Research Article Development of a Model of Chronic Kidney …downloads.hindawi.com/journals/bmri/2015/172302.pdf · 2019-07-31 · kidney disease patients [ , ]. As previously demonstrated

Research ArticleDevelopment of a Model of Chronic Kidney Disease inthe C57BL6 Mouse with Properties of Progressive Human CKD

Zahraa Mohammed-Ali1 Gaile L Cruz1 Chao Lu1 Rachel E Carlisle1 Kaitlyn E Werner1

Kjetil Ask2 and Jeffrey G Dickhout1

1Department of Medicine Division of Nephrology McMaster University and St Josephrsquos Healthcare HamiltonHamilton ON Canada L8N 4A62Department of Medicine Division of Respirology McMaster University and St Josephrsquos Healthcare HamiltonHamilton ON Canada L8N 4A6

Correspondence should be addressed to Jeffrey G Dickhout jdickhoustjoshamonca

Received 26 September 2014 Revised 16 December 2014 Accepted 29 December 2014

Academic Editor Andrea Vecchione

Copyright copy 2015 Zahraa Mohammed-Ali et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population CKD leads to endstage renal disease and increases the risk of cardiovascular disease As such it is important to study the mechanisms underlyingCKD progression To this end an animal model was developed to allow the testing of new treatment strategies or moleculartargets for CKD prevention Many underlying risk factors result in CKD but the disease itself has common features includingrenal interstitial fibrosis tubular epithelial cell loss through apoptosis glomerular damage and renal inflammation Further CKDshows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD We sought todevelop and characterize an animal model with these common features of human CKD in the C57BL6 mouse Mice of this geneticbackground have been used to produce transgenic strains that are commercially available Thus a CKDmodel in this strain wouldallow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost This paper describessuch a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers

1 Introduction

Although various models of chronic kidney disease (CKD)have been established in the rat [1] the ability to transgeni-cally manipulate the rat is not nearly as well established asin the mouse Further many genetic knockout mouse strainsincluding tissue specific and conditionally inducible knock-outs are available on the C57BL6 background [2] Howeverthis mouse has proven to be resistant to the development ofCKD C57BL6 mice have shown resistance to the inductionof CKD by standard techniques such as streptozotocin-induced diabetes [3] bovine serum albumin overload pro-teinuria [4] and reduced renal mass [5] Thus developinga mouse model of CKD on the C57BL6 background thatshares salient pathological features of human CKD allowsthe use of preexisting knockout strains Experiments on theseknockout strains would determine the effect of these genes on

the development of renal interstitial fibrosis proteinuria andthe chronic inflammatory response in CKD

A model has been developed in the C57BL6 that showsfeatures of progressive human CKD including proteinuriaand inflammation [6] In this model mice are uninephrec-tomized given Angiotensin (Ang) II infusion and deoxy-corticosterone acetate (DOCA) with 1 salt in the drinkingwaterThis model can be referred to as the Ang IIDOCA saltmouse The use of Ang II and DOCA with a high salt diet inthis model results in sodium retention and volume expansionand therefore hypertension [7] As well the reduction inrenal mass promotes hyperfiltration which contributes toproteinuria [8]The dysregulation of the Renin-Angiotensin-Aldosterone system that is stimulated in this model plays acentral role in cardiorenal syndrome [9] This fact is wellsupported by the success of angiotensin converting enzyme(ACE) inhibitors and angiotensin receptor blockers (ARBs)

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 172302 10 pageshttpdxdoiorg1011552015172302

2 BioMed Research International

as first line therapies in the treatment of hypertension andkidney disease patients [9 10] As previously demonstratedby Kirchhoff et al (2008) this mouse model of CKD inducesproteinuria and renal injury and increased systolic bloodpressure [6] In this paper we will further characterizethis model by quantifying the apoptotic responses in renaltubular epithelium as well as renal interstitial fibrosis andthe inflammatory response Further we sought to determinewhether gender alters the severity of the development of thesefeatures of CKD in this model

2 Methods and Materials

21 Model of CKD in C57BL6 Mice Ten-week-old mice(gender-balanced groups) underwent uninephrectomy (Unx)or a sham uninephrectomy under isofluraneoxygen anaes-thesia 2 weeks before the start of the experiment and wereallowed to recover (Figure 1) The Unx mice were then given1 sodium chloride in the drinking water and receivedDOCA pellet implants and Ang II infusion using osmoticminipumps Model 1004 ALZET osmotic infusion pumps(Durect) containing Ang II in sterile water were subcuta-neously implanted in the back of the necks of mice underisofluraneoxygen anaesthesia to deliver a dose of 15 ng AngII (Sigma) per minute per gram body weight At this timea 50mg 21-day release DOCA pellet (Innovative Research ofAmerica M-121) was also implanted subcutaneously Micethat underwent the sham Unx were also treated with asham procedure for subcutaneous implantation All micewere sacrificed on day 21 after implantation This animalutilization and the described procedures were approved bythe McMaster University Research Ethics Board

