from lower
sodium transport in PT-mTAL in females and PT and DCT in males. Additionally, we show
inhibition of 20-HETE via 1-aminobenzotriazole treatment evokes greater sodium excretion and
lower transporter abundance in males (not females) and shifts renal function curves leftward in
both sexes.

Physiological responses to acute hypertension
To investigate mechanisms responsible for acute pressure natriuretic response we increased total
peripheral vascular resistance through vasoconstriction, a protocol adapted from Roman and
Cowley [35]. We did not observe serial pressure ramps with step-wise vasoconstriction as
suggested by Ivy, J.R. and Bailey, M.A. [60], instead, constriction of celiac artery, superior
mesenteric artery, and abdominal aorta resulted in similar levels of hypertension whether
constricted in series or simultaneously. Baseline MAP was lower in females compared to males
which is consistent with other experimental [62, 63] and clinical [64] reports. Vasoconstriction
increased MAP equivalent levels in both females and males although the difference compared to
pre-constriction tended to be greater in females (D34 ± 4 in F and D25 ± 2 mmHg in M, P=0.07)
and could contribute to natriuresis. A significant rise in GFR five minutes post-constriction was
37
rapidly autoregulated to a similar extent in females and males. Albeit a more mild disruption, this
is consistent with early reports in males using this experimental model [35]. GFR remained 2-
fold higher than pre-constriction levels for the remainder of the experiments. The first finding of
the current study is demonstrating females have a more robust acute pressure natriuretic response
to acute hypertension that evokes a leftward shift in renal function curves (i.e., greater capacity
to excrete sodium at lower blood pressure) and greater sum-total UNaV. Despite similar MAP,
females exhibit significantly greater UNaV and UV compared to males with acute hypertension.
Together these observations culminate in a leftward shift in renal function curves in females
compared to males. In other words, females excrete more sodium and volume at lower arterial
pressures versus males. Our findings are consistent with previous reports in SHRs showing
chronic hypertension is more severe in males versus females and acute step-wise reductions in
blood pressure exhibit a leftward shift in renal function curves in females versus males [65].
Additionally, studies in FVB/N mice undergoing a similar surgery as our current study, also
show a leftward shift in female renal function curves compared to males [66]. Over 25 minutes
females had greater sum-total UNaV confirming our previous reports that females have a greater
capacity to excrete sodium [15]. Together these findings suggest baseline sexual dimorphisms in
kidney transport, RAAS, and hormonal status confer a female “head-start” to maintaining ECV
and handling a hypertensive challenge. Recent studies indicate this female head-start also
contributes to female SD rats having a greater ability to maintain sodium homeostasis during
dietary sodium challenges compared to males [67].

38
Renal function and distinct adaptation of sodium transporters
The second finding of the current study is acute hypertension provokes distinct changes in the
abundance of key Na
+
transporters and covalent modifications in females and males. Our
previous studies in males demonstrate that redistribution of proximal tubule NHE3 to the base of
the microvilli and NaPi2 and NCC to subapical endosomes plays an integral role in both acute
[11-14] and chronic [40, 41] pressure natriuresis. In these studies, we reported no change in the
total pool size of sodium transporters during acute hypertension. Contrary to our previous
reports, the current study shows acute hypertension evokes distinct changes in pool size and
covalent modifications in female and male rats. A possible explanation for these contradictory
results could be the use of subcellular fractions in previous studies versus kidney homogenates in
the current study which allow for better detection of total pool sizes. In the proximal tubule,
acute hypertension was associated with greater NHE3p (which is associated with reduced
activity [14, 68]) and lower medullary NKCC2 in both males and females. In females, not males,
medullary NHE3 and NHE3p were significantly lower with acute hypertension versus Sham. To
determine the functional implications of these transporter adaptations, endogenous lithium
clearance (a marker for volume flow leaving proximal tubule and medullary thick ascending
limb) was measured. At baseline, females (versus males) exhibit greater lithium clearance
consistent with our previous studies [15, 69]. Acute hypertension increased lithium clearance
6.6-fold in both sexes and remained 3.2 to 3.6-fold higher than baseline levels after 20-25
minutes. This is consistent with our previous studies in males, which show acute hypertension
evokes a 5- to 6-fold increase in lithium clearance after 5 min that tended to fall to 3-fold over
control after 30-min [68, 70]. While females have lower fractional sodium reabsorption at
baseline contributing to greater lithium clearance, we show the pressure natriuretic response to
39
acute hypertension is similar in males and females compared to their respective baseline. At
baseline, females (versus males) exhibit 2.5-fold greater sodium clearance, a measure of whole
nephron volume flow, mediated by lower proximal tubule transport [15]. However, acute
hypertension increased sodium clearance 9- and 11-fold in both females and males, respectively.
This increase in sodium clearance remained 6- and 9-fold higher than baseline levels in females
and males, respectively, after 20-25 minutes. While females have greater absolute excretion of
sodium, the greater increase in sodium clearance in males is likely mediated by the lower NCC
and NCCp abundance with acute hypertension. Interestingly, potassium clearance spiked after 5
minutes but rapidly returned to baseline levels by 10 minutes. This spike in potassium excretion
is likely associated with the rapid spike and autoregulation of GFR after 5 minutes of acute
hypertension. However, these observations of differential handling of sodium and potassium
suggest pressure natriuretic response is sodium selectivity. Taken together the similar increase in
lithium clearance and adaptations in the proximal tubule give credence to the hypothesis that
baseline differences in transporters confer a female head-start to adaptations to acute
hypertension. However, we also show adaptations of transporters to acute hypertension are sex-
specific.

Role of cytochrome P450 metabolites of arachidonic acid in females and males
The third finding of the current study is 1-aminobenzotriazole (ABT) treatment evoked greater
sodium excretion and lower transporter abundance in males (not females) but shifted renal
function curves leftward in both sexes. Sacerdoti D. et al. [71] provided the first evidence for the
role of CYP metabolites of AA on blood pressure by showing depletion of CYP metabolites
normalized blood pressure in SHRs. Previous studies by our lab using cobalt chloride and others
40
using ABT to inhibit 20-HETE production show attenuated pressure natriuretic response by
blunting the inhibition of Na
+
, K
+
-ATPase and sodium transport in response to increases in renal
perfusion pressure [43-47]. To investigate the potential role of CYP metabolites on pressure
natriuresis in females, we used 1-aminobenzotriazole to block 20-HETE formation before
surgical protocols. ABT treatment lowered blood pressure at baseline in both females and males
versus untreated rats, although differences only reached statistical significance in females. This is
consistent with previous reports in male rats treated with ABT [72]. We predicted ABT treatment
would blunt pressure natriuresis as previously reported in males. However, ABT treatment in
males was associated with lower sodium transporter abundance and greater sodium excretion.
While this was not observed in females, ABT treatment did shift renal function curves leftward
in both sexes. These findings are inconsistent with previous reports of blunted sodium and
volume excretion in ABT-treated male rats [43, 45, 46] or our previous reports using cobalt
chloride to inhibit CYP [47]. However, recent reports show overexpression of 20-HETE in the
proximal tubule promotes salt-sensitive hypertension, greater NCC phosphorylation, and a
rightward shift in renal function curves [48]. In the vasculature, 20-HETE promotes increases in
vascular tone, endothelial dysfunction, and renin-angiotensinogen-aldosterone system (RAAS)
activation [44, 49]. Mice overexpressing 20-HETE in vascular smooth muscle cells exhibit
hypertension and reduced vasodilatory capacity which was reversed with 20-HETE inhibition
[50]. The results of the current study provide evidence inhibition of 20-HETE shifts the blood
pressure set-point leftward in males and females. However, a limitation of the current study is
the low n’s for ABT-treated males. Taken together, the lower blood pressure and a leftward shift
of renal function curves with ABT suggest 20-HETEs pro-hypertensive role in the vascular may
predominate over the anti-hypertensive role in the kidney.
41
In summary, females exhibit more robust pressure natriuresis than males in response to
acute hypertension mediated by distinct alterations in transporters along the nephron. We provide
evidence that pressure natriuresis in females relies on proximal tubule and medullary thick
ascending limb regulation whereas males may rely on proximal tubule and distal convoluted
tubule. These findings suggest baseline sexual dimorphisms in kidney transporter [15], RAAS
[66], and hormonal status [65] confer a female “head-start” to maintaining ECV and handling a
hypertensive challenge. Together, these results suggest mediators of acute pressure natriuresis
and physiological responses to acute hypertension are sex-specific.

42
Supplemental Information

Supplemental Table 1-1. Antibody and immunoblot protocol details…………………………...43

Supplemental Figure 1-1. GFR, UV vs MAP, UKV, and UKV vs MAP in male and female SD
rats with acute hypertension……………………………………………………………..44

Supplemental Figure 1-2. Impact of ABT on UV vs MAP, UV, and UKV vs MAP in male and
female SD rats with acute hypertension…………………………………………………45

43
Supplemental Table 1-1. Antibody and immunoblot protocol details. ~kDa refers to apparent molecular weight determined by BIO-
RAD Precision Plus Protein
TM
Duel Color Standards. Ab=antibody, Mu=mouse, Rb=rabbit, Sh=sheep, O/N=overnight, DAS=donkey
anti-sheep, GAM=goat anti-mouse, and N/A=not assayed
Antibody
Target
~kDa
Protein/
lane
cortex
(µg)
Protein/
lane
medulla
(µg)
Primary
antibody
supplier
Ab
host
Dilution Time
Secondary
antibody
supplier
Host
and
target
Dilution Time Ref
γENaC
75
60
60, 30 15,7 .5 Palmer Rb 1:1000 2hr Invitrogen
GAR
680
1:5000 1 hr [73]
Na,K-
ATPase α1
~100 1, 0.5 1, 0.5
Kashgarian
(Yale)
Mu 1:200 O/N Invitrogen
GAM
680
1:5000 1 hr [74]
NaPi2
50-
75
40, 20 N/A McDonough Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [75]
NHE3 ~75 15, 7.5 8, 4 McDonough Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [68]
NHE3-
pS552
~75 5, 2.5 8, 4
Santa Cruz
(53962)
Mu 1:1000 2 hr LI-COR
GAM
680
1:5000 1 hr [76]
NCC 150 60, 30 N/A McDonough Rb 1:5000 O/N Invitrogen
GAR
680
1:5000 1 hr [42]
NCCpS71 150 20, 10 N/A
Loffing
(Zurich)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [77]
NCCpT53 150 60, 30 N/A
Loffing
(Zurich)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [77]
NKCC2 160 20, 10 8, 4
DSHB
(Iowa)
Mu 1:6000 O/N Invitrogen
GAM
800
1:5000 1 hr [78]
NKCC2p-
T96T101
160 20,10 8, 4
Forbush
(Yale)
Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [79]
SPAK
60-
70
20, 10 10, 5 Delpire S15 Rb 1:3000 2 hr Invitrogen
GAR
680
1:5000 1 hr
[80,
81]
SPAKp-
S373
60-
70
80, 40 4, 2
DSTT
(Dundee)
Sh 1:2500 2 hr Invitrogen
DAS
680
1:5000 1 hr
[80,
82]
Villin 100 5, 2.5 N/A
Santa Cruz
(sc58897)
Mu 1:2000 O/N Invitrogen
GAM
800
1:5000 1 hr [83]

44

Supplemental Figure 1-1. (A) GFR (n=10 total) with black line denoting mean ± SEM, (B)UV vs
MAP, (C) UKV, and (D) UKV vs MAP in male (n=9) and female (n=12) SD rats with acute
hypertension. Statistical comparisons were performed using GraphPad Prism (9.2.0) mixed
effects analysis followed by Sidaks multiple comparisons post hoc test or student t-test (A) or
correlation test and linear regression analysis (B,D).
Pre- 5 10 15 20 25
0
10
20
30
40
50
UKV (µmol/min)
Time post-constriction (min)
Pre- 5 10 15 20 25
Time post-constriction (min)
60 80 100 120 140
0.0
0.1
0.2
0.3
0.4
MAP (mmHg)
UV (ml/min)
r=0.54; P<0.001
r=0.77; P<0.001
M
F
Linear regression:
Slope P=0.02
80 100 120 140
0
10
20
30
50
MAP (mmHg)
UKV (µmol/min)
M
F
r=0.33; P=0.007
r=0.40; P<0.001
Linear regression:
Slope P=0.34
Elevation P=0.60
Pre- 5 10 15 20 25
0
1
2
3
4
20
40
60
GFR (ml/min)
M
F
P<0.001
Time post constriction (min)
Pre- 25 min
0
1
2
3
4
GFR (ml/min)
P<0.001

45

Supplemental Figure 1-2. Impact of ABT on (A,B) urine volume (UV) vs mean arterial pressure
(MAP), (C,E) UKV, and (D,F) UKV vs MAP in male and female SD rats with acute
hypertension. Statistical comparisons were performed using GraphPad Prism (9.2.0) correlation
test and linear regression analysis (A-B,D,F).

60 80 100 120 140
200
400
600
0
MAP (mmHg)
UV (µl/min)
M
MABT
r=0.54; P<0.0001
r=0.74; P<0.0001
Linear regression:
Slope P=0.003
Pre- 5 10 15 20 25
0
10
20
30
40
50
UKV (µmol/min)
Time post-constriction (min)
Pre- 5 10 15 20 25
0
10
20
30
40
50
UKV (µmol/min)
Time post-constriction (min)
60 80 100 120 140
200
400
600
0
MAP (mmHg)
UV (µl/min)
F
FABT
Linear regression:
Slope P=0.33
Elevation P<0.0001
r=0.77; P<0.0001
r=0.65; P<0.0001
Pre- 5 10 15 20 25
Time post-constriction (min)
Pre- 5 10 15 20 25
Time post-constriction (min)
60 80 100 120 140
0
5
10
15
20
25
50
MAP (mmHg)
UKV (µmol/min)
M
MABT
r=0.33; P=0.007
r=0.78; P<0.0001
Linear regression:
Slope P=0.93
Elevation P=0.40
60 80 100 120 140
0
5
10
15
20
25
50
MAP (mmHg)
UKV (µmol/min)
F
FABT
Linear regression:
Slope P=0.34
Elevation P=0.06
r=0.40; P<0.001
r=0.47; P=0.001

46
Chapter 2. Sex-Specific Adaptations to Obesity Preserve Kidney Function in
Female ZSF1 Rats

Abstract
Background. Obesity has reached epidemic levels and is a primary driver for parallel increases in
diabetes and hypertension. Together, diabetes and hypertension are responsible for nearly three-
fourths of all chronic kidney disease cases and strong risk factors for cardiovascular related
mortality. Significant sex disparities are evident in both disease susceptibility and outcomes
driving efforts to include sex as a biological variable. Previous studies show obesity and diabetic
kidney disease can cause alterations in kidney function and electrolyte imbalances. However, the
impact of these diseases along the nephron is poorly understood. In the current study, we aim to
define sexual dimorphisms in renal tubular responses to obesity and early diabetic kidney disease
and to determine the physiological consequences of these differences have in disease
progression.

