proximal tubule microvilli remodeling and albuminuria in the ren2 transgenic rat

Manuscript # F-00252-2006.R2

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Proximal Tubule Microvilli Remodeling and Albuminuria in the Ren2 Transgenic

Rat

Melvin R Hayden, MD1, Nazif A Chowdhury, MD1, Shawna A Cooper MS1, Adam Whaley-Connell, DO, MSPH 1,4, Javad Habibi, PhD1, Lauren Witte, BA1, Charles

Wiedmeyer, DVM, PhD5, Camila Margarita Manrique, MD1, Guido Lastra, MD1, Carlos Ferrario, MD7, Craig Stump, MD, PhD 1,2,3,6, James R Sowers, MD 1, 2,3,6

University of Missouri School of Medicine, Departments of Internal Medicine1, Medical Pharmacology and Physiology2, Divisions of Endocrinology3 and Nephrology4, College

of Veterinary Medicine5, Harry S Truman VA Medical Center6, Hypertension and Vascular Disease Unit, Bowman Gray School of Medicine Wake Forest University7

Running Title: Renal Proximal Tubule Remodeling, Oxidative Stress, and Tissue Ang-II Overexpression Word Count Abstract - 250 Text – 4,344

Corresponding author: James R. Sowers, M.D., FACE, FACP, FAHA Thomas W. and Joan F. Burns Missouri Chair in Diabetology Director of the MU Diabetes and Cardiovascular Center Professor of Medicine, Physiology and Pharmacology 1 Hospital Dr. MA410 Medical Science Bldg. Columbia, MO 65212 (573) 884-2013 (573) 884-1996 (fax) [email protected]

of 27Articles in PresS. Am J Physiol Renal Physiol (October 10, 2006). doi:10.1152/ajprenal.00252.2006

Copyright © 2006 by the American Physiological Society.


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Abstract

TG(mRen2)27 (Ren2) transgenic rats overexpress the mouse renin gene with

subsequent elevated tissue Ang-II, hypertension, and nephropathy. The proximal tubule

cell (PTC) is responsible for the reabsorption of 5-8 grams of glomerular filtered albumin

(Alb) each day. Excess filtered albumin may contribute to PTC damage and

tubulointerstitial disease. This investigation examined the role of angiotensin-II (Ang-II)

induced oxidative stress in PTC structural remodeling; whether such changes could be

modified with in vivo treatment with Ang type 1 receptor (AT1R) blockade (valsartan) or

superoxide dismutase/catalase mimetic (tempol). Male Ren2 (6-7 week old) and age-

matched Sprague-Dawley (SD) rats were treated with valsartan (Ren2-V; 30 mg/kg),

tempol (Ren2-T; 1 mmol/L), or placebo for three weeks. Systolic blood pressure (SBP),

albuminuria, n-acetyl-β-d-glucosaminidase (beta-NAG), kidney tissue malondialdehyde

(MDA) were measured and PTC microvilli structure assessed using 60K transmission

electron microscopy (TEM) images. There were significant differences in SBP,

albuminuria, lipid peroxidation (MDA and Nitrotyrosine staining), and PTC structure in

Ren2 versus SD rats (each p <0.05). Increased mean diameter of PTC microvilli in the

Ren2-C (p <0.05) correlated strongly with albuminuria (r2 = 0.83) and moderately with

MDA (r2 = 0.49), and there was an increase in the ratio of abnormal forms of microvilli

in Ren2 rats when compared to SDC (p <0.05). AT1R blockade, but not tempol

treatment, abrogated albuminuria and beta-NAG; both therapies corrected abnormalities

in oxidative stress and PTC microvilli remodeling. These data indicate that PTC

structural damage in the Ren2 rat is related to oxidative stress response to Ang-II and/or

albuminuria.

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Keywords Ang-II, Malondialdehyde, Albuminuria, Proximal Tubule Cell,

TG(mRen2)27 (Ren2) Transgenic Rat

Introduction

Chronic kidney disease (CKD) is increasing worldwide (15, 27). In the United

States, it is estimated that 372,000 persons have end-stage renal disease (ESRD), and

approximately 11% of adults are currently in the earlier stages of CKD (15). Principal

causes of CKD leading to ESRD are type 2 diabetes mellitus (T2DM) and hypertension

(HTN) (15, 27, 29, 31). Proteinuria is emerging as a major mediator of progressive renal

disease leading to renal interstitial fibrosis and progressive renal injury (15, 29). The

concept has been developed that excess protein filtered through the glomerulus exerts

direct toxicity upon epithelial cells of the kidney proximal tubule (3, 5, 8, 28). Excessive

filtered protein, particularly albumin (Alb), is thought to exert direct pro-inflammatory

and pro-fibrotic effects on tubular epithelial cells (3, 5, 8, 13, 21, 29) and cause renal

interstitial fibrosis (5, 8, 13).

Renal proximal tubular epithelial cells (PTC) are important for the reabsorption of

most of the glomerular physiologically filtered Alb (8). Approximately 5 to 8 grams of

Alb passes through the glomerular filtration barrier daily, yet only approximately 30 mg

or less appears in the urine (8). Reabsorption of filtrate in the PTC occurs primarily by

clathrin and receptor-mediated endocytosis (3, 5, 8, 13, 28, 35). Microvilli cover the

apical portion of the PTC and provide the surface responsible for receptor–mediated

endocytosis of Alb. Receptors that mediate this endocytosis include megalin and cubilin.