22 Blood Pressure Measurements Blood pressure measure-ments were obtained with tail cuff plethysmography using aCODA (Kent Scientific) blood pressure analyzer before AngIIDOCA implantation and also before sacrifice (Figure 1)Briefly animals were placed in restraint and positioned ona heating pad with a tail cuff attached to the machine Thecuff then measured systolic blood pressure diastolic bloodpressure and heart rate

23 Urinalysis Metabolic Cages and Microalbumin ELISABefore the surgical procedure and after 3 weeks on treatmentwith AngIIDOCA salt mice were placed in metabolic cagesfor 24 h urine collection (Figure 1) Urine samples were sentto our in-house laboratory to evaluate total protein concen-trations and an ELISA was used to measure mouse urinealbumin concentration (BETHYLLaboratories) to determinehypertension-induced proteinuria

24 Tissue Preparation for Histological Assessment andImmunohistochemical Analysis of Protein Cast FormationRenal Interstitial Fibrosis Apoptosis and Glomerular SclerosisRenal tissue was prepared for histological analysis The tissuewas fixed in 4 paraformaldehyde upon sacrificing theanimal The tissue was then embedded in paraffin blocks andsectioned (4 120583m) using a microtome To assess protein cast

Day 1 Day 21

CKD

dev

elopm

ent

Uninephrectomy

Ang II + DOCA

pellet implantation1 Na+ in drinkingwater

Sacrifice

∙ Metabolic cages∙ BP measurements


Day 14

Figure 1 Ang IIDOCA salt model of chronic kidney disease inthe C57BL6 mouse Graph describing the time course of CKDdevelopment

formation and glomerular injury score these tissues werestained with Periodic Acid Schiff (PAS) stain and imagedusing a light microscope (Olympus)

Collagen deposition indicating extracellular matrix accu-mulation and renal interstitial fibrosis was evaluated usingMassonrsquos trichrome stain (Sigma-Aldrich)

To assess apoptosis kidney sections were stained usingthe protocol and reagents provided by the TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit (Trevigen Cat 4812-30-K) This method is based on specific binding of TdTto 31015840-OH ends of DNA and the incorporation of biotiny-lated deoxyuridine at sites of DNA breaks This signal isthen amplified by avidin-peroxidase allowing apoptotic cellswhere DNA fragmentation has occurred to be visualizedwith light microscopy [11]

The sections were analyzed using an Olympus BX41microscope Immunohistochemistry sections were imagedwith 20x and 40x objective lens For the quantification ofprotein cast formation apoptosis and F480 staining tenmicroscopic fields were randomly sampled in each of thecortex and the medulla To score glomeruli ten microscopicfields were randomly sampled from the cortex allowing thescoring of approximately 50 glomeruli per animal Imageswere analyzed for protein cast formation using the Meta-Morph program to select and quantify PAS-stained areas asa percentage of the total area of each image The average ofprotein cast area density was then calculated for each animalTUNEL-stained sections were processed using the cell counttool in Image J software Glomerular sclerosis was assessedbased on the scale and method used in a previous study [12]

Lungs from AngIIDOCA salt and sham mice wereextracted without performing a bronchoalveolar lavage pro-cedure and fixed in 4 paraformaldehyde for 24 hoursAfter standard paraffin embedding 4 120583M thick specimenswere sectioned and stained with HampE stain to visualizelung damage Mouse hearts were weighed upon sacrifice andthis parameter was normalized to body weight in grams toprovide a measure of increase in cardiac mass The heartswere then fixed in 4 paraformaldehyde for 24 hoursembedded in paraffin sectioned and stained with PAS toevaluate areas of hypertrophy

BioMed Research International 3

25 Statistical Analysis Statistical analysis was performedusing GraphPad Prism software 119905-tests were used to comparedata between groups and significance is denoted by 119875 lt 005Bar graphs show group averages and standard error of themean as error bars