Methods. Zucker Spontaneously Hypertensive Heart Failure F1 hybrid (ZSF1) rats are either lean
or obese depending on heterozygosity or homozygosity for the leptin receptor gene mutation,
respectively. Lean rats are hypertensive without metabolic complications whereas obese rats are
hypertensive with metabolic complications that mimic human disease. Two cohorts of ZSF1 rats
were studied: 1) lean male (LM), obese male (OM), lean female (LF), and obese female (OF)
ZSF1 rats (10 weeks old; n=4/group) and 2) obese male ZSF1 rats (n=5-6/group) fed either a
0.3% Na
+
diet or 0.1% Na
+
diet for 10 weeks. Both were assessed using metabolic cages
collections, flame photometry, blood pressure by tail-cuff sphygmomanometer, and semi-

47
quantitative immunoblotting of kidney transporters, channels, claudins, and regulatory factors
(collectively called transporters).

Results. Both lean and obese rats exhibit hypertension independent of sex. While OM were more
hyperphagic and exhibited greater leptinemia, OF exhibited greater obesity respective to leans
rats. OM, not OF, develop hyperglycemia, glycosuria, and glomerular hyperfiltration. Both OM
and OF exhibit a 2-fold increase in sodium glucose transporter 1 and hyperinsulinemia
independent of hyperglycemia. While both OM and OF develop progressively worsening
proteinuria, it is more severe in males consistent with more severe glomerulopathy evident by
COLIV and trichrome staining. Transporters along the nephron in lean ZSF1 rats exhibit similar
sexual dimorphic transporter pattern as previously described in normotensive SD with lower
proximal tubule transporters and greater distal convoluted tubule transporters in females versus
males. OM and OF similarly exhibit lower abundance of key Na
+
transporters versus lean
counterparts: cortical NHE3, NHE3p, claudin 2, SPAKp, NKCC2, NKCC2p, NCC, NCCp,
claudin 7, 8, and 10 and medullary NHE3, NHE3p NKCC2, and NKCC2p. OM, not OF, exhibit
lower AQP1 and renin-angiotensin-aldosterone system components (i.e. ACE1 and ACE2)
versus LM. OF, not OM, exhibit greater proximal tubule NBCe-1A, villin, NaPi2, and OAT1
versus LF. Lower dietary sodium intake in OM blunted proteinuria progression and lowered
glycosuria but tended to increase hyperglycemia.

Conclusion. Obesity provokes a lower abundance of key Na
+
transporters along the nephron
independent of hyperglycemia or baseline sexual dimorphic patterns. Female advantage is

48
partially mediated by baseline sex-specific differences along the nephron that give females a
head-start in renal adaptations to hypertension and metabolic challenges.

49
Introduction
The global prevalence of obesity has tripled over the last five decades, reaching epidemic
levels [16, 84]. These patterns are even starker in the United States where an estimated 74% of
American adults are overweight or obese [17]. Obesity is associated with a range of
cardiometabolic disorders most notably the two leading causes of chronic kidney disease (CKD)
– hypertension and diabetes – and is now considered an independent risk factor for kidney
disease [20, 21]. Approximately 40% of diabetic patients will develop diabetic kidney disease
(DKD) which increases the risk of progression to end-stage kidney disease (ESKD) and
cardiovascular-related mortality [85, 86]. As the prevalence of obesity-driven DKD and
hypertension continue to rise it heightens the demand on researchers to better understand the
underlying pathophysiology and develop more effective therapeutics. Both obesity and diabetes
have previously been shown to alter hemodynamics, activate RAAS, activate the sympathetic
nervous system, increase sodium reabsorption, and cause kidney pathology [24, 84, 87].
Premenopausal women have a lower prevalence of diabetes and hypertension despite greater
obesity compared to men indicating sex-specific differences in disease susceptibility [88, 89].
Although advances in diagnostic capabilities and treatments have extended the lives of obese and
diabetic patients, the increasing prevalence indicates these metabolic disorders remain clinically
unmet needs.
Recent clinical trials indicating the beneficial effects of sodium-glucose cotransporter
(SGLT) 2 inhibitors emphasize the potential of targeted therapeutics and the necessity for a
better understanding of kidney adaptations associated with obesity and diabetes [90, 91].
Previous studies into the impact of diabetes or obesity along the nephron have primarily focused
on the glomerulus, glucose transporters, or a subset of Na
+
transporters [92, 93]. In addition,

50
studies have involved numerous diabetic models each with their limitations in translatability to
the human disease [32, 94, 95]. In 2000, Tofovic et al [96] were the first to characterize Zucker
Diabetic Fatty Spontaneously Hypertensive Heart Failure F1 hybrid (ZSF1) rats generated from
crossing Zucker Diabetic Fatty rats (+/fa) and Spontaneously Hypertensive Heart Failure rats
(+/fa
cp
). The F1 progeny are either lean or obese depending on heterozygosity or homozygosity
for the leptin receptor gene mutation, respectively [97, 98]. Obese (fa/fa
cp
) male ZSF1 rats are a
unique model, both genetically hyperphagic and hypertensive which together contribute to the
development of diabetes that progresses to ESKD. The similarity to the progressive human
disease suggests this model provides high translatable value [33, 34]. In obese female ZSF1,
there are conflicting reports of euglycemia [99-101] and hyperglycemia [102]. Nonetheless,
obese female ZSF1 rats seem to be comparatively protected from the development of
hyperglycemia but exhibit diabetic risk factors of obesity, hypertension, and dyslipidemia.
However, the impact of these diseases along the nephron is ill-defined in either sex. The current
study aimed to define sexual dimorphisms in renal tubular responses to obesity and early diabetic
kidney disease and to determine the physiological consequences of these differences on disease
progression.

51
Methods
All animal procedures were approved by the Institutional Animal Care and Use Committee of the
Keck School of Medicine of the University of Southern California and were conducted in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Rats. Schematic of experimental protocols are provided in Supplemental Figure 1.

Experimental protocol 1
Experiments were performed on obese male and female Zucker Diabetic Fatty Spontaneously
Hypertensive Heart Failure F1 hybrid (ZSF1) rats and their lean littermates (n=4/group; 16 rats
total – 8 weeks of age), purchased from Charles River Laboratories (Wilmington, MA). Rats
were acclimated and housed under 12-hour light-dark cycles with ab libitum access to food
(Rodent diet 5001, LabDiet) and water. Body weights were measured weekly. At 10, 16, and 18
weeks of age, blood was collected via tail snip under brief anesthesia (2% isoflurane) and rats
were placed in metabolic cages for 24-hour urine collections with ab libitum access to food and
water which were also measured. At 18 weeks of age, rats were anesthetized intramuscularly
with ketamine (80 mg/kg, Phoenix Pharmaceuticals, St. Joseph, MO):xylazine (8 mg/kg, Lloyd
laboratories, Shenandoah, IA) in a 1:1 volume ratio. Vaginal smears were obtained by lavage
(0.3 ml sterile saline – 0.9% NaCl w/v) and cytology via 10X objective (Nikon TMS, Japan)
used to determine estrus cycle in females. A blood sample was collected from the tail for
measurement of hematocrit in Micro-Cal heparinized hematocrit capillary tubes. After clamping
renal arteries both kidneys were excised. One kidney was decapsulated, dissected into cortex and
medulla, and immediately homogenized (Ultra-Turrax T25) in isolation buffer [5% sorbitol, 0.5
mM disodium EDTA, and 5 mM histidine-imidazole buffer, pH = 7.5, with the addition of 0.2

52
mM phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 µl/ml of phosphatase inhibitor
cocktail (sigma P0044)]. Homogenates were quick frozen in liquid nitrogen and stored at -80°C
as single-use aliquots. The other kidney was cut along the coronal plane with one half bathed in
ice-cold periodate-lysine-paraformaldehyde fixative (2% paraformaldehyde, 75 mM lysine, and
10 mM Na-periodate, pH 7.4) for 4 hours before being cryoprotected overnight in 30% sucrose-
PBS, embedded in Tissue-Tek OCT Compound, and frozen at -80ºC. The other half was bathed
in 10% formalin for 24 hours before embedding in paraffin. There was no animal attrition during
the protocol. Protocol summarized in Figure 2-1A.

Experimental protocol 2
Experiments were performed on obese male ZSF1 rats (n=5-6/group; 12 rats total – 7 weeks of
age), purchased from Charles River Laboratories (Wilmington, MA). Rats were acclimated and
housed under 12-hour light-dark cycles with ab libitum access to food and water. Rats were fed
either 0.3% Na
+
diet (Envigo TD.7012) or 0.1% Na
+
diet (Envigo TD.7034) for 10 weeks during
the age window when obese ZSF1 males developed progressive albuminuria in animal protocol
1. Body weights were measured weekly. Blood glucose was measured at prior to (0 weeks), 5
weeks, and 10 weeks of feeding respective diets. Rats were placed in metabolic cages at 1, 3, 6,
8, and 10 weeks on diet for 24 hours urine collections with ab libitum access to food and water
which were also measured. One animal was excluded due to a significant difference in body
weight and baseline macroalbuminuria upon arrival from vendor. Protocol summarized in Figure
2-1B.

53

Figure 2-1. (A) Experimental protocol schematic for set 1 study in lean, obese and male, female
ZSF1 rats. (B) Protocol schematic for set 2 study in obese rats on 0.3% Na or 0.1% Na diet.

Treatment
Control Sodium
(0.3% Na
+
diet)
N=6
Lowered Sodium
(0.1% Na
+
diet)
n=5
10 Weeks
Continuous
measurements
Body weight
Blood pressure
Blood glucose
Food/water intake
24-hr urine collection
Plasma
A
B
Groups (n=4/group)
Lean males (LM)
Obese males (OM)
Lean females (LF)
Obese females (OF)
10-18 wks of age
Continuous
measurements
Body weight
Blood pressure
Blood glucose
Food/water intake
24-hr urine
collection
Plasma
8 week old animals

54
Blood pressure measurement
Systolic blood pressure was measured noninvasively by tail cuff plethysmography (Visitech
Systems BP2000, Apex, NC). Rats were acclimated to the system for one week, blood pressure
was measured over second week, and values for the last 4 days were averaged for each time
point.

Electrolyte, hormone, and glucose analysis
Urine volumes were measured with pipettes. [Na
+
], [K
+
], and [Li
+
] in plasma and urine were
measured by flame photometry (Cole-Parmer, model 02655-10. IL, USA). Endogenous lithium
clearance (CLi), an estimate of volume flow leaving the proximal tubule, was calculated
classically as (urinary [Li
+
] X urine volume)/plasma [Li
+
]. Sodium clearance (CNa), an estimate
of overall sodium reabsorption along nephron, was calculated as (urinary [Na
+
] X urine
volume)/Plasma [Na
+
]. Creatinine was measured in urine and plasma using capillary
electrophoresis (UT Southwest Medical Center O’Brein Kidney Research Core) and creatinine
clearance (CCr), an estimate of GFR, was calculated as (urinary [Cr] X urine volume)/plasma
[Cr]. Plasma leptin and insulin were measured using by DORI Metabolic Core at USC using
MILLIPLEX MAP Rat Metabolic Hormone Magnetic Bead Panel - Metabolism Multiplex Assay
(Millipore Sigma, St. Louis, MO). Blood glucose was measured using OWell Contour Next ONE
blood glucose system from tail prick in conscious rats. Urine glucose was measured using
Glucose Colorimetric Assay Kit (Cayman Chemical #10009582).

55
Histology and immunofluorescence
For histological staining, paraffin embedded kidneys were sectioned to 4 µm thickness and
Masson’s trichrome staining was performed using a Masson’s Trichrome Stain Kit
(Polysciences, Warrington, PA #25088) following the manufacturer’s instructions. For
immunofluorescence, paraffin embedded kidneys were again sectioned to 4 µm thickness.
Sections were incubated for 10 minutes in 0.1% Triton X-100 (Sigma T8787) in PBS before
heat-induced antigen retrieval by boiling sections in 10 mM sodium citrate (pH 6.0) for 8
minutes. To reduce nonspecific binding of antibodies, sections were blocked in donkey serum
(1:20) for 30 minutes. Slides were incubated in anti-collagen IV (1:100 Abcam ab6586) primary
antibody diluted in blocking buffer for 2 hours then Alexa Fluor 594 donkey anti-rabbit IgG
(Invitrogen A21207) for 1 hour. Sections were mounted using DAPI containing Vectashield
mounting medium. Sections were examined using the Leica TCS SP8 DIVE (Leica
Microsystems, Wetzlar, Germany) confocal/multiphoton laser scanning microscope system.

Semiquantitative immunoblotting
Protein concentration in each homogenate was determined using a bicinchoninic acid (BCA)
assay. Homogenates with equal protein concentration were denatured in Laemmli sample buffer
for 20 min at 60°C. Uniform sample preparation was confirmed by densitometry of Coomassie
stained gels loaded with equal amounts of protein as previously described [61] (Supplemental
Figure 2-1A) To assess the linearity of the detection system, 1X and ½X amounts of sample were
loaded in parallel and quantitated (Supplemental Figure 2-1B). Normalized values of 1X and ½X
runs were averaged for statistical analysis. Supplemental Table 2-1 provides loading amounts,

56
antibody information, dilutions and incubation times. Signals were detected and quantified with
the Odyssey Infrared Imaging System and accompanying software (LI-COR, Lincoln, NE).

K
+
-Dependent p-nitrophenyl phosphatase (K-pNPPase) activity
K
+
-pNPPase activity, estimate of Na
+
,K
+
ATPase activity [11, 47], was measured in kidney
cortical and medullary homogenates. K
+
-pNPPase reaction product, p-nitrophenyl, is bright
yellow which can be determined colorimetrically. A sodium solution [100 mM TRIS, 20 mM
MgCl2, 10 mM EDTA, and 200 mM NaCl], potassium solution [100 mM TRIS, 20 mM MgCl2,
10 mM EDTA, and 200 mM KCl], and quench solution [1 N NaOH, 50 mM EDTA, and 0.1%
Triton] were made fresh. In 15-second intervals, 10 µl of cortical or medullary homogenates
were added to 0.5 ml of sodium or potassium solution in triplicate. After 30 minutes, the reaction
was stopped by addition of 2 ml of quench solution in 15 second intervals. Sample absorbance
was read using a 96-well plate and plate reader at 410 nm wavelength. µmole Pi was calculated
by [(potassium absorbancesample - sodium absorbancesample) – (potassium absorbancebackground –
sodium absorbancebackground] X [(1000 µmole/L)/extinction coefficient)] X final reaction volume
(L). Total K
+
-pNPPase activity was calculated by µmole Pi/[(protein concentrationsample (mg/ul))
X Time (hr) X sample volume assayed (ul)].

Statistical analysis
Data represented as individual values and reported as means ± SEM. Statistical analyses were
performed using GraphPad Prism 9.2.0 (San Diego, CA). To evaluate sex, obesity, and time
variables simultaneously (e.g., clearance, KpNPPase activity, non-fasted blood glucose, and
albuminuria), differences between groups were compared by three-way ANOVA followed by a

57
Tukeys multiple comparisons post hoc test. Urine glucose from 10 weeks to 18 weeks of age
(two variables: sex, time), differences between groups were compared by two-way ANOVA
followed by Tukeys multiple comparison post hoc test. Differences between groups for 18-week
parameters only (e.g., Table 1-2) and all kidney proteins were compared by one-way ANOVA
followed by sidak or Tukeys multiple comparisons post hoc test. For protocol 2, differences
between 0.3% Na
+
diet and 0.1% Na
+
diet were compared by parametric unpaired students t-test.
P values < 0.05 was considered statistically significant.