Cytoskeletal remodeling involving actin and myosin mediated contraction facilitates the

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formation of endocytic vesicles (3, 5, 8, 13, 28, 35). Alb is dissociated from these

receptors and transported to lysosomes for degradation to amino acids, which are released

through the basolateral membranes and absorbed into interstitial capillaries.

The mechanism by which exposure of excessive Alb leads to PTC injury, and the

morphological and functional nature of this injury remains poorly understood.

Compensatory hypertrophy of PTC in response to renal damage caused by HTN,

hypoxia, and metabolic abnormalities appear to be an early process in the progression to

tubular atrophy and tubulointerstitial fibrosis (18, 38). This hypertrophic process may be

followed by apoptosis and other mechanisms leading to loss of PTC and/or microvilli

(18, 20, 37, 38). There is accumulating evidence that the renal renin-angiotensin system

(RAS) plays a pivotal roll in PTC maladaptive responses to various types of renal injury

(4, 9, 16, 17). PTC produce and secrete angiotensin-II (Ang-II) into the lumen where its

concentration (6-10 nmol/L) is at least 10 times higher than that in plasma (17). Ang-II

(AT1 and AT2) receptors are located on both basolateral and luminal sides of the PTC (4,

9, 16). The critical location of Ang-II receptors and the high concentration of Ang-II

suggest an autocrine/paracrine role for the RAS in the PTC. Further, in pathophysiologic

conditions of albuminuria, as in T2DM or HTN, treatment with blockers of the AT1R

reduce the progression of renal disease (15, 27, 29, 43), as well as urine Alb loss (36).

However, the precise mechanisms by which Ang-II mediates renal tubular damage

remains poorly understood.

Excessive albuminuria has been reported to induce Ang-II production and

increase Ang-II uptake by an AT1R-mediated process (17). Increased renal Ang-II has

been reported in systemically Ang-II infused rats and in the transgenic TG (mRen2)27

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(Ren2) rat (19, 22, 41). The Ren2 rat overexpresses Ang-II in tissues and develops HTN,

insulin resistance, and albuminuria (2, 22, 36). Investigations conducted in this and other

hypertensive, insulin resistant, proteinuric rodent models has shown that Ang-II induced

oxidative stress contributes to development of insulin resistance, HTN, albuminuria, and

renal injury (2, 22, 36), and that these effects can be abrogated by AT1R blockade and

anti-oxidant therapy (2, 26, 30, 36). Accordingly, in the present investigation we

hypothesized that 1) the Ren2 rat would manifest oxidative stress mediated PTC injury in

relation to albuminuria; 2) treatment of this model of Ang-II overexpression in vivo with

either an AT1R blocker or a cell-permeable superoxide dismutase (SOD)/catalase

mimetic (1, 32) would attenuate this Ang-II induced oxidative stress mediated PTC

injury.

Materials and Methods Animals and treatments

Male transgenic Ren2 rats and Sprague-Dawley controls (SDC) were obtained at

6 weeks of age and fed rat chow throughout the investigation. They were randomly

assigned to treatment with valsartan (Ren2-V, SDV), tempol (Ren2-T, SDT) or placebo

treated groups (Ren2-C, SDC). The treatment animals received valsartan (30 mg/kg/day)

or tempol (1 mmol/L) in drinking water based on previous work (2), for 21 days prior to

sacrifice. All protocols were approved by the University of Missouri and Harry S.

Truman Veterans medical center animal care and use committee and housed/harvested in

accordance with NIH guidelines.

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Determination of Weight, Systolic Blood Pressure (SBP), Albuminuria, and N-Acetyl-β-

D- Glucosaminidase (beta-NAG)

Restraint conditioning was initiated on the day of initial SBP measurement and

then measured in triplicate, on separate occasions throughout the day, using the tail-cuff

method (Harvard Systems, Student Oscillometric Recorder) on days 19 or 20 of treatment

prior to sacrifice (2, 36) in addition to weights obtained.

A commercially available kit for rat urine Alb (Nephrat, Exocell Inc.,

Philadelphia, PA) was used to measure urine Alb. Urine was collected over a 24 hour

period at the end of treatment and measured for levels of Alb normalized to creatinine.

The Jaffe reaction on an automated chemistry analyzer (Olympus AU400, Olympus

America, Dallas, TX) was used to measure urine creatinine (Cr). Results are reported as

a ratio of urine Alb to Cr (mg/mg).

N-acetyl-β-d-glucosaminidase (beta-NAG) is a 140 kDa lysosomal enzyme

present in high concentrations in PTC, but not typically excreted into the urine by normal

intact PTC (25). Thus, increased beta-NAG in the urine is a potential marker for PTC

injury (25). Urine excretion of beta-NAG was determined by colorimetric assay from

Roche Diagnostics (Indianapolis, IN). Urine samples were first fractionated on a gel

filtration column to remove NAG inhibitors and then assayed for NAG activity via a

colorimetric assay on an automated chemistry analyzer (Olympus AU400, Olympus

America, Dallas, TX). Beta-NAG excretion is expressed as mU of NAG activity.

Tissue Malondialdehyde (MDA):

To measure lipid peroxidation, tissue MDA levels were determined as a surrogate

end-marker for oxidative stress (36). Butylated hydroxytoluene (BHT; 5 mM) was added

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to kidney cortical tissue (100 mg) to prevent new lipid peroxidation. Samples were then

homogenized in a buffer solution (0.25 M sucrose, 0.5 mM EDTA, 50 mM HEPES,

protease inhibitors) on ice and centrifuged at 4° C at 15,000 rpm for 10 minutes. The

supernatant was collected for MDA and O-phthaldialdehyde (OPA) protein assays.