3 Results

31 Development of Hypertensive Proteinuria in Ang IIDOCASalt Model Blood pressure measurements were taken usingtail cuffmeasurements before the subcutaneous implantationof the osmotic pump containing Ang II and the DOCA pelletas well as at the end of the model before sacrificing the miceAng IIDOCA salt mice experienced a significant increasein systolic (Figure 2(a) 119875 lt 0001 119873 = 14) and diastolicblood pressure (Figure 2(b) 119875 lt 0001119873 = 14) 21 days afterimplantation Ang IIDOCA salt mice also had significantlyhigher systolic (119875 lt 0001119873 = 14) and diastolic (119875 lt 0001119873 = 14) blood pressure compared to sham operated controls21 days after implantation This hypertensive response wasaccompanied by an increase in total protein (Figure 2(c))and albumin (Figure 2(d)) excreted in the urine over 24 hAng IIDOCA saltmice experienced significantly higher totalprotein (119875 = 0009 119873 = 14) and total albumin (119875 lt 0001119873 = 14) in 24 h urine compared to measurements obtainedbefore implantation Total 24 h urine protein (119875 = 0006119873 =14) and albumin (119875 lt 0001119873 = 14) were also significantlyhigher with Ang IIDOCA salt treatment compared to shamcontrols In order to assess CKD progression in this modelthe time course of the evolution of proteinuria was followedat days 0 7 14 18 and 21 after Ang IIDOCA salt treatmentProteinuria was significantly elevated at days 18 and 21(Figure 2(e))

32 Characteristics of Renal Tissue Damage in Response toAng IIDOCA Salt Treatment The proteinuria data was con-sistent with the immunohistological analysis of PAS-stainedkidney sections showing the percentage of protein cast for-mation as compared to sham controls (Figure 3(a)) Kidneysfrom Ang IIDOCA salt mice showed a significantly higherpercentage of protein cast formation as compared to shamcontrols PAS staining images show increased protein castformation in renal tubules of the cortex (119875 = 0003119873 = 14)and medulla (119875 = 001 119873 = 14) in AngIIDOCA salt micecompared to sham controls (Figure 3(b)) Higher magnifi-cation images of PAS staining showed increased glomerularsclerosis in the cortex of Ang IIDOCA salt mice comparedto sham mice as indicated by the arrows (Figure 3(c))Quantification of glomerular sclerosis by two independentassessors utilizing the method of Raij et al [12] indicated thatthe Ang IIDOCA salt mice displayed significantly elevatedglomerular sclerosis scores (119875 lt 0001119873 = 10) compared toage-matched sham operated controls (Figure 3(d))

In order to assess renal cell loss we performed TUNELstaining TUNEL staining images demonstrated increasedapoptosis (Figure 3(e) arrows) in kidneymicrographs of AngIIDOCA salt mice compared to sham animals (Figure 3(f)119875 lt 0001 119873 = 10) Immunohistochemical stainingfor F480 a highly specific macrophage cell surface marker

[13] demonstrated a significant increase in macrophageinfiltration density (Figure 3(g)) in response toAng IIDOCAsalt treatment compared to sham controls (Figure 3(h) 119875 =002119873 = 5) To examine renal interstitial fibrosis we stainedkidney sections with Massonrsquos trichrome stain Indeed inour Ang IIDOCA salt model we saw increased collagendeposition as indicated by blue-stained fibres in the renalinterstitium of areas showing kidney damage in the AngIIDOCA salt mice compared to sham controls (Figure 3(i))

33 Impact of CKD on Cardiac and Lung Function andMorphology Microscope images of heart cross-section por-trayed right and left ventricle hypertrophy in response to AngIIDOCA salt treatment (Figure 4(a)) Cardiacmuscle hyper-trophy is an adaption to fluid retention and hypervolemiaresulting from the model This phenomenon is confirmed bythe increase in heart weights (mgg of body weight) (119875 lt0001119873 = 14) observed in mice treated with Ang IIDOCAsalt compared to sham controls (Figure 4(b)) Lung sectionsderived fromAng IIDOCA salt mice showed features of pul-monary edema inflammatory infiltrates and thickening ofthe alveoli Signs of increased vessel thickness and capillariescongestedwith red blood cells are also observed (Figure 4(c))Although further characterization is required it is likelythat the observed pulmonary changes are driven by sodiumretention and the severe hypervolemic changes observed inthis model

34 Impact of Gender on Renal Tissue Damage Induced by AngIIDOCA Salt Model Both male (119875 = 002 119873 = 7) andfemale (119875 = 004119873 = 7) Ang IIDOCA salt mice developedproteinuria compared to their respective sham controls as aresult of the CKD model However male Ang IIDOCA saltmice showed significantly higher total protein in 24 h urine(119875 = 004 119873 = 7) compared to female Ang IIDOCA saltmice (Figure 5(a)) Total 24 h urine albumin measurementsshowed a similar trend where male Ang IIDOCA salt mice(119875 lt 0001 119873 = 6) experience a higher level of albumin inthe urine compared to their sham controls and compared tofemale Ang IIDOCA salt mice (119875 = 003119873 = 6) Althoughfemale Ang IIDOCA salt mice had a higher albumin levelin 24 h urine (119875 = 011 119873 = 6) than their sham controlsthis increase was not significant (Figure 5(b)) Protein castformation was significantly higher in the cortex (119875 = 002119873 = 7) and medulla (119875 = 004 119873 = 7) of Ang IIDOCAsalt male mice and in the cortex (119875 = 0004 119873 = 7) andmedulla (119875 = 001119873 = 7) of Ang IIDOCA salt female micecompared to their respective sham controls The percentageof PAS-stained area was higher in the cortex (119875 = 0048119873 = 7) and medulla (119875 = 0145119873 = 7) of male mice treatedwithAng IIDOCA salt compared to femalemice treatedwithAng IIDOCA salt (Figure 5(c)) TUNEL staining was usedto assess the influence of gender on kidney cell death andultimately nephron loss Although female Ang IIDOCA saltmice experienced an increase in apoptosis (119875 = 003119873 = 5)compared to female sham controls the apoptosis observed inmale mice treated with Ang IIDOCA salt was significantlyhigher (119875 = 0002 119873 = 5) compared to Ang IIDOCA salttreated females (Figure 5(d)) In additionMassonrsquos trichrome