58
Results
Effects of obesity and diabetes on physiological parameters
Among lean rats, there was no evidence of sex-specific differences in any measured parameter
except body weight which was greater in lean males (LM) compared to lean females (LF). At 18
weeks of age, hyperphagic obese rats exhibit greater food intake (75% greater in males and 50%
greater in females) and body weight versus lean rats (Table 2-1). When normalizing for body
weight, obese males (OM) and females (OF) similarly eat 71 grams food per kg body weight and
exhibit pronounced perirenal fat and lipidemia (Supplemental Figure 2-2). All groups (male,
female, lean, and obese) were hypertensive and exhibit similar plasma electrolytes levels. OM,
not OF, develop hyperglycemia and glycosuria. Plasma creatinine was 35% lower in OM versus
LM (P<0.001) and 23% lower in OF versus LF (LF; P<0.001) which may reflect greater GFR.
Plasma osmolality was significantly greater in OM (P=0.001) likely a consequence of osmotic
diuresis driven by elevated tubular glucose. Plasma osmolality tended to be greater in OF
compared to leans rats. OM (versus LM) exhibit 3.2-fold greater urine volume (UV), 72%
greater natriuresis (UNaV), and 93% greater kaliuresis (UKV) consistent with greater food and
water intake (all P<0.001). In OF (versus LF) there was no detectable difference in water intake,
UV, UNaV, UKV, or glycosuria despite greater food intake. Urine osmolality (Uosm) was not
significantly different between groups but correcting for 24 hr urine volume, urinary osmoles
excreted (UosmV) was 2.5-fold greater in OM versus LM (P<0.001) with no detectable
difference in females. Collectively these results exhibit greater alterations in physiological
parameters in OM versus LM compared to OF versus LF. This is potentially a consequence of
hyperglycemia in OM.

59
Table 2-1. Physiological parameters in ZSF1 rats at 18 weeks of age.
Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism
(9.2.0) one-way ANOVA followed by Sidak multiple comparisons post hoc test. P<0.05
considered significant *versus LM, ^versus LF, and #versus OM.

Parameter
Lean Male – LM
(N=4)
Obese Male – OM
(N=4)
Fold
Change
Lean Female - LF
(N=4)
Obese Female – OF
(N=4)
Fold
Change
Body weight (BW, g) 378 ± 5 573 ± 12* 1.5 230 ± 4* 420 ± 8^# 1.8
Systolic Blood Pressure
(SBP, mmHg)
168 ± 4 156 ± 5 NS 166 ± 7 155 ± 5 NS
Food Intake (g/24hrs) 23 ± 1 41 ± 2* 1.8 20 ± 1 30 ± 5# NS
Water Intake (ml/24hrs) 34 ± 1 66 ± 8* 1.9 34 ± 1 35 ± 7# NS
Plasma Sodium (mM) 131 ± 1 127 ± 1 NS 132 ± 2 127 ± 3 NS
Plasma Potassium (mM) 3.9 ± 0.4 4.3 ± 0.2 NS 3.6 ± 0.2 3.7 ± 0.2 NS
Plasma Lithium (mM) 0.23 ± 0.01 0.22 ± 0.01 NS 0.22 ± 0.01 0.22 ± 0.01 NS
Plasma Creatinine (µM) 20 ± 1 13 ± 1* 0.7 22 ± 1 17 ± 1^# 0.8
Plasma Osmolality (mM) 279 ± 1 295 ± 3* 1.1 279 ± 2 287 ± 3 NS
Blood Glucose (mg/dl) 93 ± 8 198 ± 31 2.1 112 ± 5 115 ± 10# NS
24-hr Urine Volume (UV, ml) 13 ± 2 42 ± 7* 3.2 14 ± 1 16 ± 3# NS
Urine Sodium Excretion
(UNaV, mmol/24 hrs)
2.5 ± 0.1 4.3 ± 0.3* 1.7 2.3 ± 0.1 2.6 ± 0.3# NS
Urine Potassium Excretion
(UKV, mmol/24 hrs)
4.6 ± 0.3 8.9 ± 0.7* 1.9 4.3 ± 0.2 5.3 ± 0.7# NS
Urine Creatinine Excretion
(UCrV, µmol/24 hrs)
51 ± 4 59.7 ± 0.4 NS 40 ± 2* 43 ± 2# NS
Urine Glucose (mg/dl) 9.0 ± 0.6 5978 ± 331* 664 4.3 ± 0.5 7.6 ± 0.8# NS
Urine Osmolality
(Uosm, mOsm/kg H2O)
1845 ± 183 1467 ± 140 NS 1727 ± 55 1731 ± 131 NS
Urine Osmoles Excretion
(UosmV, mOsm/24 hr)
22956 ± 1203 58316 ± 5202* 2.5 23963 ± 1032 26910 ± 3816# NS

60
LM (versus LF) exhibit 45-70% greater heart, kidney, and liver weight (Table 2-2, all
P<0.01) due to overall greater body mass and skeletal structure as evidence by tibia length (4.01
± 0.09 in LM vs 3.63 ± 0.05 in LF, P=0.05). OM (versus LM) exhibit 19-21% greater heart and
kidney weights whereas OF (versus LF) exhibit 40% greater heart and kidney weights (Table 2-
2). Suggesting that OF (versus OM) exhibit 2-fold greater heart and kidney hypertrophy. Both
OM and OF exhibit similar liver hypertrophy (2.7- and 2.4-fold greater than leans rats,
respectively).

Table 2-2. Organ weights and tibia lengths of ZSF1 rats at 18 weeks.

Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism
(9.2.0) one-way ANOVA followed by Sidak multiple comparisons post hoc test. P<0.05
considered significant *versus LM, ^versus LF, and #versus OM.
Parameter
Lean Male – LM
(N=4)
Obese Male – OM
(N=4)
Fold
Change
Lean Female - LF
(N=4)
Obese Female – OF
(N=4)
Fold
Change
Heart (g) 1.25 ± 0.06 1.49 ± 0.05* 1.2 0.86 ± 0.01* 1.21 ± 0.01^# 1.4
Kidney (g) 1.66 ± 0.04 2.01 ± 0.08* 1.2 1.02 ± 0.03* 1.43 ± 0.06^# 1.4
Liver (g) 11.7 ± 0.9 32.0 ± 1* 2.7 6.9 ± 0.3* 16.3 ± 0.6^# 2.4
Tibia (cm) 4.03 ± 0.09 4.08 ± 0.09 NS 3.63 ± 0.05* 3.73 ± 0.1# NS

61
OM develop progressively worsening hyperglycemia and glycosuria not detected in OF despite
similar alterations in glucose transporters
At 10 weeks of age, there was no detectable difference in non-fasted blood glucose between
groups (Figure 2-2A). By 16 weeks, OM develop pronounced hyperglycemia (P<0.001 versus 10
weeks, Figure 2-2A) that was not observed in OF which was confirmed by 18-week fasted blood
glucose (Figure 2-2B). Blood glucose strongly correlated with albuminuria (r=0.70 and P<0.001,
Figure 2-2C). Interestingly, OM exhibit pronounced glycosuria at 10 weeks despite lack of
detectable hyperglycemia (Figure 2-2D). Although glycosuria was considered within normal
reference range for LM, LF, and OF, LM tend to have greater glycosuria versus LF and OF tend
to have greater glycosuria than LF (P=ns). At 18 weeks, OM exhibit 5-fold increase in
glycosuria (versus 10 weeks) and three orders of magnitude greater than LM (both P<0.001).
Independent of hyperglycemia, plasma insulin was 23- and 20-fold greater in OF (P=0.03) and
OM (P<0.001) versus leans rats, respectively (Figure 2-2F). Under normal physiological
conditions, glucose is freely filtered and ~100% reabsorbed in the proximal tubule via sodium
glucose cotransporters (SGLT). There was no detectable difference among all groups in RAU of
high capacity, low affinity SGLT2 which is responsible for 97% of glucose reabsorption in the
proximal tubule (Figure 2-2G-H). Abundance of downstream, low capacity, high affinity cortical
SGLT1 was 3-fold greater in LF (versus LM), 50% greater in OM (versus LM), and 25% lower
in OF (versus LF). Medullary SGLT1 abundance was 70-100% greater in OM and OF compared
to leans rats, independent of glycemia (both P<0.001) (Figure 2-2G-H).

62
Figure 2-2. OM, not females develop, develop hyperglycemia despite hyperinsulinemia and
greater SGLT1 abundance in both sexes. (A) Non-fasted and (B) 7 hour fasted blood glucose
(n=4/group) measured using OWell Contour Next ONE blood glucose system. (C) correlation
between blood glucose and albuminuria. Urine glucose measured in (D) OM and females at 10,
16, and 18 weeks of age and (E) LM, female and obese male, female at 18 weeks of age using
Glucose Colorimetric Assay Kit (Cayman Chemical #10009582). (F) Plasma insulin was
measured MILLIPLEX MAP Rat Metabolic Hormone Magnetic Bead Panel - Metabolism
Multiplex Assay. (G) Immunoblots for sodium-glucose cotransporter 2 (SGLT2), cortical and
medullary sodium-glucose cotransporter 1 (SGLT1) were performed in kidney cortex (c prefix)
and medulla (m prefix) homogenates, respectively, at 18 weeks of age. Data expressed as mean ±
SEM and P<0.05 *versus LM and ^versus LF. 1X and 1/2X amounts were assayed to confirm
linearity of detection system (one amount shown); assay and immunoblot details are provided in
Supplemental Table 1. (H) Individual values plotted as relative abundance normalized to LM = 1
and error bars denoting mean ± SEM. P values provided. Statistical comparisons were performed
using GraphPad Prism (9.2.0) three-way ANOVA (A and D) or one-way ANOVA (B, E-G)
followed by Tukeys multiple comparisons post hoc test. Statistical comparisons for blood
glucose vs albuminuria (C) were performed using GraphPad Prism correlation test.

-75
-50
SGLT2
-75
mSGLT1
-50
Male
Lean Obese
Female
Lean Obese
kD
1.00 ± 0.03 0.98 ± 0.03 0.97 ± 0.03 1.05 ± 0.03
1.00 ± 0.04 1.99 ± 0.07* 0.90 ± 0.05 1.7 ± 0.1^
LM LF OM OF
0
5
10
15
6000
8000
10000
18 week
Urine glucose (mg/dl)
P<0.0001 P<0.0001
LM LF OM OF
0
100
200
300
400
500
18 wk Fasted
Blood Glucose (mg/dl)
P=0.003 P=0.02
100 250 400
0
5
10
15
20
Blood Glucose (mg/dl)
Albuminuria
(arbitrary density units)
r = 0.70
P < 0.0001
10 16 10 16
0
100
200
300
400
500
Non-fasted
Blood Glucose (mg/dl)
Lean Obese
Weeks
P=0.0002
Three-way ANOVA Results:
Time P=0.006
Obesity P<0.0001
Sex P=0.0003
Male
Female
1.0 ± 0.1 1.45 ± 0.08 2.9 ± 0.2* 2.2 ± 0.1^#
cSGLT1
-75
-50
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
SGLT2
Rel. Abundance (LM=1)
Male
Female
Lean Obese
0
1
2
3
4
cSGLT1
P<0.001
P=0.01
P=0.02
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
mSGLT1
P<0.0001
P=0.0002
LM LF OM OF
0
2
4
6
8
10
18 wk Fasted
Plasma Insulin (ng/ml)
P<0.001
P=0.03
10 16 18
0
1000
2000
4000
6000
8000
10000
Weeks
Urine Glucose (mg/dl)
Two-way ANOVA Results:
Interaction P<0.001
Age P<0.001
Sex P<0.001
P=0.001 P<0.001
P<0.001
P<0.001
10 16 10 16
0
100
200
300
400
500
Weeks
Non-fasted
Blood Glucose (mg/dl)
Lean Obese
P=0.002
Three-way ANOVA Results:
Age P=0.006
Obesity P<0.001
Sex P=0.003
A B C
D E F
G H

63
Obesity is associated with glomerular injury and exacerbated by hyperglycemia in OM
Under basal conditions little to no proteins are present in the urine. To investigate glomerular
injury we used semi-quantitative immunoblot technique to measure urinary biomarkers albumin,
angiotensinogen (A’ogen), and plasminogen in 24-hour UV (Figure 2-3A). We predicted obese
rats will have greater albuminuria so we loaded 0.0025% of 24 hr UV for obese rats and 0.025%
of 24 hr UV for lean rats. Quantitation was performed using densitometry and reported as
relative abundance units (RAU). At 10 weeks of age, obese versus leans rats exhibited 15- to 20-
fold greater albuminuria in OF and OM, respectively (Figure 2-3A, both P<0.001). OM (16 ± 4
RAU) exhibited 2-fold greater albuminuria compared to OF (7 ± 3 RAU, P<0.001). By 18
weeks, albuminuria increased 75-fold in OM (vs LM, P<0.001) and 21-fold in OF (150 ± 96
RAU, P=ns). Urinary A’ogen and plasminogen were below detection limit at 10 weeks of age
with the volume of UV loaded but were detectable by 16 weeks of age and directly correlated
with increases in albuminuria (Figure 2-3-B). The proximal tubule has high capacity to reabsorb
filtered proteins via endocytic receptors, megalin and cubilin. Relative abundance of megalin and
cubilin were 36% lower and 30% greater in LF and OF versus LM and OM (Figure 2-3C-D).
Cortical tissue albumin was 5- and 3-fold greater in OM (P=0.001) and OF (P=ns), respectively,
compared to leans rats (Figure 2-3C-D). Immunofluorescence staining of fibrosis with collagen
IV (COLIV; red) showed greater glomerular fibrosis in LM (versus LF). In both OM and OF,
COLIV staining exhibits significantly greater glomerular fibrosis versus leans rats and expansion
of fibrosis into the interstitium. Trichrome staining confirms these patterns seen with COLIV and
also exhibits more pronounced fibrosis and tubular atrophy in OM (versus OF). These results
suggest progressively worsening proteinuria is a consequence of glomerular injury in OM and
OF.