Supernatant (200 μL) was used to measure free MDA using a MDA-586

spectrophotometric assay kit (OxisResearch Biotech, Portland OR). Total protein was

measured by OPA fluorometric assay and FLX-800 fluorometer (BioTek, Winooski,

VT).

Immunostaining of PTC with Nitrotyrosine:

Nitrotyrosine staining was used to corroborate levels of lipid peroxidation in the

PTC. Sections of kidney were deparaffinized, rehydrated, and epitopes retrieved in

citrate buffer. Endogenous peroxidases were quenched with 3% H2O2 and non-specific

binding cites were blocked with avidin, biotin, and finally with protein block (Dako,

Carpinteria, CA). Sections were incubated with 1:200 primary antibody, rabbit

polyclonal anti-Nitrotyrosine antibody (Chemicon, Cat# AB5411) then washed,

incubated with the secondary antibodies, linked, and labeled (Strepavidin, Dako, LSAB+

Kit K0690) for 30 min each. After several rinses with distilled water, diaminobenzidine

(DBA Dako, K3466) was applied for 10 min. Sections were again rinsed with distilled

water, then stained with hematoxilyn for 1 min, rehydrated, and mounted with a

permanent media. Slides were evaluated under a bright field (Nikon 50i) microscope and

40X images were captured with a cool snapcf camera. Images were analyzed and signal

intensities measured with MetaVue (Boyce Scientific Inc. Gary Summit, MO).

Transmission Electron Microscopy (TEM) Methods

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Renal cortical tissue from excised kidneys was thinly sliced and placed

immediately in primary EM fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M

Na cadodylate buffer, pH 7.35). A Pelco 3440 Laboratory Microwave Over was utilized

for secondary fixation, with acetone dehydration and Epon-Spurr’s resin infiltration.

Specimens were placed on a rocker overnight at room temperature, embedded the

following morning, and polymerized at 60 degrees Centigrade for 24 hours. A Leica

Ultracut UCT microtome with a 45 degree Diatome diamond knife was used to prepare

85 nm thin sections. The specimens were then stained with 5% uranyl acetate and Sato’s

Triple lead stain. A JOEL 1200-EX transmission microscope was utilized to view all

renal samples.

Three glomerular/proximal tubule fields were randomly chosen per rat in order to

obtain three 60K images/kidney containing cross sectional views of the microvilli. PTC

were visualized adjacent to glomeruli and were identified on the basis of their continuous

microvilli at the apical surface. Then five (450 nm X 450 nm) images were cropped out

of each 60K image obtained. These cropped images were then analyzed using Image J (a

public domain Java image processing program made possible by the NIH). By viewing

the microvilli in cross section in each cropped image, the diameter of the microvilli were

measured and recorded in a blinded fashion (38). Additionally, abnormal forms as

defined by a diameter greater than 100 nm were counted per each field (Figure 3). The

value of 100 nm was chosen as a value approximately greater than 2 standard deviations

above the upper limit of the range of 76.6 to 86.9 nm of normal proximal tubules

observed in control animals.

RNA Isolation and Reverse Transcriptase/Real-time Polymerase Chain Reaction

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RNA was isolated from renal tissue and measured for angiotensinogen, renin,

angiotensin-converting enzyme (ACE), ACE2, neprilysin, AT1R, and the mas receptor

using the TRIZOL reagent (GIBCO Invitrogen, Carlsbad, CA), as previously reported

(34).

Statistical Analysis

All values are expressed as mean ± standard error. Statistical analyses were

performed in SPSS 13.0 (SPSS Inc., Chicago IL). Abnormal forms were evaluated via

nonparametric binomial testing with SDC control values as the test parameter and a cut

point of 100 as a value approximately greater than 2 standard deviations above the upper

limit of the range of 76.6 to 86.9 nm of normal proximal tubules. All other variables

were evaluated parametrically via ANOVA with Fisher’s LSD, as appropriate. Data from

PTC measurements were then plotted against final systolic blood pressure, albuminuria,

and MDA and then natural log (ln) transformed as appropriate for linear regression

analysis. P <0.05 was considered statistically significant.

Results

Systolic Blood Pressure (SBP), Albuminuria, and beta-NAG

SBP were significantly higher in Ren2-C (n = 6) (192 ± 5.8 mmHg) compared to

SDC (n = 4) (123 ± 5.5 mmHg) (p <0.05) (Table 1), and significantly lower with

valsartan treatment (Ren2-V) (n = 6) (147 ± 6.1 mmHg) (p <0.05) but not improved with

tempol treatment (Ren2-T) (n = 4) (188 ± 8.4 mmHg) (p >0.05). Consistent with prior

observations that the Ren2 is insulin resistant, measured glucoses were higher in the Ren2

than SD (p <0.05). Similarly, albumin/creatinine ratio (Table 1) was higher in Ren2-C

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(0.27 ± 0.02 mg/mg) versus SDC (0.06 ± 0.01 mg/mg) (p <0.05), and decreased with

valsartan treatment (0.05 ± 0.02 mg/mg, p <0.05), but not tempol (0.31 ± 0.07 mg/mg) (p

>0.05). Beta-NAG was not significantly elevated in the Ren2-C animal compared to

SDC. However there were significant reductions seen with valsartan treatment (Table 1).

Oxidative stress:

A marker of lipid peroxidation, MDA levels were elevated in Ren2 (Ren2-C)

kidney cortical tissue (0.56 ± 0.17 µg of MDA/mg of protein) compared to SDC (0.05 ±

0.01 µg of MDA/mg of protein) (p <0.05), and decreased in the Ren2-V (0.10 ± 0.01 µg

of MDA/mg of protein) and Ren2-T (0.22 ± 0.12 ug MDA/mg protein) (p <0.05) (Table

1). Similar to MDA, there were significant increases in grey scale intensity measures of

Nitrotyrsoine, another marker of lipid peroxidation, on PTC cross sections in the Ren2-C

(53.2 ± 5.6 arbitrary units) compared to SDC (20.6 ± 3.6 arbitrary units) (p <0.05) that

were decreased in both the Ren2-V (22.2 ± 1.8 arbitrary units) and Ren2-T (32.4 ± 5

arbitrary units) (each p <0.05) (Figure 1A and B).

Ultrastructure analysis:

The diameters of PTC microvilli were measured in cross section and compared

between treatment groups (Figure 2 and 3). Ren2-C exhibited a significantly increased

mean microvilli diameter (86.94 ± 0.25 nm) (Figure 3A) associated with albuminuria (r2

= 0.83) and levels of MDA (r2 = 0.49) (Figure 3B) when compared to SDC (76.58 ± 0.19

nm) (p <0.05). Treatment with valsartan (Ren2-V) and tempol (Ren2-T) resulted in a

reduction of the mean diameter (75.85 ± 0.14 nm and 80.57 ± 0.22 nm; p <0.05,

respectively). Even with the exclusion of abnormal forms from diameter analyses, the

relationships among the groups remained significant (data not shown). Thus, the presence

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of abnormal forms alone did not account for the diameter increase seen in Ren2-C

animals. The number of abnormal forms as a ratio of abnormal forms/total number of

microvilli was increased in the untreated Ren2-C rats compared to SDC (0.14 ± 0.01 vs.

0.03 ± 0.01) (p <0.05) (Figure 4B). Treatment groups Ren2-V and Ren2-T demonstrated

a significant reduction in the number of abnormal forms (0.03 ± 0.002 and 0.05 ± 0.01) (p

<0.05).

Tissue mRNA for RAS: There were expected increases in mRNA transcripts for

AT1R (1.79 ± 0.20) and mas receptor (2.43 ± 0.04) in Ren2-C when compared to SDC (p

<0.05) and decreases with AT1R blockade (1.28 ± 0.18 and 1.38 ± 0.13, respectively; p

<0.05) and also tempol treatment (mas receptor, 1.87 ± 0.16; p <0.05). Further, there

were significant improvements with AT1R blockade in the Ren2-C mRNA transcripts of

ACE2 (1.80 ± 0.20), renin (5.41 ± 1.10), angiotensinogen (1.43 ± 0.11), and neprilysin

(1.61 ± 0.07) (each p <0.05). Similar improvements were seen with tempol treatment in

ACE2 (1.97 ± 0.18), neprilysin (1.81 ±0.22), and angiotensinogen (1.57 ± 0.1) mRNA

transcripts (each p <0.05).

Discussion

This investigation demonstrated that the Ren2 rat, which manifests enhanced

tissue RAS (2, 22, 36, 41), develops renal tubular structural abnormalities in conjunction

with development of HTN and albuminuria. TEM analysis revealed that the proximal

tubules of the Ren2 rat displayed substantial microvilli abnormalities when compared

with age-matched SD littermates. These abnormalities were generally characterized by

increased mean diameter and increased numbers of abnormal microvilli structures.

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Further, the extent of the abnormalities of the Ren2 microvilli were directly related to

quantitative increases in urinary albumin. These observations are in concert with prior

reports that albuminuria may cause PTC injury (3, 5, 8, 13, 28).

Our current understanding of PTC damage in response to albuminuria or other

insults (i.e. hypoxia and metabolic abnormalities) is very limited. However, the TEM

abnormalities in the Ren2 rat are potentially consistent with loss of the cytoskeletal

integrity necessary for maintenance of erect tubules (20, 37, 38). Indeed, it has been

previously observed that actin and myosin microfilaments undergo depolymerization,

fragmentation, and basolateral retraction from the apical region of the microvilli in

conditions of ischemia and HTN injury (20, 37, 39). This loss of cytoskeletal integrity

likely contributes to flaccid/folded microvilli. This alteration, in conjunction with

apoptosis/necrosis, could account for the loss of microvilli and widening of remaining

microvillar structures observed in this study. Finally, the observation that oxidative stress

increased in the Ren2 as measured by MDA and confirmed with staining of nitrotyrosine

in the PTC and that in vivo treatment with both valsartan and tempol abrogated the

microvilli structural abnormalities may suggest a role for oxidative stress as a common

converging process mediating PTC injury to both Ang-II and albumin excess in the Ren2

rat.