64

Figure 2-3. Obese ZFF1 rats exhibit progressively worsening proteinuria, tissue albumin, and
glomerular fibrosis. (A) Immunoblots (n=4/group) for albuminuria, angiotensinogen (A’ogen),
and plasminogen were performed on 0.025% (lean) and 0.0025% (obese) 24-hr urines at 10 and
18 weeks of age. 1X and 1/2X amounts were assayed to confirm linearity of detection system
(one amount shown); assay and immunoblot details are provided in Supplemental Table 1. (B)
Correlation between urinary A’ogen and plasminogen against albuminuria. (C) Abundance of
megalin, cubilin, and albumin determined by semi-quantitative immunoblot in kidney cortex
homogenates at 18 weeks of age. 1X and 1/2X amounts were assayed to confirm linearity of
detection system (one amount shown); assay and immunoblot details are provided in
Supplemental Table 1. Data expressed as mean ± SEM. (D) Individual values (black circles –
LM, blue circles – OM, grey triangles – LM, and red triangles – OF) plotted as relative
abundance normalized to LM = 1 and error bars denoting mean ± SEM. Glomerular and
Male Female
10 week
Male Female
18 week
%24hr UV
0.025%
0.0025%
Lean
Obese
Albumin
Lean
Obese
A’ogen
0.025%
0.0025%
0.025%
0.0025%
Lean
Obese
Plasminogen
MW
-75
-50
-75
-50
-75
-100
Albumin
Male
Lean Obese
Female
Lean Obese
-250
Megalin
-250
Cubilin
-50
kD
1.00 ± 0.04 1.04 ± 0.03 0.64 ± 0.04* 0.65 ± 0.05#
1.00 ± 0.07 0.95 ± 0.04 1.30 ± 0.08* 1.34 ± 0.08#
1.00 ± 0.03 5.29 ± 0.06* 1.3 ± 0.1 3.4 ± 0.6
-75
-100
-50
-50
Lean Obese
0
2
4
6
8
Albumin
P=0.001
Lean Obese
0.0
0.5
1.0
1.5
Megalin
Rel. Abundance (LM=1)
P=0.0005 P=0.001
Lean Obese
0.0
0.5
1.0
1.5
Cubilin
P=0.003
P=0.02
0
10
20
30
40
Angiotensinogen
(Rel. Abundance)
LM
OM
LF
OF
r=0.98
P<0.001
0 50 100 150 200
0
100
200
300
400
500
Plasminogen
(Rel. Abundance)
r=0.99
P<0.001
Albuminuria (Rel. Abundance)
Lean Male Lean Female
Obese Male Obese Female
A B
C D
E F

65
interstitial fibrosis were evaluated by (E) immunofluorescence microscopy of COL IV (red;
Abcam ab6586 1:100) and (F) Masson trichrome staining in LM (top left), LF (top right), OM
(bottom left) and OF (bottom right). Statistical comparisons were performed using GraphPad
Prism (9.2.0) one-way ANOVA followed by Tukeys multiple comparisons post hoc test or
correlation test. P values provided when applicable.

66
Altered kidney function in OM but not OF
Endogenous creatinine clearance (CCr) was used to estimate GFR (Figure 2-4A). At 10 weeks,
there was no detectable difference among all groups. By 18 weeks, CCr in OM (versus 10-week
CCr) increased 44% (P<0.001), evidence for hyperfiltration. Whereas LF, LM, and OF CCr
tended to fall due to increases plasma creatinine potentially a consequence of body growth or
evidence of lower GFR. K
+
-dependent ATPase activity was measured using K
+
-dependent p-
nitrophenyl phosphatase (K-pNPPase) enzymatic assay as previously described [11, 47]. K
+

dependent ATPase activity was greater in medullary kidney tissue compared with cortical tissue
with no sex- or disease-specific effect (Figure 2-4B). In lean rats there was no detectable sex-
specific difference in endogenous lithium clearance (CLi, Figure 3C), an estimate of volume
flow leaving the proximal tubule and medullary thick ascending limb, or sodium clearance (CNa,
Figure 3D), an estimate of overall sodium reabsorption along the nephron. At 10 weeks, OM
exhibit greater CLi and CNa compared to LM and OF (P=0.004 and 0.03, respectively). By 18
weeks, CLi increases 54% in OM versus 10 weeks (P<0.001) and 40% in OF versus 10 weeks
(P=ns) (Figure 2-4C). By 18 weeks, CNa also increases 15% in OM versus 10 weeks and 31% in
OF versus 10 weeks (both P=ns) (Figure 2-4D). Together these results show major alterations in
renal function in response to obesity and these changes that are exacerbated by hyperglycemia in
OM.

67
Figure 2-4. Altered renal function with time in OM but not OF. Kidney tissue, plasma, and urine
(n=4/group) were used to assess renal function. (A) Raw creatinine clearance (top) and
normalized to tibia length (bottom). Creatinine was measured using capillary electrophoresis (UT
Southwest Medical Center O’Brein Kidney Research Core) and creatinine clearance (CCr), an
estimate of GFR, was calculated using the following equation: CCr = (UCr x UV)/PCr. (B) K+-
dependent p-nitrophenyl phosphatase (K-pNPPase) activity, estimate of K
+
dependent ATPase
activity, was measured in kidney cortex and medullary homogenates (see method for details). (C)
Raw lithium clearance (top) and normalized to tibia length (bottom). Endogenous lithium
clearance (CLi), an estimate of volume flow leaving the proximal tubule, was calculated
classically as CLi = ([U/P]Li × UV]). (D) Raw sodium clearance (top) and normalized to tibia
length (bottom). Sodium clearance (CNa) was calculated as CNa = ([U/P]Na × UV]). P=plasma,
U=urine, and UV=urine volume. Statistical comparisons were performed using GraphPad Prism
(9.2.0) mixed-effects analysis (A) or three-way ANOVA (B-D) followed by Tukeys multiple
comparisons post hoc test. P values provided.
0
2
4
6
Creatinine Clearance
(L/24hr)
Lean Obese
Male Female
Three-way ANOVA Results:
Age P=0.18
Obesity P<0.001
Sex P<0.001
P<0.001
P=0.02
P<0.001
P<0.001
0
100
200
300
Lithium Clearance
(ml/24hr)
Lean
Obese
Three-way ANOVA Results:
Age P<0.001
Obesity P<0.001
Sex P=0.001
P=0.03
P<0.001
P=0.004
P<0.001
Cortex Medulla Cortex Medulla
0.0
0.5
1.0
1.5
2.0
K-pNPPase activity
(umole Pi/mg*hr)
Lean Obese
Three-way ANOVA Results:
Region P<0.0001
Obesity P=0.14
Sex P=0.16
0
10
20
30
40
Sodium Clearance
(ml/24hr)
Lean Obese
Three-way ANOVA Results:
Age P<0.001
Obesity P<0.001
Sex P=0.004
P<0.001
P<0.001
P<0.001
P<0.001
P=0.03
10 18 10 18
0
20
40
60
80
Weeks
Lithium Clearance
(ml/min/cm TL)
P<0.001
P<0.001
P=0.003
P=0.009
Three-way ANOVA Results:
Age P<0.001
Obesity P<0.001
Sex P=0.003
10 18 10 18
0.0
0.5
1.0
1.5
Weeks
Creatinine Clearance
(L/24hr/cm TL)
P<0.001
P=0.02
P<0.001
P<0.001
Three-way ANOVA Results:
Age P=0.15
Obesity P=0.002
Sex P=0.03
10 18 10 18
0
2
4
6
8
10
Weeks
Sodium Clearance
(ml/min/cm TL)
P=0.003
P<0.001
P<0.001
P=0.002
Three-way ANOVA Results:
Age P<0.001
Obesity P<0.001
Sex P=0.002
A B
C D

68
Sex- and disease-specific adaptations in proximal tubule to medullary thick ascending limb
transporters in obese rats
Lean rats exhibit baseline sex-specific differences, some previously reported [15, 103-106]
(Figure 2-5A-B). Our previous studies exhibit how these transporter abundance patterns
contribute to lower sodium transport in the proximal tubule in females compared to males. This
contributes to females having a greater ability to excrete a saline load compared to males. The
abundance profile of these transporters suggests females may have a head-start to handle
metabolic challenges. LF (vs LM) exhibit overall lower abundance of proximal tubule
transporters, reaching significance for villin, Na
+
dependent phosphate cotransporter 2 (NaPi2),
aquaporin 1 water channel, organic anion transporter 1 (OAT1), A’ogen, angiotensin converting
enzyme (ACE) 2, and collectrin (all P<0.05). LF (vs LM) exhibit greater abundance of proximal
tubule phosphorylation of Na
+
,H
+
exchanger isoform 3 (NHE3p; associated with inactivation),
Dipeptidyl peptidase-4 (DPP-IV), and Na
+
,K
+
-ATPase (NKA) b1 (all P<0.05). In the medullary
thick ascending limb, LF (vs LM) exhibit greater abundance of NHE3, NHE3p, STE20/SPS1-
related proline/alanine-rich kinase (SPAK) phosphorylation, Na
+
,K
+
,2Cl
-
cotransporter isoform 2
(NKCC2) phosphorylation at T96T101 and S87 but only reached significance for NHE3. There
were no detectable sex-specific differences in abundance of cortical NHE3, Na
+
,H
+
exchanger
regulatory factor (NHERF1), heme-oxygenase 1 (HO-1), NKA α1 or medullary SPAK, NKCC2,
NKA α1b1, and uromodulin (Figure 2-5A-B). Obese rats (vs respective leans rats) similarly
exhibit lower proximal tubule abundance of NHE3, NHE3p, DPP-IV (only significant in OF),
and claudin 2 (Figure 4). OM but not OF, additionally exhibit trends of lower Na
+
, HCO
3-

cotransporter (NBCe1-A), NHERF1, aquaporin 1, OAT1, ACE1&2, and collectrin but only
reached significance for AQP1, ACE1&2, and collectrin. Interestingly, OF (vs LF) exhibit

69
greater NBCE1-A, villin, NaPi2, and OAT1. These patterns may be adaptations to preserve
effective circulating volume or blunt potential metabolic acidosis. In the medullary thick
ascending limb, obese rats (vs respective leans rats) similarly exhibit lower NHE3, NHE3p,
SPAKp, and NKCC2p abundance. There were no detectable differences in cortical HO-1 and
NKAα1, or medullary SPAK with obesity.

70

A
B
-75
-100
NHE3
NHE3p
-75
-100
-75
-100 DPPIV
-75
-100
Villin
-50
NHERF1
-75
NaPi2
-37
-25
-20
AQP1
-75
OAT1
-25
-20 Cldn2
Cldn10
-25
-20
-250
-150
ACE1
-150
-100
ACE2
-37
Collectrin
NBCe1A
HO-1
-37
A’ogen
-50
-100
-150
Male
Lean Obese
Female
Lean Obese
kD
Male
Lean Obese
Female
Lean Obese
kD
1.00 ± 0.02 0.65 ± 0.02* 1.09 ± 0.03 0.83 ± 0.02^#
1.00 ± 0.04 0.51 ± 0.03* 1.5 ± 0.1* 0.96 ± 0.08^#
1.00 ± 0.06 0.78 ± 0.03 0.78 ± 0.02* 1.12 ± 0.04^#
1.00 ± 0.02 0.89 ± 0.03 0.73 ± 0.04* 0.90 ± 0.05^
1.00 ± 0.06 0.75 ± 0.04 1.14 ± 0.08 1.07 ± 0.09
1.00 ± 0.02 0.84 ± 0.03 1.84 ± 0.05 1.60 ± 0.07^#
1.00 ± 0.02 1.01 ± 0.05 0.77 ± 0.03* 0.95 ± 0.04
1.00 ± 0.04 0.76 ± 0.02* 0.51 ± 0.02* 0.58 ± 0.02#
1.00 ± 0.02 0.80 ± 0.01* 0.75 ± 0.02* 0.70 ± 0.04#
1.00 ± 0.08 0.75 ± 0.03 0.40 ± 0.03* 0.50 ± 0.04
1.00 ± 0.02 0.97 ± 0.03 1.13 ± 0.04 1.15 ± 0.06
1.00 ± 0.06 0.68 ± 0.04* 0.85 ± 0.04 0.55 ± 0.05^
1.00 ± 0.06 0.87 ± 0.04 1.69 ± 0.06* 0.95 ± 0.08^
1.00 ± 0.02 0.96 ± 0.06 0.64 ± 0.05* 0.86 ± 0.06
1.00 ± 0.04 0.73 ± 0.02* 1.84 ± 0.05* 1.84 ± 0.04#
1.00 ± 0.02 0.76 ± 0.03* 0.39 ± 0.01* 0.37 ± 0.03#
1.00 ± 0.02 0.67 ± 0.03* 0.76 ± 0.04* 0.80 ± 0.02
-75
mNHE3
mNHE3p
-75
-75
-50
mSPAKp
-150
-250
mNKCC2
-150
-250
mNKCC2p-
T96T101
-150
mNKCC2p-
S87
-250
mUMOD
-100
-75
mSPAK
-50
-100
-75
-50
mNKAβ1
mNKAα1
1.00 ± 0.01 0.86 ± 0.01 1.39 ± 0.07 0.97 ± 0.05^
1.00 ± 0.09 0.45 ± 0.07 1.52 ± 0.21 0.7 ± 0.1
1.00 ± 0.04 1.05 ± 0.02 1.04 ± 0.02 1.1 ± 0.4
1.00 ± 0.08 0.67 ± 0.06 1.4 ± 0.1 1.2 ± 0.3
1.00 ± 0.02 1.00 ± 0.09 1.10 ± 0.05 0.91 ± 0.04
1.0 ± 0.2 0.34 ± 0.05 1.4 ± 0.3 0.8 ± 0.2
1.00 ± 0.04 0.44 ± 0.02 1.4 ± 0.2 0.9 ± 0.2
1.00 ± 0.02 1.02 ± 0.05 1.01 ± 0.05 1.00 ± 0.07
1.00 ± 0.05 1.05 ± 0.06 1.08 ± 0.06 1.2 ± 0.1
1.00 ± 0.01 1.06 ± 0.03 1.01 ± 0.07 0.86 ± 0.09
Lean Obese
0.0
0.5
1.0
1.5
2.0
NHE3
P<0.0001
P=0.0006
P=0.01
Male Female
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
Villin
P=0.01
P=0.01
Lean Obese
0.0
0.5
1.0
1.5
2.0
Cldn2
P=0.003
P=0.007
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
Angiotensinogen
P=0.005
Lean Obese
0.0
0.5
1.0
1.5
2.0
NHE3p
P=0.02
P=0.01
P=0.03
P=0.02
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
ACE1
P=0.02
P<0.0001
P<0.0001
Lean Obese
0.0
0.5
1.0
1.5
NBCe1-A
P=0.003
P=0.046 P=0.003
P=0.051
Lean Obese
0.0
0.5
1.0
1.5
NaPi2
P=0.02
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
ACE2
P=0.0003
P<0.0001
P<0.0001
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
AQP1 - 35 kD
P=0.0005
P=0.007
P<0.0001
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
AQP1 - 24 kD
P<0.0001
P=0.01
P<0.0001
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
OAT1
P=0.06
P<0.0001
P=0.07
Relative abundance (Lean M = 1)

71
Figure 2-5. Impact of obesity and diabetes on transporters, channels, claudins, and regulators in
the proximal tubule to medullary thick ascending limb. 1X and 1/2X amounts were assayed to
confirm linearity of detection system (one amount shown); assay and immunoblot details are
provided in Supplemental Table 1. Arbitrary density values Relative abundances (arbitrary
density values) were normalized to LM = 1, data expressed as mean ± SEM, and P<0.05 *versus
LM, ^versus LF, and #versus obese male. kD indicated apparent molecular weight of the stained
markers and indicated lane loaded with prestained protein ladders (BioRad). (A) Immunoblots
for proximal tubule and medullary thick ascending limb (m prefix). (B) Selected graphs
expressing individual values plotted as relative abundance normalized to LM = 1 and error bars
denoting mean ± SEM. P values provided. Statistical comparisons were performed using
GraphPad Prism (9.2.0) one-way ANOVA followed by Tukeys multiple comparisons post hoc
test. Abbreviations: ACE: angiotensin converting enzyme -1 and -2; A’ogen: angiotensinogen;
AQP1: aquaporin 1; Cldn: claudin -2 and -10; DPPIV: Dipeptidyl Peptidase IV; HO-1: heme
oxygenase -1; NaPi2: Na
+
-phosphate cotransporter 2A; NBCe1-A: sodium bicarbonate
cotransporter 1A; NHE3: Na
+
/H
+
exchanger isoform 3; NHE3p: NHE3 phosphorylated at S552;
NHERF1: Na
+
/H
+
Exchanger Regulatory Factor; NKCC2: Na
+
-K
+
-2Cl
-
cotransporter isoform 2;
NKCC2p -S87 -T39T101: NKCC2 phosphorylated at S87, T39, T101; OAT1: organic anion
transporter 1; SPAK: Ste/SPS-1 related proline-alanine rich kinase; SPAKp: SPAK
phosphorylated at S373; UMOD: uromodulin.