As previously observed in skeletal tissue (2) and cardiovascular tissue (34), there

were increased oxidative changes (MDA) in the renal cortical tissue of the Ren2 rat. The

HTN (22, 41), insulin resistance (2, 12, 34), and albuminuria (36) in this rodent (2, 12,

34) model are related to excess tissue Ang-II signaling through the AT1R to increase

oxidative stress. Our observed increases in lipid peroxidation, both in cortical tissue

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MDA levels and more specifically on Nitrotyrosine staining of the PTC in the Ren2,

suggests a role for oxidative stress in the PTC remodeling. That both AT1R blockade and

tempol treatment decreased renal lipid oxidation, as well as PTC microvilli structural

abnormalities, provides evidence for Ang-II/albumin induced oxidative stress as a

convergent pathway in the pathogenesis of PTC injury in this rodent model of tissue RAS

overexpression. Furthermore, it is also important to consider ACE2 and neprilysin

activity in opposing the effects of Ang-II stimulation of the AT1R (7, 10, 36). We have

previously observed that valsartan increased expression of ACE2 and neprilysin in the

renal cortical tissue of Ren2 animals (36). We report here similar effects noted following

in vivo treatment with tempol as well. Collectively, these observations suggest that

diminishing oxidative stress improves relative ACE2 signaling in renal cortical tissue.

Ang-II generates superoxide anion (O2-) and hydrogen peroxide (H2O2) in

cardiovascular tissue (34) and the kidney (14, 26, 36). At normal physiologic

concentrations, O2- and H2O2 can act as second messengers in Ang-II mediated signaling

pathways, but in excess they mediate inflammation and cellular dysfunction (33). The

tubular structural alterations were related to levels of MDA and albuminuria, and damage

was ameliorated by either 3 weeks of tempol or valsartan treatment. Tempol is a

SOD/catatase mimetic, that attenuates development of HTN and vascular injury via

scavenging of ROS, both O2- and H2O2 (1, 30, 32). Indeed, previous in vivo treatment

with tempol has been shown to normalize vascular superoxide levels, blood pressure, and

renal inflammatory responses in Ang-II infused hypertensive rats (1, 24). Although

valsartan treatment abrogated HTN, albuminuria, and PTC injury; tempol exerted its

protective effects on PTC despite not significantly lowering systolic blood pressure or

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albuminuria. Ang-II has previously been shown to selectively increase O2- and H2O2 in

PTC via NADPH oxidase activation, independent of mitochondrial O2- production (6,

11). In PTC, the flavoprotein inhibitor diphenylene iodinium, as well as, the antioxidant

N-acetylcysteine, prevented membrane-bound NADPH oxidase activation and generation

of ROS (6, 11). These data collectively suggest that an increase in ROS is the convergent

pathway by which increased Ang-II and/or albuminuria causes PTC injury.

One limitation in the interpretation of these results is the lack of increase in beta-

NAG in the Ren2 which has recently been reported as an indicator or tubular injury.

However, results from other studies have been inconclusive on the contribution of beta-

NAG to proximal tubule injury and the most promising papers have been in the area of

diabetic nephropathy. Further, that beta-NAG was not increased in the Ren2 despite the

structural changes may reflect its relative insensitivity in detecting early tubular injury.

Another possible limitation is drawing a conclusion regarding the correlation between

oxidative stress (as measured by MDA) and PTC injury with an r2 of 0.49. Although not

as strong of an association as seen with albuminuria (r2 = 0.85), there is a modest

correlation between level of MDA and PTC structural changes. Furthermore, our

staining of the PTC with Nitrotyrosine, specific for oxidation, does support our

conclusions. However, these associations are best corroborated with in vitro studies.

Thus, we are currently investigating the impact of ROS generation on structural and

functional alterations in proximal tubule cells in culture (23).

Acknowledgements

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This research was supported by NIH (R01 HL73101-01A1), the Veterans Affairs

Merit System (0018), and Novartis Pharmaceuticals. Male transgenic Ren2 rats and male

Sprague-Dawley (SD) controls were kindly provided by Dr. Carlos M. Ferrario, Wake

Forest University School of Medicine, Winston-Salem, North Carolina through the

Transgenic Core Facility supported in part by NIH grand HL-51952. Special thanks to

Allison Farris for assistance in preparing this manuscript. The authors wish to also

acknowledge the electron microscope core facility (Cheryl Jensen, electron microscopy

specialist) for their excellent help and preparation of transmission electron micrographs.

References

1. Beswick RA, Zhang H, Marable D, Catravas JD, Hill WD, Webb RC. Long-

term antioxidant administration attenuates mineralocorticoid hypertension and

renal inflammatory response. Hypertension 2001;37(2 Part 2):781-786.

2. Blendea MC, Jacobs D, Stump CS, McFarlane SI, Ogrin C, Bahtyiar G, Stas

S, Kumar P, Sha Q, Ferrario CM, Sowers JR. Abrogation of oxidative stress

improves insulin sensitivity in the Ren-2 rat model of tissue angiotensin II

overexpression. Am J Physiol Endocrinol Metab 2005;288(2): E353-E359.

3. Brunskill NJ, Cockcroft N, Nahorski S, Walls J. Albumin endocytosis is

regulated by heterotrimeric GTP-binding protein G alpha i-3 in opossum kidney

cells. Am J Physiol 1996;271(2 Pt 2):F356-F364.

4. Caruso-Neves C, Kwon SH, Guggino WB. Albumin endocytosis in proximal

tubule cells is modulated by angiotensin II through an AT2 receptor-mediated

of 27


16

protein kinase B activation. Proc Natl Acad Sci U S A 2005;102(48):17513-

17518.

5. Eddy AA. Proteinuria and interstitial injury. Nephrol Dial Transplant

2004;19(2):277-281.

6. Feliers D, Gorin Y, Ghosh-Choudhury G, Abboud HE, Kasinath BS.

Angiotensin II stimulation of VEGF mRNA translation requires production of

reactive oxygen species. Am J Physiol Renal Physiol 2006;290(4):F927-F936.

7. Ferrario CM, Jessup J, Chappell MC, Averill DB, Brosnihan KB, Tallant

EA, Diz DI, Gallagher PE. Effect of angiotensin-converting enzyme inhibition

and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2.

Circulation. 2005;111(20):2605-2610.

8. Gekle M. Renal tubule albumin transport. Annu Rev Physiol 2005;67:573-594.

9. Guo DF, Tardif V, Ghelima K, Chan JS, Ingelfinger JR, Chen X, Chenier I.

A novel angiotensin II type 1 receptor-associated protein induces cellular

hypertrophy in rat vascular smooth muscle and renal proximal tubular cells. J Biol

Chem. 2004;279(20):21109-21120.

10. Handa RK. Angiotensin-(1-7) can interact with the rat proximal tubule AT(4)

receptor system. Am J Physiol. 1999;277(1 Pt 2):F75-F83.

11. Hannken T, Schroeder R, Zahner G, Stahl RA, Wolf G. Reactive oxygen

species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1):

role in angiotensin II-mediated hypertrophy of proximal tubular cells. J Am Soc

Nephrol. 2000;11(8):1387-1397.

of 27


17

12. Holness MJ, Sugden MC. The impact of genetic hypertension on insulin

secretion and glucoregulatory control in vivo: studies with the TGR(mRen2)27

transgenic rat. J Hypertens. 1998;16(3):369-376.

13. Hayden MR, Chowdhury NA, Witte L, Sowers JR. Microalbuminuria and

proximal tubule remodeling in the cardiometabolic syndrome. Journal of the

CardioMetabolic Syndrome 2006;1(2):107-114.

14. Hayden MR, Whaley-Connell A, Sowers JR. Renal redox stress and

remodeling in metabolic syndrome, type 2 diabetes mellitus, and diabetic

nephropathy: paying homage to the podocyte. Am J Nephrol. 2005;25(6):553-

569.

15. Jaber BL, Madias NE. Progression of chronic kidney disease: can it be

prevented or arrested? Am J Med 2005;118(12):1323-1330.

16. Kobori H, Prieto-Carrasquero MC, Ozawa Y, Navar LG. AT1 receptor

mediated augmentation of intrarenal angiotensinogen in angiotensin II-dependent

hypertension. Hypertension 2004;43(5):1126-1132.

17. Li XC, Carretero OA, Navar LG, Zhuo JL. AT1 Receptor-Mediated

Accumulation of Extracellular Angiotensin II in Proximal Tubule Cells: Role of

Cytoskeleton Microtubules and Tyrosine Phosphatases. Am J Physiol Renal

Physiol 2006;291(2):F375-83.

18. Liu B, Preisig PA. Compensatory renal hypertrophy is mediated by a cell cycle-

dependent mechanism. Kidney Int 2002;62(5):1650-1658.

of 27


18

19. Mitchell KD, Jacinto SM, Mullins JJ. Proximal tubular fluid, kidney, and

plasma levels of angiotensin II in hypertensive ren-2 transgenic rats. Am J Physiol

1997;273(2 Pt 2):F246-F253.

20. Molitoris BA. Ischemia-induced loss of epithelial polarity: potential role of the

actin cytoskeleton. Am J Physiol. 1991;260(6 Pt 2):F769-78.

21. Morigi M, Macconi D, Zoja C, Donadelli R, Buelli S, Zanchi C, Ghilardi M,

Remuzzi G. Protein overload-induced NF-kappaB activation in proximal tubular

cells requires H(2)O(2) through a PKC-dependent pathway. J Am Soc Nephrol

2002;13(5):1179-1189.

22. Moriguchi A, Brosnihan KB, Kumagai H, Ganten D, Ferrario CM.

Mechanisms of hypertension in transgenic rats expressing the mouse Ren-2 gene.

Am J Physiol 1994;266(4 Pt 2):R1273-R1279.

23. Morris EM, Whaley-Connell A, Rehmer N, Cipporah JC, Clark SE,

Wiedmeyer CE, Resch ZT, Stump CS, Sowers JR. The Effect of HMG-CoA

Reductase Inhibition on Oxidative Stress in Zucker Obese Rat Kidney and

Opossum Proximal Tubule Cells. 2006 ENDO, Annual meeting of the Endocrine

Society Abstract # PI-286.

24. Nishiyama A, Fukui T, Fujisawa Y, Rahman M, Tian RX, Kimura S, Abe Y.

Systemic and Regional Hemodynamic Responses to Tempol in Angiotensin II-

Infused Hypertensive Rats. Hypertension. 2001;37(1):77-83.

25. Oktem F, Ozguner F, Yilmaz HR, Uz E, Dundar B. Melatonin reduces urinary

excretion of N-acetyl-beta-D-glucosaminidase, albumin and renal oxidative

markers in diabetic rats. Clin Exp Pharmacol Physiol. 2006;33(1-2):95-101

of 27


19

26. Onozato ML, Tojo A, Goto A, Fujita T, Wilcox CS. Oxidative stress and nitric

oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int.

2002;61(1):186-194.