72
Sex- and disease-specific adaptations in distal convoluted tubule to collecting duct transporters
in obese rats
In the distal convoluted tubule to cortical collecting duct, LF (vs LM) exhibit greater abundances
of key sodium transporters, channels, claudins, and regulators which is consistent with our
previous reports [15, 103-106]. greater uromodulin (UMOD), calbindin, SPAK, SPAKp,
NKCC2, NKCC2p, inwardly rectifying potassium channel (Kir4.1), Na
+
,Cl
-
cotransporter
(NCC), NCC phosphorylation (NCCp) at S71 and T53, glycosylated renal outer medullary
potassium channel (ROMK), epithelial Na
+
channel (ENaC) αb subunits, claudin (cldn) -7, -8,
and -10, aquaporin 2 (AQP2), and AQP2 phosphorylation (Figure 2-6A-B). There were no
detectable sex-specific differences in abundance of cortical plasmin(ogen), core ROMK, and
gENaC-full length or medullary cldn -4 and -10 (Figure 2-6A-B). Obese rats (vs respective leans
rats) similarly exhibit lower abundance of SPAKp, NKCC2, NKCC2p, NCCpT53, glycosylated
ROMK, cldn -7, -8, -10. SPAKp, NCCp, and cldn-10 although not all reached statistical
significance in both sexes. Obese rats (vs respective leans rats) similarly exhibit greater
abundance of UMOD, plasmin(ogen), ALG-2-interacting protein X (Alix), AQP2, and AQP2p.
Differences only reach statistical significance for plasminogen, Alix, and AQP2p-24 kD in OM
and UMOD and AQP2 in OF. In the medullary collecting duct, obese rats (vs respective leans
rats) exhibit lower CD63 and tendency towards greater AQP2 and AQP2p (Figure 2-6A-B).
Statistical significance was only reached for CD63 in both obese groups and AQP2-24 kD in OF.
Changes in kidney transporters along the nephron are summarized in Figure 2-7 and a complete
summary in Index Figure 2-1. Overall, LF versus LM exhibit similar transporter abundance
patterns as previous reported in normotensive SD rats [15]. These patterns of lower proximal
tubule sodium transporter abundance and greater distal nephron transporter abundance may

73
contribute to a female head-start to handle challenges from metabolic complications. Obese rats
exhibit similarly lower abundance of sodium transporters, channels, and claudins along the
nephron in attempt to maintain ECV by facilitating natriuresis and diuresis.

74
-75
Alix
-75
-100
-20
-25
-20
-37
-25
-20
1.00 ± 0.01 1.17 ± 0.02* 1.43 ± 0.02* 1.44 ± 0.03#
1.00 ± 0.03 1.43 ± 0.04* 1.07 ± 0.07* 0.96 ± 0.07#
1.00 ± 0.09 1.39 ± 0.05* 1.08 ± 0.03 1.18 ± 0.06
1.00 ± 0.04 0.74 ± 0.02* 1.48 ± 0.04 1.13 ± 0.04^#
1.00 ± 0.08 0.71 ± 0.02* 1.38 ± 0.04* 1.05 ± 0.04^#
1.00 ± 0.05 1.19 ± 0.09 1.19 ± 0.06 1.5 ± 0.1
1.00 ± 0.04 1.16 ± 0.05 1.50 ± 0.03* 1.71 ± 0.05#
-75
UMOD
-100
-25
-20
Calbindin
SPAK
-75
-50
-50
-75
SPAKp
NKCC2 -150
NKCC2p-
T96T101
-150
-150
-250
-250
NKCC2p-
S87
Kir4.1
-50
-37
-150 NCC
NCCpS71 -150
-250
NCCpT53 -150
ROMK
Gly-
Core-
-50
-37
Plasminogen
-75
-100
αENaC-Fl
à
αENaC-Cl
βENaC
γENaC
-75
-37
-25
-75
-100
Cldn7
Cldn8
AQP2
-37
-25
-20
AQP2p
Male
Lean Obese
Female
Lean Obese
kD
1.00 ± 0.04 1.06 ± 0.03 1.08 ± 0.04* 1.17 ± 0.05^#
1.00 ± 0.02 0.96 ± 0.05 1.64 ± 0.04* 1.51 ± 0.03#
1.00 ± 0.06 1.26 ± 0.06 1.35 ± 0.05 1.16 ± 0.07
1.0 ± 0.1 0.81 ± 0.06 2.3 ± 0.1 1.4 ± 0.1^
1.00 ± 0.04 0.75 ± 0.02* 1.69 ± 0.06* 1.07 ± 0.04^#
1.00 ± 0.04 0.78 ± 0.02 1.7 ± 0.1* 1.18 ± 0.09#
1.00 ± 0.04 0.44 ± 0.02 1.4 ± 0.2 0.90 ± 0.2
1.00 ± 0.04 1.02 ± 0.05 1.18 ± 0.05* 1.27 ± 0.04#
1.00 ± 0.04 0.93 ± 0.02 1.38 ± 0.05* 1.41 ± 0.07#
1.0 ± 0.1 1.20 ± 0.08 2.5 ± 0.1* 1.98 ± 0.2#
1.00 ± 0.08 0.82 ± 0.03 1.9 ± 0.1* 1.25 ± 0.09^
Male
Lean Obese
Female
Lean Obese kD
1.00 ± 0.01 0.71 ± 0.04* 1.5 ± 0.1* 1.24 ± 0.07^#
1.00 ± 0.06 1.06 ± 0.04 1.06 ± 0.04 1.02 ± 0.04
1.00 ± 0.06 1.76 ± 0.04* 0.99 ± 0.02 1.5 ± 0.2
1.00 ± 0.08 1.0 ± 0.1 3.32 ± 0.04* 2.2 ± 0.1^#
1.00 ± 0.08 1.02 ± 0.05 1.30 ± 0.08 1.47 ± 0.05#
1.0 ± 0.1 1.17 ± 0.05 1.17 ± 0.06 1.5 ± 0.2
1.00 ± 0.08 1.44 ± 0.09 1.73 ± 0.07 2.4 ± 0.3#
-25
mCldn4
-20
mCldn10
mCD63
-25
-20
-50
-25
-20
-37
mAQP2
-25
-20
-37
mAQP2p
1.00 ± 0.04 1.17 ± 0.06 1.08 ± 0.03 0.95 ± 0.03
1.00 ± 0.03 0.99 ± 0.04 1.04 ± 0.05 0.92 ± 0.05
1.00 ± 0.06 0.52 ± 0.04* 1.42 ± 0.08* 0.82 ± 0.07^
1.00 ± 0.04 1.16 ± 0.07 1.25 ± 0.04 1.12 ± 0.07
1.00 ± 0.03 1.04 ± 0.07 1.20 ± 0.04 0.96 ± 0.02
1.0 ± 0.2 1.4 ± 0.1 1.2 ± 0.1 2.6 ± 0.6
1.0 ± 0.2 1.43 ± 0.05 1.5 ± 0.1 2.4 ± 0.5
Cldn10
-25
-20
1.00 ± 0.06 0.87 ± 0.04 1.69 ± 0.06* 0.95 ± 0.08^
Lean Obese
0.0
0.5
1.0
1.5
2.0
Calbindin
P<0.0001
P<0.0001
Male Female
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
NKCC2
P=0.02
P<0.0001
P<0.0001
P=0.003
Lean Obese
0
1
2
3
4
5
αENaC - Fl
P=0.02
P=0.0002
P=0.047
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
NKCC2pT96T101
P=0.006
P=0.04
Lean Obese
0.0
0.5
1.0
1.5
2.0
Cldn7
P=0.01
P=0.0005
P<0.0001
P=0.001
Lean Obese
0
1
2
3
4
5
αENaC - Cl
P=0.04
Lean Obese
0
1
2
3
4
SPAKp
P=0.0007
P=0.02
Lean Obese
0
1
2
3
NCC
P=0.004
P=0.0006
Lean Obese
0.0
0.5
1.0
1.5
2.0
βENaC
P=0.007
P<0.0001 P=0.0002
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
Kir4.1
P=0.04
P=0.007
Lean Obese
0
1
2
3
NCCpT53
P=0.004
P=0.03
Lean Obese
0.0
0.5
1.0
1.5
2.0
2.5
Plasminogen
P=0.02
Lean Obese
0.0
0.5
1.0
1.5
2.0
γENaC - Fl
P=0.003
P=0.001
Lean Obese
0.0
0.5
1.0
1.5
2.0
Cldn10
P=0.0001
P<0.0001
Relative abundance (Lean M = 1)
NKA β1
NKA α1
-100
-75
-50
1.00 ± 0.05 0.96 ± 0.02* 1.19 ± 0.06* 1.06 ± 0.10
1.00 ± 0.06 1.13 ± 0.06* 1.50 ± 0.05* 1.16 ± 0.03^
A
B

75
Figure 2-6. Impact of hyperglycemia and obesity on transporters, channels, claudins, and
regulators along the distal tubule and collecting duct. 1X and 1/2X amounts were assayed to
confirm linearity of detection system (one amount shown); assay and immunoblot details are
provided in Supplemental Table 1. Arbitrary density values Relative abundances (arbitrary
density values) were normalized to LM = 1, data expressed as mean ± SEM, and P<0.05 *versus
LM, ^versus LF, and #versus obese male. kD indicated apparent molecular weight of the stained
markers and indicated lane loaded with prestained protein ladders (BioRad). (A) Immunoblots
for distal tubule to medullary collecting duct (m- prefix denoting medullary). (B) selected graphs
expressing Individual values plotted as relative abundance normalized to LM = 1 and error bars
denoting mean ± SEM. P values provided. Statistical comparisons were performed using
GraphPad Prism (9.2.0) one-way ANOV A followed by Tukeys multiple comparisons post hoc
test. Abbreviations: Alix: ALG-2-interacting protein X; AQP1: aquaporin 2; AQP2
phosphorylated at S256 (AQP2p); Cldn: claudin -4, -7, -8, and -10; ENaC: epithelial Na
+
channel
alpha α, β, and γ full length (Fl) or cleaved (Cl); Kir4.1: inwardly rectifying K
+
channel 4.1;
NCC: Na
+
-Cl
-
cotransporter; NCCp -S71 –T53: NCC phosphorylated at S71 and T53; NKA:
Na
+
,K
+
-ATPase alpha and beta subunit; NKCC2: Na
+
-K
+
-2Cl
-
cotransporter isoform 2; NKCC2p
-S87 -T39T101: NKCC2 phosphorylated at S87, T39, T101; ROMK: renal outer medullary K
+
channel core and glycosylated (gly) forms; SPAK: Ste/SPS-1 related proline-alanine rich kinase;
SPAKp: SPAK phosphorylated at S373; UMOD: uromodulin.

76

Figure 2-7. Summary of key transporters, channels, claudins, and regulators along the nephron in
ZSF1 rats. (A) Comparison of baseline sex-specific protein expression in LM and LF normalized
to LM =1 (solid lines). *P<0.05 compared to LM. (B) Comparison effect of hyperglycemia and
obesity on protein expression in OM and OF normalized to respective lean rats = 1 (solid line).
*P<0.05 compared to respective leans. White stars at the base of the column denote P<0.05
between normalized OM vs. OF. Data expressed as mean ± SEM (n=4/group) and statistical
comparisons were performed using GraphPad Prism (9.2.0) multiple parametric unpaired
Student’s t-test.

NHE3
NHE3p
DPPIV
NBCe1-A
Villin
NHERF1
NaPi2
SGLT2
AQP1-35 kD
AQP1-24 kD
OAT1
Cldn 2
A'ogen
ACE 1
ACE 2
Collectrin
NKA α1
NKA β1
NHE3
NHE3p
SGLT1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NKA α1
NKA β1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NCC
NCCpS71
NCCpT53
ROMK Gly.
ROMK Core
Plasminogen
αENaC - Fl
αENaC -Cl
βENaC
γENaC - Fl
Cldn 7
Cldn 8
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
0.0
0.5
1.0
1.5
2.0
3.0
6.0
Relative Abundance (LM = 1)
*
LM LF
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NHE3
NHE3p
DPPIV
NBCe1-A
Villin
NHERF1
NaPi2
SGLT2
AQP1-35 kD
AQP1-24 kD
OAT1
Cldn 2
A'ogen
ACE 1
ACE 2
Collectrin
NKA α1
NKA β1
NHE3
NHE3p
SGLT1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NKA α1
NKA β1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NCC
NCCpS71
NCCpT53
ROMK Gly.
ROMK Core
Plasminogen
αENaC - Fl
αENaC -Cl
βENaC
γENaC - Fl
Cldn 7
Cldn 8
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
0.0
0.5
1.0
1.5
2.0
3.0
6.0
Relative Abundance (Lean = 1)
OM OF
**
**
*
*
*
*
*
**
* *
*
*
*
*
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* **
*
*
Cortex Medulla Cortex

77
Lower dietary sodium alters electrolyte handling and tends to blunts albuminuria progression
OM exhibit 78% greater food intake versus LM and 37% greater food intake versus OF. Greater
food intake also means OM have greater sodium intake compared to LM and OF. To investigate
the role of high sodium intake on disease progression, a cohort of OM ZSF1 rats (n=5-6/group)
were fed either control diet (0.3% Na
+
; ENVIGO T.7012) or equivalent diet with lower sodium
(0.1% Na
+
; ENVIGO T.7034) for 10 weeks. The hypothesis was lowering sodium intake, while
leaving calorie intake the same, would slow disease progression in OM. Physiological
parameters after 10 weeks on respective diet are summarized in Table 2-3. Surprisingly, rats fed
0.1% Na
+
diet (vs 0.3% Na
+
) did not exhibit lower blood pressure. Low sodium fed rats
expectedly leads to 42% lower water intake and 24-hour UV. Rats fed 0.1% Na
+
(vs 0.3% Na
+
)
also exhibit 35% lower CLi, 79% lower CNa, and 76% lower UNaV. Interestingly, lower salt
intake exhibits 46% lower glycosuria concentration and 80% lower glucose excretion versus rats
fed 0.3% Na
+
. Lower glucose excretion tends to increase blood glucose concentration likely a
consequence of lower GFR (not measured). Rats fed 0.1% Na
+
(vs 0.3% Na
+
) exhibit blunted
albuminuria progression and urinary plasminogen excretion although these differences did not
reach statistical significance (Figure 2-8A). It is clear that 10 weeks on 0.1% Na
+
diet, 2/5 rats
develop albuminuria compared to 5/6 rats on 0.3% Na
+
. Put another way, 60% of 0.1% Na
+
fed
rats exhibit an order of magnitude lower albuminuria compared to 0.3% Na
+
fed rats also evident
by 17% lower area under the curve. These patterns were similar for plasminogen but were
statistical significance (Figure 2-8B). Together these findings support our hypothesis that
lowering sodium intake did modestly blunt proteinuria progression suggesting lower glomerular
injury.