27. Rahman M, Pressel S, Davis BR, Nwachuku C, Wright JT Jr, Whelton PK,

Barzilay J, Batuman V, Eckfeldt JH, Farber MA, Franklin S, Henriquez M,

Kopyt N, Louis GT, Saklayen M, Stanford C, Walworth C, Ward H,

Wiegmann T; ALLHAT Collaborative Research Group. Cardiovascular

outcomes in high-risk hypertensive patients stratified by baseline glomerular

filtration rate. Ann Intern Med 2006;144(3):172-180.

28. Reich H, Tritchler D, Herzenberg AM, Kassiri Z, Zhou X, Gao W, Scholey

JW. Albumin activates ERK via EGF receptor in human renal epithelial cells. J

Am Soc Nephrol 2005;16(5):1266-1278.

29. Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies. N Engl J

Med 1998;339(20):1448-1456.

30. Rodriguez-Iturbe B, Zhan CD, Quiroz Y, Sindhu RK, Vaziri ND.

Antioxidant-rich diet relieves hypertension and reduces renal immune infiltration

in spontaneously hypertensive rats. Hypertension 2003;41(2):341-346.

31. Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B, Hamm LL,

McCullough PA, Kasiske BL, Kelepouris E, Klag MJ, Parfrey P, Pfeffer M,

Raij L, Spinosa DJ, Wilson PW; American Heart Association Councils on

Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical

Cardiology, and Epidemiology and Prevention. Kidney disease as a risk factor for

development of cardiovascular disease: a statement from the American Heart

of 27


20

Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure

Research, Clinical Cardiology, and Epidemiology and Prevention. Hypertension.

2003;42(5):1050-1065.

32. Schnackenberg CG, Wilcox CS. Two-week administration of tempol attenuates

both hypertension and renal excretion of 8-Iso prostaglandin f2alpha.

Hypertension. 1999;33(1 Pt 2):424-428.

33. Sowers JR. Hypertension, angiotensin II, and oxidative stress. N Engl J Med.

2002;346(25):1999-2001.

34. Sowers JR. Insulin resistance and hypertension. Am J Physiol Heart Circ Physiol.

2004;286(5):H1597-H1602.

35. Verroust PJ, Christensen EI. Megalin and cubilin--the story of two

multipurpose receptors unfolds. Nephrol Dial Transplant 2002;17(11):1867-1871.

36. Whaley-Connell A, Chowdhury NA, Hayden MR, Stump CS, Habibi J,

Wiedmeyer CE, Gallagher PE, Tallant EA, Cooper SA, Link CD, Ferrario C,

Sowers JR. Oxidative stress and glomerular filtration barrier injury: Role of the

renin-angiotensin system in the Ren2 transgenic Rat. Am J Physiol Renal Physio

2006 June [Epub ahead of print].

37. White P, Doctor RB, Dahl RH, Chen J. Coincident microvillar actin bundle

disruption and perinuclear actin sequestration in anoxic proximal tubule. Am J

Physiol Renal Physiol 2000;278(6):F886-F893.

38. Wolf G, Neilson EG. Molecular mechanisms of tubulointerstitial hypertrophy

and hyperplasia. Kidney Int 1991;39(3):401-420.

of 27


21

39. Yang LE, Maunsbach AB, Leong PK, McDonough AA. Redistribution of

myosin VI from top to base of proximal tubule microvilli during acute

hypertension. J Am Soc Nephrol. 2005;16(10):2890-2896.

40. Zhai XY, Birn H, Jensen KB, Jesper S. Thomsen JS, Andreasen A, Erik I.

Christensen EL. Digital Three-Dimensional Reconstruction and Ultrastructure

of the Mouse Proximal Tubule. J Am Soc Nephrol. 2003;14:611-619.

41. Zhuo J, Ohishi M, Mendelsohn FA. Roles of AT1 and AT2 receptors in the

hypertensive Ren-2 gene transgenic rat kidney. Hypertension 1999;33(1 Pt

2):347-353.

42. Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG. Ang II

accumulation in rat renal endosomes during Ang II-induced hypertension: role of

AT(1) receptor. Hypertension 2002;39(1):116-121.

43. Zou LX, Imig JD, Hymel A, Navar LG. Renal uptake of circulating angiotensin

II in Val5-angiotensin II infused rats is mediated by AT1 receptor. Am J

Hypertens 1998;11(5):570-578.

Figure Legends

Figure 1A and B: A) demonstrates immunostaining with Nitrotyrosine as a marker of

lipid peroxidation on cross sections of Proximal Tubule Cells (PTC). B) demonstrates

grey-scale measures of intensity of the PTC images in A). *, p<0.05 Ren2-C vs. SDC;

**, p<0.05 Ren2-C vs. Ren2-V and Ren2-T.

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Figure 2A through D. Cross sections of proximal tubule cells (PTC) microvilli for

animal from each treatment group: A) Sprague-Dawley Control (SDC) - Note the

uniformity of the microvilli of the PTC. B) Ren2 Control (Ren2-C) - Numerous

abnormal microvilli forms present. C) Ren2 Valsartan (Ren2-V) - Number and size of

abnormal forms reduced. D) Ren2 Tempol (Ren2-T) - Abnormal forms are reduced, but

to a lesser degree.

Figure 3A and B. A) Microvilli mean diameter is significantly increased in Ren2-C vs.

SDC and is attenuated in the Ren2-V and to a lesser degree in the Ren2-T. *, p<0.05

Ren2-C vs. SDC; **, p<0.05 Ren2-C vs. Ren2-V and Ren2-T. B) Linear regression

analysis of natural log (ln) transformed values for albuminuria (Alb/Cr ratio) and

malondialdehyde (MDA), a surrogate marker for oxidative stress, versus microvilli

diameter (MVD).