78
Table 2-3. Physiological Parameters after 10 weeks of 0.3% Na
+
or 0.1% Na
+
diet

Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism
(9.2.0) parametric unpaired Student’s t test. *P<0.05 versus 0.3% Na diet.

Parameter
0.3% Na
+
N=6
0.1% Na
+
N=5
Fold
Change
Body Weight (BW, g) 534 ± 6 523 ± 10 NS
Blood Pressure (BP, mmHg) 157 ± 4 159 ± 3 NS
Food Intake (g/24 hrs) 37 ± 3 31 ± 7 NS
Water Intake (ml/24 hrs) 62 ± 8 26 ± 5* 0.4
Blood Glucose (mg/dl) 188 ± 16 196 ± 20 NS
Plasma Sodium (mM) 125 ± 1 128 ± 1 NS
Plasma Potassium (mM) 4.7 ± 0.2 4.3 ± 0.3 NS
Lithium Clearance
(CLi, ml/min/kg BW)
237 ± 22 129 ± 11* 0.5
Sodium Clearance
(CNa, ml/min/kg BW)
38 ± 2 8 ± 1* 0.2
24-hr Urine Volume (UV, ml) 40 ± 7 23 ± 10* 0.6
Urine Glucose (mg/dl) 7576 ± 1327 4092 ± 1093* 0.5
Urinary Glucose Excretion (mg) 3182 ± 736 653 ± 270* 0.2
Urinary Sodium Excretion
(UNaV, mmol/24 hrs)
2.5 ± 0.2 0.55 ± 0.04* 0.2
Urinary Potassium Excretion
(UKV, mmol/24 hrs)
5.2 ± 0.5 4.1 ± 0.3 NS

79

Figure 2-8. 10 weeks of 0.1% Na
+
intake versus 0.3% Na
+
tends to blunt proteinuria. 10 weeks of
lower sodium intake leads to lower onset of macroalbuminuria and urinary excretion of
plasminogen in obese male ZSF1 rats (n=5-6/group; 0.3% Na ● and 0.1% Na ▲). (A) Individual
values (top left graph) and mean ± SEM (bottom left graph) quantification of albuminuria
immunoblots performed on 0.0025% 24-hour urine at 1, 3, 6, 8, and 10 weeks on diet. (B)
Individual values (top right graph) and mean ± SEM (bottom right graph) quantification of
urinary plasminogen immunoblots performed on 0.0025% 24-hour urine at 1, 3, 6, 8, and 10
weeks on diet. Statistical comparisons were performed using GraphPad Prism (9.2.0) one-way
ANOVA and area under the curve (AUC). AUC provided.

0.3 Na 0.1 Na
0.0025% 24hr urine
75-
50-
75-
50-
75-
50-
75-
50-
75-
50-
75-
50-
1 3 6 8 10 1 3 6 8 10 Weeks
kD
Weeks
0.3 Na 0.1 Na
1 3 6 8 10 1 3 6 8 10
0.0025% 24hr urine
100-
kD
75-
100-
75-
100-
75-
100-
75-
100-
75-
100-
75-
0.1
1
10
100
1000
10000
0.3 Na 0.1 Na
1 3 6 8 10
0.1
1
10
100
1000
10000
Weeks
AUC: 805 units x wks (0.3 Na)
AUC: 669 units x wks (0.1 Na)
Log
10
Albuminuria (1wk = 1)
0.1
1
10
100
1 3 6 8 10
0.1
1
10
100
Weeks
AUC: 31 units x wks (0.3 Na)
AUC: 23 units x wks (0.1 Na)
Log
10
Plasminogen (1wk = 1)

80
Discussion
The current study aimed to define sexual dimorphisms in renal tubular response to obesity and
early diabetic kidney disease and determine the physiological consequences these differences
have in disease progression. Despite recent advances in therapeutics (i.e. GIP/GLP1 agonists and
SGLT2 inhibitors), the intrarenal mechanisms and pathogenesis involved in diabetic kidney
disease and obesity pathogenesis remain incompletely understood. This is partly due to the lack
of animal models that mimic human disease. Here we use the highly translatable ZSF1 rat model
previously reported, in males, to meet all criteria put forth by the AMDCC and shown to mimic
disease progression seen in humans [33, 34].
Lean ZSF1 rats are hypertensive (due to parental SHHR genetics) but do not develop
obesity or diabetic complications. The female advantage or head-start in regards to abundance
and covalent modification patterns along the nephron, as previously described in this
dissertation, was also evident in ZSF1 rats. Abundance patterns of lower proximal tubule
transporters and greater distal nephron transporters as previously shown in SD rats [15], may
again confer a female head-start in the handling of metabolic challenges. Notably, While we
report no sex-specific differences in endogenous lithium clearance or sodium clearance in Figure
2-4, normalizing these parameters to kidney weight or body weight as shown in previous reports
[15, 69] does show LF have greater clearance than LM. These sex-specific differences are
consistent with and expand on our previous reports in normotensive SD rats that females have
lower expression of proximal tubule to thick ascending limb transporters and greater distal tubule
to collecting duct transporters that contribute to enhanced capability to excrete a saline load
compared to age-matched males [15].

81
Impact of diabetic kidney disease in OM versus LM
Here we show OM exhibit lower protein abundance of many transporters, channels, claudins,
and regulators along the nephron that functionally leads to greater lithium clearance (a measure
of volume leaving the proximal tubule and medullar thick ascending limb) and sodium clearance
(a measure of overall sodium reabsorption) compared to LM. While we did not observe a
difference in Na
+
,K
+
-ATPase subunit abundance or K-pNPPase activity (a measure of K
+
dependent ATPase activity), OM exhibited a lower abundance of cortical and medullary NHE3
and NHE3p. This is consistent with previous reports in ZDF rats [107] and potentially, in part, a
consequence of lipotoxicity from PT albumin accumulation [108]. Previous studies implicate
overactivation of RAAS as a contributor to hypertension in diabetic rats [93] and RAAS
inhibition is shown to be nephroprotective [109]. We observe a lower abundance of ACE and
ACE2 in OM and no change in angiotensinogen abundance suggesting suppression of intrarenal
RAAS components, consistent with reports in other diabetic models [110-112]. While greater
urinary angiotensinogen could be evidence for overactivation of circulating RAAS, suppression
of intrarenal RAAS is likely to contribute to natriuresis and diuresis to maintain volume
homeostasis. Lower medullary and cortical thick ascending NKCC2p-T96T101 and -S87, distal
tubule NCC and NCCpT53, and glycosylated ROMK (important for K
+
recycling abundance),
contribute to greater natriuresis, diuresis and lower blood pressure in OM versus LM [113]. OM
exhibit a greater abundance of b ENaC and full-length g ENaC but this is likely an intracellular
accumulation of these proteins that are not participating in transport at the apical membrane. We
did not observe an increase in α or g ENaC cleavage despite increases in urinary and tissue
plasmin(ogen); previously shown to activate (cleave) g ENaC [114]. Our findings are consistent
with other reports in high-fat-fed mice [115], db/db mice [116], and ZDF rats [117]. Lower

82
abundance of cldn2-10, AQP1, OAT1, and collectrin - involved in paracellular sodium
reabsorption, transcellular water reabsorption, and amino acid transport - likely facilitate
natriuresis and diuresis in OM (versus LM). Consistent with previous reports, AQP2 and AQP2p
abundance tended to be greater in OM, reaching significance in AQP2pS256 - 24 kD, to prevent
dehydration in the face of polyuria [118].
One of the most common comorbidities of obesity is hypertension which is thought to
contribute to worse kidney disease outcomes and progression [84]. Previous reports on blood
pressure in obese ZSF1 rats are inconclusive, showing lower blood pressure using radiotelemetry
[119], no difference using carotid artery cannulation [96, 120, 121], or greater blood pressure
using undefined methods [100] in obese rats. We observe a ~10 mmHg lower systolic blood
pressure in obese rats, independent of sex, using tail-cuff plethysmography. This suggests the
lower abundance of sodium transporters in obese rats (vs lean rats) may contribute to lower
blood pressure by facilitating natriuresis and diuresis. At 10 weeks of age, OM exhibit modest
glycosuria that seems to proceed hyperglycemia potentially facilitated by obesity-induced
hyperinsulinemia driving glucose excretion [122, 123] or early hyperfiltration driving increased
glucose filtration that we are unable to detect via creatinine clearance. OM develop significant
hyperglycemia by 16 weeks and hyperfiltration by 18 weeks of age. Polyuria observed OM and
most likely greater plasma osmolality is driven by greater vascular and tubular glucose
concentrations causing plasma hypertonicity and osmotic diuresis, respectively. Under
physiological conditions 97% of freely filtered glucose is reabsorbed by high capacity, low-
affinity SGLT2 in the S1 and S2 segments of the proximal tubule while the low-capacity, high-
affinity SGLT1 reabsorbs the remaining 3% in the S3 segment that together leads to near zero
urinary loss of glucose [92]. Previous reports of protein expression of SGLTs in the kidney are

83
inconsistent [124-127]. In the current study, we observe no difference in abundance of SGLT2
between sex or with hyperglycemia. However, hyperglycemia increases cortical SGLT1 45%
and medullary SGLT1 by 2-fold in OM versus LM. Previous studies indicate that under
euglycemic conditions, SGLT2 operates at 50% capacity [128]. Our results suggest once the
transport capacity of SGLT2 is exceeded, the burden of glucose reabsorption is shifted
downstream to SGLT1, similar to studies of SGLT2 inhibition [129], with no change in SGLT2
abundance. Proximal tubule megalin and cubilin are responsible for the reabsorption of filtered
proteins and previous studies in ZDF rats [130] and db/db mice [131] (models with similar
genetic mutations as ZSF1 rats) suggest lower expression of megalin and cubilin contribute to
albuminuria in diabetic rats. In the current study, OM develop progressively worsening
proteinuria. However, we see no detectable change in megalin and cubilin abundance in OM
versus LM, and additionally, see greater cortical tissue albumin accumulation in OM. This has
previously been shown to cause tubular toxicity [132]. This suggests proteinuria was a
consequence of glomerular damage rather than impaired proximal tubule handling. This was
confirmed by histological and immunofluorescence staining of fibrosis which showed markedly
more glomerular fibrosis and interstitial fibrosis in OM versus LM. Given proteinuria preceded
hyperglycemia in OM versus LM, it seems that initial obesity-related glomerular damage was
then exacerbated by hyperglycemia and hyperfiltration. Blood glucose, urinary plasminogen, and
urinary angiotensinogen are directly correlated with urinary albumin which is consistent with
reports of maladaptive glomerulosclerosis in the presence of hyperglycemia [24]. Collectively
we show that hyperglycemia in OM ZSF1 rats is associated with lower pool sizes of transporters,
channels, claudins, and regulators along the entire nephron. These adaptations are necessary to

84
maintain volume and electrolyte homeostasis in the face of hyperphagia and the metabolic
challenges of diabetes.
Dietary interventions for controlling hypertension have traditionally focused on lowering
sodium intake. However, there is a paradoxical relationship between sodium intake and
glomerular filtration rate in diabetes [133]. The “salt paradox” in diabetes is lower dietary
sodium intake is associated with greater GFR and renal blood flow (RBF) whereas higher dietary
sodium intake is associated with lower GFR and RBF [92, 133]. While the relationship between
dietary sodium and glucose homeostasis remains unclear, recent studies provide evidence that
sodium intake can directly regulate glucose homeostasis [134]. To investigate the role of sodium
intake on disease progression, we fed OM ZSF1 rats’ control (0.3% Na
+
) diet or lower sodium
(0.1% Na
+
) diet. This allowed us to investigate the impact of sodium independent of calorie
intake. Lowering dietary sodium had expected adaptations of lower water intake, urine volume,
and sodium excretion. Interestingly, lowering dietary sodium significantly lowered urinary
glucose excretion and, although it did not reach statistical significance, elevated blood glucose.
This may be a function of lower glomerular filtration rate or greater fractional sodium
reabsorption in the proximal tubule. Our results go along with studies in diabetic WBN/Kob
diabetic fatty rats which show high salt diet ameliorates hyperglycemia and insulin resistance
[135]. Lowering dietary sodium did lower the severity of albuminuria which could again be a
function of lowering GFR or greater proximal tubule reabsorption. These results suggest
lowering dietary sodium intake in diabetic OM, lowers albuminuria but may maladaptively
contribute to worsening hyperglycemia by lowering glucose excretion.

85
Obesity-induced kidney pathology in female ZSF1 and comparisons with diabetic males
Previous experimental and clinical studies suggest obesity and diabetes-induced kidney disease
are associated with similar pathological changes in the kidney [136]. Studies also implicate
estrogen as a contributor to a lower incidence of diabetes via enhanced insulin sensitivity and
proper maintenance of glucose homeostasis [30, 137]. Consistent with previous studies in ZSF1
rats, OF were resistant to hyperglycemia [99-101]. Additionally, we did not observe differences
in physiological parameters despite severe obesity and 50% greater food (P=NS) in OF
compared to LF. However, OF did exhibit obesity-associated hyperinsulinemia and
glomerulopathy. We did not observe obesity-associated alterations in renal function (e.g.,
hyperfiltration) as previously reported in other obese models [84, 136, 138]. These observations
may be partially explained by recent findings in high fat-fed SD rats showing increases in
glomerular capillary pressure are associated with obesity-associated glomerulopathy and
attenuated tubular glomerular feedback, independent of changes in GFR [139].
Epidemiological studies in humans show women have a greater prevalence of obesity
compared to men [140]. In the current study, bodyweight is 80% greater in OF and 52% greater
in OM compared to lean rats. Additionally, kidney and heart hypertrophy is greater in OF than
OM compared to respective lean rats. Together suggests that OF exhibit more severe obesity
versus OM. Although to a lesser extent, OF exhibit similar patterns of progressively worsening
proteinuria and glomerular pathology. Greater levels of urinary albumin, angiotensinogen, and
plasmin(ogen) along with greater glomerular and interstitial fibrosis provide evidence of
pronounced renal injury independent of hyperglycemia.
Along the proximal tubule, OF display distinct patterns of kidney transporters. Contrary
to OM, OF exhibit greater NBCe1A, NaPi2, AQP1-35 kD, OAT1, and angiotensinogen

86
abundance compared to lean rats. Potential explanations for this pattern could include: 1)
adaptations to maintain euvolemia in the face of overall lower sodium transport along the
nephron; 2) proximal tubular hyperplasia due to tubular injury previously observed in chronic
obesity or early stages of diabetes [92, 141]; 3) tubular hypertrophy or greater tubular diameter;
or 4) a genetic artifact in ZSF1 strain that alters the abundance of the aforementioned proteins or
differences in tubular morphology. More investigation is necessary to understand this phenotype.
Overall OF (versus LF) exhibit similar patterns of lower abundance of key Na
+
transporters
along the nephron as previously described in diabetic OM (versus LM). Given the pattern of
lower proximal tubule transporters and fractional sodium reabsorption in LF versus LM, it
appears OF may have a head-start to renal adaptions of lowering transporter pool sizes in the
face of metabolic challenges. However, we do not observe significant differences in CLi or CNa.
This could be a limitation of N’s or possibility a function of the distinct patterns observed in the
proximal tubule. These patterns are better summarized in Figure 2-7 for lean rats (Figure 2-7A)
and obese rats (Figure 2-7B). White stars at the base of columns in Figure 2-7B denote
statistically significant differences between OM versus OF whereas the asterisks denote
significant differences with respect to lean rats. However, this study is not without limitations.
While leptin impairment exists in humans, leptin receptor deficiency is rare [142] but potentially
underdiagnosed [143]. This limits the current study to an investigation of obesity independent of
potential maladaptive effects of hyperleptinemia. Additionally, diabetes typically occurs in older
populations of humans. Thus metabolic and pathophysiology are confounded by aging and
postmenopausal changes [144]. However as we see the age of disease diagnosis continue to
lower and the prevalence of childhood obesity continues to rise, this may not be the case for long
[145].

87
In summary, we report sexual dimorphisms in renal transporters, channels, claudins, and
regulatory factors and provide potential (patho)physiological implications of these patterns on
disease progression. While OF exhibit resistance to the development of hyperglycemia, the
results summarized here suggest they are quite susceptible to obesity-induced kidney pathology.
While factors contributing to the female advantage to disease pathology are undoubtedly
multifactorial, we provide evidence that baseline sexually dimorphic patterns of transporters
along the nephron may confer a head-start to renal adaptations to metabolic challenges in
females. This head-start likely contributes to the maintenance of homeostasis and slower disease
pathogenesis in females versus males. Additionally, studies investigating the potential impact of
sex steroids in this model would provide valuable insight to better understand the differences
summarized here. Our findings build on the evidence for the high translatability of OM
pathophysiology to that observed in human diabetic kidney disease and potentially show the
usefulness of OF as a model of obesity-induced kidney disease independent of hyperglycemia.

88
Supplemental Information

Supplemental Table 2-1. Antibody and immunoblot protocol details………………………..89-92

Supplemental Figure 2-1. Assessment of equal loading by protein staining of loading gel and
linearity of the detection system…………………………………………………………93

Supplemental Figure 2-3. Pronounced perirenal fat, lipidemia, and hyperleptinemia in obese
rats…………………………………………………………………………….......……..94

89
Supplemental Table 2-1. Antibody and immunoblot protocol details. ~kDa refers to apparent molecular weight determined by BIO-
RAD Precision Plus Protein
TM
Duel Color Standards. Ab=antibody, Gt=goat, Mu=mouse, Rb=rabbit, Sh=sheep, O/N=overnight,
DAG=donkey anti-goat, DAS=donkey anti-sheep, GAM=goat anti-mouse, GAR=goat anti-rabbit, N/A=not assayed
Antibody
Target
~kDa
Protein/
lane
cortex
(µg)
Protein/
lane
medulla
(µg)
Primary
antibody
supplier
Ab
host
Dilution Time
Secondary
antibody
supplier
Host
and
target
Dilution Time Ref
Kidney immunoblot details
ACE 1 190 5, 2.5 N/A
Santa Cruz
(sc12187)
Gt 1:1000 2 hr Invitrogen
DAG
680
1:5000 1 hr [146]
ACE 2 ~92 20, 10 N/A Abcam Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [147]
Albumin ~66 30, 15 N/A
Santa Cruz
(sc271605)
Mu 1:2000 O/N Invitrogen
GAM
680
1:5000 1 hr [148]
Alix
(1A12)
95 80, 40 5, 2.5
Santa Cruz
(sc53540)
Mu 1:1000 O/N Invitrogen
GAM
680
1:5000 1 hr [149]
Angiotens-
inogen
140 10, 5 N/A
Abcam
(ab213705)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [150]
AQP1
37
23
10, 5 N/A
Maunsbach
(Denmark)
Rb 1:1000 1 hr Invitrogen
GAR
680
1:5000 1 hr [151]
AQP2
37
23
20, 10 13, 6.5
Santa Cruz
C-
17(sc9882)
Gt 1:500 O/N Invitrogen
DAG
680
1:5000 1 hr [152]
AQP2p-
S256
~37
~23
20, 10 4, 2
Invitrogen
(PA5-38407)
Rb 1:1000 2 hr Invitrogen
GAR
680
1:5000 1 hr [153]
Calbindin
D28K
28 5, 2.5 N/A
Santa Cruz
(sc365360)
Mu 1:2000 O/N Invitrogen
GAM
680
1:5000 1 hr [154]
CD26
(5E8)
“DPPIV”
100 15, 7.5 N/A
Santa Cruz
(sc52642)
Mu 1:1000 2 hr Invitrogen
GAM
680
1:5000 1 hr [155]
CD63 53 80, 40 10, 5
BD
Biosciences
Mu 1:1000 2 hr Invitrogen
GAM
680
1:5000 1 hr [156]

90
Claudin 2 20 5, 2.5 N/A
ThermoFisher
(#32-5600)
Mu 1:2000 O/N Invitrogen
GAM
800
1:5000 1 hr [157]
Claudin 4 20 4, 2 4, 2
ThermoFisher
(#364800)
Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [158]
Claudin 7 20 5, 2.5 N/A
ThermoFisher
(#34-9100)
Rb 1:1000 O/N Invitrogen
GAR
680
1:5000 1 hr [69]
Claudin 8 20 60, 30 20, 10
ThermoFisher
(#40-0700Z)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [159]
Claudin 10 20 4, 2 4, 2
ThermoFisher
(#38-8400)
Rb 1:1000 2 hr Invitrogen
GAR
680
1:5000 1 hr [160]
Collectrin ~40 5, 2.5 N/A
Thu Le (U.
Rochester)
Rb 1:1000 2 hr Invitrogen
GAR
680
1:5000 1 hr [161]
Cubilin 460 5, 2.5 N/A
R&D
Systems
Biotechne
Sh 1:4000 O/N Invitrogen
DAS
680
1:5000 1 hr
[162,
163]
αENaC
~80
~30
80, 40 N/A
Loffing
(Zurich)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [164]
βENaC ~80 60, 30 N/A
Loffing
(Zurich)
Rb 1:15,000 O/N Invitrogen
GAR
680
1:5000 1 hr [164]
γENaC
75
60
60, 30 15, 7.5 Palmer Rb 1:1000 2hr Invitrogen
GAR
680
1:5000 1 hr [73]
HO-1 32 80, 40 N/A
Abcam
(ab13243)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [165]
Kir 4.1 40 40, 20 NA
Alomone
APC-035
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [166]
Megalin 500 10, 5 N/A
Farquhar
(UCSD)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [167]
Na,K-
ATPase α1
~100 1, 0.5 1, 0.5
Kashgarian
(Yale)
Mu 1:200 O/N Invitrogen
GAM
680
1:5000 1 hr [74]
Na,K-
ATPase β1
37-
50
1, 0.5 2, 1
P. Martin-
Vasallo
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [168]

91
NaPi2
50-
75
60,30 N/A McDonough Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [75]
NBCe1-A ~130 15, 7.5 N/A
I. Kurtz
(UCLA)
Rb 1:5000 O/N Invitrogen
GAR
680
1:5000 1 hr [169]
NHERF1 50 15, 7.5 N/A
Weinman (U.
Maryland)
Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [170]
NCC 150 60, 30 N/A McDonough Rb 1:5000 O/N Invitrogen
GAR
680
1:5000 1 hr [42]
NCCpS71 150 20, 10 N/A
Loffing
(Zurich)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [77]
NCCpT53 150 60, 30 N/A
Loffing
(Zurich)
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [77]
NHE3 ~75 15, 7.5 8, 4 McDonough Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [68]
NHE3-
pS552
~75 5, 2.5 8, 4
Santa Cruz
(53962)
Mu 1:1000 2 hr LI-COR
GAM
680
1:5000 1 hr [76]
NKCC2 160 15, 7.5 8, 4
DSHB
(Iowa)
Mu 1:6000 O/N Invitrogen
GAM
800
1:5000 1 hr [78]
NKCC2p-
T96T101
160 15, 7.5 8, 4
Forbush
(Yale)
Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [79]
NKCC2p-
S87
160 60, 30 10, 5
DSTT
(Dundee)
Sh 1:2500 2 hr Invitrogen
DAS
680
1:5000 1 hr [171]
OAT 1 74 30, 15 N/A
Alpha
Diagnostic
Rb 1:5000 2 hr Invitrogen
GAR
680
1:5000 1 hr [172]
Plasmino-
gen
90 40, 20 N/A
Abcam
(ab154560)
Rb 1:5000 O/N Invitrogen
GAR
680
1:5000 1 hr [173]

92
ROMK
50
37
40, 20 N/A Novus Rb 1:2000 2 hr Invitrogen
GAR
680
1:5000 1 hr [174]
SPAK
60-
70
20, 10 10, 5 Delpire S15 Rb 1:3000 2 hr Invitrogen
GAR
680
1:5000 1 hr
[80,
81]
SPAKp-
S373
60-
70
80, 40 4, 2
DSTT
(Dundee)
Sh 1:2500 2 hr Invitrogen
DAS
680
1:5000 1 hr
[80,
82]
SGLT1 ~70 80, 40 5, 2.5
Koepsell
(Würzburg)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [175]
SGLT2 ~60 30, 15 N/A
Koepsell
(Würzburg)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [176]
Uromod-
ulin
~100 30, 15 10, 5 Meridian Sh 1:1000 O/N Invitrogen
DAS
680
1:5000 1 hr [177]
Villin
(1D2C3)
100 5, 2.5 N/A
Santa Cruz
(sc58897)
Mu 1:2000 O/N Invitrogen
GAM
800
1:5000 1 hr [83]
Urine immunoblot details
Albumin ~66
0.05-0.0025% of
24hr urine volume
Santa Cruz
(sc271605)
Mu 1:2000 O/N Invitrogen
GAM
680
1:5000 1 hr [148]
Angiotens-
inogen
140
0.05-0.0025% of
24hr urine volume
Abcam
(ab213705)
Rb 1:2000 O/N Invitrogen
GAR
680
1:5000 1 hr [150]
Plasmino-
gen
90
0.05-0.0025% of
24hr urine volume
Abcam
(ab154560)
Rb 1:5000 O/N Invitrogen
GAR
680
1:5000 1 hr [173]

93

Supplemental Figure 2-1. Assessment of equal loading by protein staining of loading gel and
linearity of the detection system. (A) Coomassie stained post SDS-PAGE protein gel (top figure)
with equivalent renal medulla homogenate protein loaded per lane (n=4/group; 16 total). Boxes
outline 3 arbitrarily chosen unidentified protein bands and quantification of the relative density
for each of the 3 protein bands (bottom graph) to illustrate equal loading per lane. (B) 1X protein
concentration and 1/2X protein concentration (see Supplemental Table 1 for amounts) were
loaded to confirm linearity of detection system for each protein. Examples illustrated here
include cortical Na
+
,H
+
exchanger isoform 3 (NHE3), medullary Na
+
,K
+
-ATPase α1 (mNKA
α1), and Na
+
,Cl
-
cotransporter (NCC). Average linearity ratio is 2.0.

Male
Lean Obese
Female
Lean Obese
kD
-75
-100
-75
-100
-75
-100
-100
-250
-100
-250
Cntrl Cntrl
-75
-100
NHE3
1X
1/2X
1X
1/2X
mNKAα1
NCC
1X
1/2X
0
1000
2000
3000
4000
5000
6000
0 5 10 15
Arbitrary Density Units
Lanes on Gel
Protein Loading Gel
Band 1 Band 2 Band 3

94

Supplemental Figure 2-2. Pronounced perirenal fat, lipidemia, and hyperleptinemia in obese rats.
(A) body size, kidney, (B) plasma images, and (C) plasma leptin in ZSF1 rats at 18 weeks of age.

Male
Lean Obese
Female
Lean Obese
LM LF OM OF
A B
LM LF OM OF
0
20
40
60
80
Plasma Leptin (ng/ml)
P<0.001 P=0.002
C

95
General conclusions
The rise in obesity and its comorbidity with high blood pressure and type 2 diabetes mellitus
imposes a significant financial burden on the health system but also on the lives of billions of
people globally. It is now well established that sex-specific differences in kidney function
manifest during reproductive years that lead to lower blood pressure, the prevalence of diabetes,
and risk of cardiovascular and kidney disease in pre-menopausal women compared to age-
matched men. Mechanisms accounting for this female advantage remain an important gap in
renal physiology. This dissertation focused on defining sex-specific intrarenal mechanisms to
obesity and hypertension to better understand this female advantage.
First, the current dissertation aimed to define intrarenal mechanisms governing acute
pressure natriuresis in female SD rats. We tested the hypothesis that females (versus males)
would have more robust pressure natriuresis mediated by baseline lower fractional Na
+

reabsorption in the proximal tubule and suppression of medullary sodium reabsorption. We show
females (versus males) have a more robust acute pressure natriuretic response that evokes a
leftward shift in renal function curves and greater sum-total UNaV that is mediated by distinct
transporter patterns. We provide evidence that natriuresis stems from proximal tubule to
medullary thick ascending limb inhibition in females whereas in males natriuresis seems to stem
from proximal tubule and distal convoluted tubule. Second, the current dissertation aimed to
define sexual dimorphisms in renal tubular response to obesity and early diabetic kidney disease
and determine the physiological consequences these differences have in disease progression. Our
results provide evidence for the female head-start in transporter abundance patterns along the
nephron in lean ZSF1 rats. Obese rats exhibit a similarly lower abundance of key Na
+

transporters, channels, and claudins along the nephron respective to lean rats. This similar

96
response between OM and OF suggests the baseline female head-start contributes to slower
disease progression in females. Despite this head-start, obesity induces hypertrophy,
hyperinsulinemia, lipidemia, progressively worsening proteinuria, and greater mSGLT1 in both
males and females. We also provide evidence that sodium intake, independent of calorie intake,
contributes to disease progression in OM. Collectively the current dissertation provides evidence
that the female advantage is partially mediated by baseline sex-specific differences along the
nephron that give females a head-start in renal adaptations to hypertension and metabolic
challenges.
There are a number of unanswered questions and potential implications of the current
dissertation. Future studies should investigate the role of hormones in disease pathology. Induce
menopause and repeating these studies would yield interesting contributions to the understanding
of female physiology. In ZSF1 rats, investigating intrarenal adaptions in hyperglycemic females
whether induced (i.e., high fat diet) versus euglycemic females would complement the findings
summarized here. Lastly, does empagliflozin treatment (SGLT2 inhibitor) shift renal function
curves in males to match females? Answering this question may have clinically relevant
implications for treatment of hypertension and kidney disease. In the current dissertation, we
provide evidence that obese female ZSF1 rats may serve as a clinically relevant model for
obesity-induced kidney disease model. Additionally, we provide evidence that SGLT1 may be a
useful therapeutic target for obesity. Inhibition of SGLT1 in concert with obesogenic therapies
(i.e. GIP, GLP agonists) may delay disease progression and onset of diabetes.