Figure 4A and B. Abnormal forms of microvilli in the Ren2 animal model. The

abnormal forms in the male 11 week old Ren2 model are highlighted by black

arrowheads. These elongated forms of micorvilli in the Ren2 Model are noted to contain

actin cytoskeletal filaments at both poles (white arrows in the 120K image insert). These

findings may indicate that the microvilli have folded over during the cellular remodeling

of the actin cytoskeleton. B) Ratio of abnormal to normal forms is significantly increased

in Ren2-C vs. SDC and is similarly attenuated by treatment with Ren2-V and Ren2-T. *,

p<0.05 Ren2-C vs. SDC; **, p<0.05 Ren2-C vs. Ren2-V and Ren2-T

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Table 1: Experimental ParametersSDC SDV SDT Ren2-C Ren2-V Ren2-T

Weight (g) 365.5 ± 4.4 347.5 ± 11.5 349.8 ± 10.9 350.8 ± 13.6 388.2 ± 23.7 344 ± 16.3Systolic Blood Pressure (mmHg) 123 ± 5.5 117.5 ± 3.2 100.8 ± 3.9 192.5 ± 5.8* 146.8 ± 6.1** 188 ± 8.4Albumin/Creatinine (mg/mg) 0.06 ± 0.01 0.07 ± 0.02 0.06 ± 0.02 0.27 ± 0.02 * 0.05 ±0.02** 0.31 ± 0.07N-Acetyl-β-D-Glucosaminidase (mU) 0.24 ± 0.01 0.16 ± 0.05# 0.23 ± 0.02 0.24 ± 0.04 0.16 ±0.05# 0.28 ± 0.03Kidney Malonyldialdehyde (uM/mg) 0.05 ± 0.01 0.19 ± 0.05 0.20 ± 0.01 0.56 ± 0.17* 0.10 ± 0.01** 0.22 ± 0.01**

*, p<0.05 Ren2-C vs. SDC; **, p<0.05 Ren2-V, Ren2-T vs. Ren2-C; #, p<0.05 Control vs. Valsartan

Table 1: Experimental ParametersSDC SDV SDT Ren2-C Ren2-V Ren2-T

Weight (g) 365.5 ± 4.4 347.5 ± 11.5 349.8 ± 10.9 350.8 ± 13.6 388.2 ± 23.7 344 ± 16.3Systolic Blood Pressure (mmHg) 123 ± 5.5 117.5 ± 3.2 100.8 ± 3.9 192.5 ± 5.8* 146.8 ± 6.1** 188 ± 8.4Albumin/Creatinine (mg/mg) 0.06 ± 0.01 0.07 ± 0.02 0.06 ± 0.02 0.27 ± 0.02 * 0.05 ±0.02** 0.31 ± 0.07N-Acetyl-β-D-Glucosaminidase (mU) 0.24 ± 0.01 0.16 ± 0.05# 0.23 ± 0.02 0.24 ± 0.04 0.16 ±0.05# 0.28 ± 0.03Kidney Malonyldialdehyde (uM/mg) 0.05 ± 0.01 0.19 ± 0.05 0.20 ± 0.01 0.56 ± 0.17* 0.10 ± 0.01** 0.22 ± 0.01**

*, p<0.05 Ren2-C vs. SDC; **, p<0.05 Ren2-V, Ren2-T vs. Ren2-C; #, p<0.05 Control vs. Valsartan

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Ren2-CA: SDC

Ren2-V Ren2-T

Ren2-CRen2-CA: SDC

Ren2-V

A: SDC

Ren2-V Ren2-TRen2-T

B:

0

10

20

30

40

50

60

70

SDC Ren2-C Ren2-V Ren2-T

Ave

rage

Gra

y Sc

ale

Inte

nsiti

es

****

*

B:

0

10

20

30

40

50

60

70

SDC Ren2-C Ren2-V Ren2-T

Ave

rage

Gra

y Sc

ale

Inte

nsiti

es

****

*

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DC

A

B A

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A

65

70

75

80

85

90

SDC SDV SDT Ren2-C Ren2-V Ren2-T

nm

*

****

*

B

y = 0.0727x + 4.4943R2 = 0.4868

4.2

4.3

4.4

4.5

4.6

-3 -2.5 -2 -1.5 -1 -0.5 0

ln MDA

ln MV

D

y = 0.0874x + 4.5969R2 = 0.8338

4.2

4.3

4.4

4.5

4.6

-4.5 -3.5 -2.5 -1.5 -0.5

ln Alb/Cr

ln MV

D

A

65

70

75

80

85

90


nm

*

****

*

B

y = 0.0727x + 4.4943R2 = 0.4868

4.2

4.3

4.4

4.5

4.6

-3 -2.5 -2 -1.5 -1 -0.5 0

ln MDA

ln MV

D

y = 0.0874x + 4.5969R2 = 0.8338

4.2

4.3

4.4

4.5

4.6

-4.5 -3.5 -2.5 -1.5 -0.5

ln Alb/Cr

ln MV

D

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B

0

0.04

0.08

0.12

0.16


*

****

B

0

0.04

0.08

0.12

0.16


*

****

120 K120K

A

120 K120K120 K120K

A

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proximal tubule microvilli remodeling and albuminuria in the ren2 transgenic rat

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