97
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Appendix

Appendix Figure 2-1. Summary of transporters, channels, claudins, and regulators along the
nephron in SD females and lean ZSF1 females normalized to respective males………113
Appendix Figure 2-2. Summary of full transporters, channels, claudins, and regulators along the
nephron in ZSF1 rats……………………………………………………………………114
Appendix Figure 2-2. Full longitudinal analysis of physiological parameters 1……………….115
Appendix Figure 2-3. Full longitudinal analysis of physiological parameters 2……………….117

113

Appendix Figure 2-1. Summary of transporters, channels, claudins, and regulators along the
nephron in SD females and lean ZSF1 females normalized to respective males. Data expressed as
mean ± SEM (n=4-6/group) and statistical comparisons were performed using GraphPad Prism
(9.2.0) multiple parametric unpaired Student’s t-test.

Cortex Medulla Cortex Medulla
NHE3
NHE3p
NBCe1-A
Villin
NHERF1
DPPIV
NaPi2
SGLT2
AQP1-35 kD
AQP1-24 kD
Megalin
HO-1
Cldn 2
Collectrin
NKA α1
NHE3
NHE3p
SGLT1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NKA α1
NKA β1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NCC
NCCpS71
NCCpT53
ROMK Gly.
ROMK Core
αENaC - Fl
αENaC -Cl
βENaC
γENaC - Fl
Cldn 7
Cldn 8
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
0.0
0.5
1.0
1.5
2.0
3.0
6.0
Relative Abundance (Male = 1)
SD Female ZSF1 LF
*
*
* * *
*
*
*
**
*
**
**
**
*
*
* *
*
*
*
*
*
**
**
**
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*

114

Appendix Figure 2-2. Full summary key transporters, channels, claudins, and regulators along
the nephron. (A) Comparison of baseline sex-specific protein expression in LM and LF
normalized to LM =1 (solid lines). *P<0.05 compared to LM. (B) Comparison effect of
hyperglycemia and obesity on protein expression in OM and OF normalized to respective lean
rats = 1 (solid line). *P<0.05 compared to respective leans. White stars at the base of the column
denote P<0.05 between normalized OM vs. OF. Data expressed as mean ± SEM (n=4/group) and
statistical comparisons were performed using GraphPad Prism (9.2.0) multiple parametric
unpaired Student’s t-test.

NHE3
NHE3p
DPPIV
NBCe1-A
Villin
NHERF1
NaPi2
SGLT2
SGLT1
AQP1-35 kD
AQP1-24 kD
Megalin
Cubilin
Albumin
OAT1
HO-1
Cldn 2
A'ogen
ACE 1
ACE 2
Collectrin
NKA α1
NKA β1
NHE3
NHE3p
SGLT1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NKA α1
NKA β1
Uromodulin
Uromodulin
Calbindin
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
Kir4.1
NCC
NCCpS71
NCCpT53
ROMK Gly.
ROMK Core
Plasminogen
αENaC - Fl
αENaC -Cl
βENaC
γENaC - Fl
Alix
Cldn 7
Cldn 8
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
Cldn 4
Cldn 10
CD63
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
0.0
0.5
1.0
1.5
2.0
3.0
6.0
Relative Abundance (LM = 1)
*
LM LF
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NHE3
NHE3p
DPPIV
NBCe1-A
Villin
NHERF1
NaPi2
SGLT2
SGLT1
AQP1-35 kD
AQP1-24 kD
Megalin
Cubilin
Albumin
OAT1
HO-1
Cldn 2
A'ogen
ACE 1
ACE 2
Collectrin
NKA α1
NKA β1
NHE3
NHE3p
SGLT1
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
NKA α1
NKA β1
Uromodulin
Uromodulin
Calbindin
SPAK
SPAKp
NKCC2
NKCC2pT96T101
NKCC2pS87
Kir4.1
NCC
NCCpS71
NCCpT53
ROMK Gly.
ROMK Core
Plasminogen
αENaC - Fl
αENaC -Cl
βENaC
γENaC - Fl
Alix
Cldn 7
Cldn 8
Cldn 10
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
Cldn 4
Cldn 10
CD63
AQP2-37 kD
AQP2-24 kD
AQP2p-37 kD
AQP2p-24 kD
0.0
0.5
1.0
1.5
2.0
3.0
6.0
Relative Abundance (Lean = 1)
OM OF
**
* *
*
*
*
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
**
*
*
*
**
*
*
*
*
****
*
*
**
*
*
*
*
*
*
Cortex Medulla Cortex Medulla
A
B

115

Appendix Figure 2-3. Complete longitudinal analysis of (A) body weight, (B) systolic blood
pressure, (C) non-fasted blood glucose, (D) 18 week fasted blood glucose, (E) urine glucose in
10 16 18 10 16 18
0
200
400
600
800
Weeks
Body Weight (g)
Lean Obese
Male Female
Three-way ANOVA Results:
Time P<0.001
Obesity P<0.001
Sex P<0.001
all comparisons P<0.001
10 16 10 16
0
100
200
300
400
500
Weeks
Non-fasted
Blood Glucose (mg/dl)
Lean Obese
P=0.002
Three-way ANOVA Results:
Time P=0.006
Obesity P<0.001
Sex P=0.003
10 16 18
0
1000
2000
4000
6000
8000
10000
Weeks
Urine Glucose (mg/dl)
Two-way ANOVA Results:
Interaction P<0.001
Time P<0.001
Sex P<0.001
P=0.001 P<0.001
P<0.001
P<0.001
10 16 10 16
100
150
200
250
Weeks
SBP (mmHg)
Lean Obese
Three-way ANOVA Results:
Time P=0.93
Obesity P=0.0004
Sex P=0.87
LM LF OM OF
0
100
200
300
400
500
18 wk Fasted
Blood Glucose (mg/dl)
P=0.003 P=0.02
LM LF OM OF
0
5
10
15
7000
10000
18 week
Urine glucose (mg/dl)
P<0.001 P<0.001
10 16 18 10 16 18
0
5
10
15
20
25
Weeks
Plasma Creatinine (µM)
Lean Obese
Three-way ANOVA Results:
Time P<0.001
Obesity P<0.001
Sex P=0.06
P=0.02
P<0.001
P<0.001
P<0.001
P=0.001
P<0.001
P<0.001
P<0.001
P=0.001
P=0.001
LM LF OM OF
250
275
300
325
350
Plasma Osmolality
(mOsm/kg H
2
O)
P=0.001
10 16 18 10 16 18
110
120
130
140
150
160
Weeks
Plasma Na (mM)
Ln Ob
Three-way ANOVA Results:
Time P<0.001
Obesity P=0.25
Sex P=0.06
P<0.001
P=0.04
P=0.01
P=0.02
P=0.002
P<0.001
P<0.001
10 16 18 10 16 18
0
2
4
6
8
Weeks
Plasma K (mM)
Lean Obese
Three-way ANOVA Results:
Time P<0.001
Obesity P=0.01
Sex P=0.32
P=0.001
P=0.005
P<0.001
P=0.02
P<0.001
P=0.003
P=0.002

116
obese rats, (F) 18 week urine glucose in lean and obese rats, (G) plasma creatinine, (H) plasma
osmolality, (I) plasma [Na], and (J) plasma [K]. Statistical comparisons were performed using
GraphPad Prism (9.2.0) one-way ANOVA (D, F, and H) or three-way ANOVA (A-C, E, G, I-J)
followed by Tukeys multiple comparisons post hoc test. P values provided.

117

Appendix Figure 2-4. Complete longitudinal analysis of (A) food intake, (B) water intake, (C)
urine volume, (D) creatinine clearance, (E) lithium clearance, (F) sodium clearance, (G) urinary
sodium excretion, (H) urinary potassium excretion, (I) urinary osmoles, and (J) urinary osmolar
10 16 18 10 16 18
0
20
40
60
Weeks
Food Intake (g/24 hr)
Lean Obese
Three-way ANOVA Results:
Time P=0.005
Obesity P<0.001
Sex P=0.002
P=0.008
P=0.006
P<0.001
Males Females
10 16 18 10 16 18
0
20
40
60
80
Weeks
Urine Volume (ml/24 hr)
Lean
Obese
Three-way ANOVA Results:
Time P=0.004
Obesity P=0.0001
Sex P=0.001
P<0.001
P<0.001
P<0.001
P=0.003
10 16 18 10 16 18
0
100
200
300
400
Weeks
Lithium Clearance
(ml/24hr)
Lean Obese
Three-way ANOVA Results:
Time P<0.001
Obesity P<0.001
Sex P=0.005
P=0.02
P<0.001
P<0.001
P<0.001
P<0.001
P=0.002
10 16 18 10 16 18
0
2
4
6
Weeks
UNaV (mmol/24hr)
Lean Obese
Three-way ANOVA Results:
Time P<0.001
Obesity P<0.001
Sex P<0.001
P<0.001
P<0.001
P<0.001
P=0.04
P<0.001
10 16 18 10 16 18
0
20
40
60
80
100
Weeks
Water Intake (ml/24 hr)
Lean
Obese
Three-way ANOVA Results:
Time P=0.001
Obesity P=0.006
Sex P=0.007
P=0.04
P=0.008
P=0.04
P=0.006
P=0.008
10 16 18 10 16 18
0
2
4
6
Weeks
Creatinine Clearance
(L/24hr)
Lean
Obese
Three-way ANOVA Results:
Time P=0.38
Obesity P<0.001
Sex P<0.001
P<0.001
P<0.001
P=0.02
P=0.006
P=0.003
P=0.04
10 16 18 10 16 18
0
20
40
60
Weeks
Sodium Clearance
(ml/24hr)
Lean
Obese
Three-way ANOVA Results:
Time P<0.001
Obesity P<0.001
Sex P=0.004
P<0.001
P<0.001
P<0.001
P=0.002
P=0.006
10 16 18 10 16 18
0
5
10
15
Weeks
UKV (mmol/24hr)
Lean Obese
Three-way ANOVA Results:
Time P=0.006
Obesity P<0.001
Sex P<0.001
P<0.001
P<0.001
P<0.001
P=0.005
P<0.001
10 16 18 10 16 18
0
1000
2000
3000
4000
Weeks
Uosm
(mosm/kg H
2
O)
Lean Obese
Three-way ANOVA Results:
Time P=0.16
Obesity P=0.009
Sex P=0.56
10 16 18 10 16 18
0
20000
40000
60000
80000
Weeks
UosmV
(mosm/24 hr UV)
Lean Obese
Three-way ANOVA Results:
Time P=0.006
Obesity P<0.001
Sex P<0.001
P<0.001
P<0.001
P<0.001
P<0.001

118
excretion. Statistical comparisons were performed using GraphPad Prism (9.2.0) three-way
ANOVA followed by Tukeys multiple comparisons post hoc test. P values provided.

Abstract (if available)

Abstract In the United States, it’s estimated that 74% of American adults are overweight or obese which is associated with a range of cardiometabolic disorders including the two leading causes of chronic kidney disease – hypertension and diabetes – and is now considered an independent risk factor for kidney disease. Hypertension and type 2 diabetes mellitus together account for three-fourths of all chronic kidney disease cases. Furthermore, these two risk factors have high comorbidity that exacerbates disease progression and increases the risk of cardiovascular-related mortality. It is established that pre-menopausal women exhibit lower blood pressure, the prevalence of diabetes, and the risk of cardiovascular and kidney disease compared to age-matched men. Our lab has previously highlighted sexual dimorphic patterns of kidney transporters and electrolyte handling that may confer a “head-start” in kidney adaptations to disease. However, mechanisms accounting for this “female advantage” remain an important gap in renal physiology. This dissertation aimed to define intrarenal sexual dimorphisms to hypertension and obesity with the goal to provide a foundation for optimizing sex-specific therapies and provide disease-specific targets for future therapeutics.
Pressure natriuresis is a powerful mechanism used by the kidneys for acute and chronic maintenance of effective circulating volume. Our lab previously showed that redistribution of proximal tubule Na+,H+ exchanger isoform 3 (NHE3) from within the microvilli to the microvillar base (inactive) plays an important role in pressure natriuresis response to vasoconstriction. Additional studies show NHE3 is localized to the base of the microvilli in females as baseline. In Chapter 1, we aimed to define the physiological and molecular responses contributing to pressure natriuresis in female SD rats. We determined that acutely increasing vascular resistance via vasoconstriction leads to similar levels of hypertension between sexes but females exhibit more robust pressure natriuresis mediated by baseline sex-specific transporter patterns and complemented by further inhibition of proximal tubule to medullary thick ascending limb transporters. In males, distinct patterns of lower proximal tubule and distal tubule transporters contribute to pressure natriuresis. We additionally show that inhibition of arachidonic acid metabolites with 1-aminobenzotraizole (ABT) similarly lowers blood pressure and shifts renal function curves to the left in both sexes. In males, not females, ABT treatment also lowered sodium transporter abundance contributing to the leftward shift in renal function curves. Together these findings suggest sex-specific changes in pool size of Na+ transporters that contribute to pressure natriuresis.
In Chapter 2, we aimed to define sexual dimorphisms in renal tubular response to obesity and early diabetic kidney disease and to determine the physiological consequences of the differences on disease progression. To accomplish this aim, experimental protocols were performed using the highly translatable ZSF1 rat model. Our findings show both obese males and females develop metabolic complications including hypertrophy, lipidemia, hyperinsulinemia, and glomerular fibrosis; only obese males develop hyperglycemia and glycosuria. We provide evidence that hyperglycemia may exacerbate kidney pathology, but obesity, independent of hyperglycemia, may induce initial kidney pathology. Both obese males and females similarly exhibit patterns of lower pool sizes of key Na+ transporters. In both chapters, we show the female advantage to slower disease progression is partially mediated by baseline sex-specific differences along the nephron that give females a head-start in kidney adaptations to hypertension and metabolic challenges.

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Asset Metadata

Creator McFarlin, Brandon Eugene (author)

Core Title Defining intrarenal sexual dimorphisms to obesity and hypertension: understanding the female advantage

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Medical Biology

Degree Conferral Date 2022-05

Publication Date 04/11/2022

Defense Date 03/10/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag diabetic kidney disease,hypertension,OAI-PMH Harvest,obesity,pressure natriuresis,sex differences,sodium transport

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Li, Zhongwei (committee chair), Peti-Peterdi, Janos (committee chair), McDonough, Alicia A. (committee member), Perin, Laura (committee member), Youn, Jang-Hyun (committee member)

Creator Email bmcfarli@usc.edu,bmcfarli@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC110920055

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