Ang IIDOCA Sham120

140

160

180

200

Systo

lic b

lood

pre

ssur

e (m

mH

g) lowast

lowast

(a)

Ang IIDOCA Sham100

120

140

160

Dia

stolic

blo

od p

ress

ure (

mm

Hg)

lowast

lowast

(b)

Ang IIDOCA Sham0

10

20

30

40

Tota

l pro

tein

24

h (m

g)

lowast

lowast

(c)

Ang IIDOCA Sham0

10

20

30

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(d)

0 7 14 18 210

20

40

60

Ang IIDOCASham

Days after implantation

Tota

l pro

tein

24

h (m

g) lowast

lowast

(e)

Figure 2 Development of hypertensive proteinuria in Ang IIDOCA salt model of CKD in the C57BL6 mouse For all graphs lowast indicates asignificant difference between two groups where119875 lt 005 For (a)ndash(d) ◻ signifies pretreatment whereas ◼ signifies 21-day posttreatment withAng IIDOCA (a) (b) Changes in systolic and diastolic blood pressure in response to Ang IIDOCA (c) (d) Total 24 h urinary protein andalbumin excretion with Ang IIDOCA treatment (e) Time-course development of proteinuria expressed as total 24-hour protein excretionat days 7 14 18 and 21 posttreatment with Ang IIDOCA

staining showed a higher level of collagen deposition andrenal interstitial fibrosis (indicated by blue staining) in maleAng IIDOCA salt mice compared to female Ang IIDOCAsalt mice (Figure 5(e))

4 Discussion

CKD is characterized by reduction in glomerular filtrationrate (GFR) albuminuria and structural or functional abnor-malities of the kidney [14] CKD is increasing in prevalenceglobally and its comorbidities include cardiovascular disease

increased all-cause and cardiovascular mortality kidneydisease progression to end stage renal disease (ESRD) andacute kidney injury [15 16] The financial impact of CKDplaces a large burden on health care systems with highcosts associated with renal replacement therapy dialysisand cardiovascular complications [15] The C57BL6 mousehas been the most preferred strain for the generation oftransgenic and knockout animal models and will be utilizedto develop a genome-wide panel of knockout animals tocharacterize gene function [2]The Ang IIDOCA salt modeldescribed here shows a robust CKD response that closely


Cortex MedullaA

ng II

DO

CASh

am

(a)

Cortex Medulla0

2

4

6

8

Ang IIDOCA Sham

Prot

ein

cast

form

atio

n (

) lowast

lowast

(b)

Ang IIDOCA Sham

(c)

Ang IIDOCA Sham0

1

2

3

4

Glo

mer

ulos

clero

sis (m

ean

scor

e)lowast

(d)

Ang IIDOCA Sham

(e)

Ang IIDOCA Sham0

5

10

15

20

TUN

EL p

ositi

ve ce

llsm

m2

lowast

(f)

(g)

Ang IIDOCA Sham00

05

10

15

20

F48

0 sta

inin

g in

kid

ney

(are

a ) lowast

(h)

Figure 3 Continued


40x 40x

20x 20x

(i)

Figure 3 Renal tissue damage in response to Ang IIDOCA salt CKD mouse model For all graphs lowast indicates a significant differencebetween two groups where 119875 lt 005 Effect of Ang IIDOCA salt model on (a) (b) protein cast formation in the cortex and medulla (c) (d)glomerulosclerosis (e) (f) apoptosis (g) (h) macrophage (F480+ cells) infiltration and (i) interstitial fibrosis (Massonrsquos trichrome staining)

LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)

Figure 4 Effect of Ang IIDOCA salt model on cardiac and lung tissue (a) Images of heart cross-section cardiac hypertrophy in AngIIDOCA salt model (b) Graph showing increase in heart weights (mgg of body weight) in response to Ang IIDOCA where lowast indicates asignificant difference between two groups (119875 lt 005) (c) Lung damage (20x 40x) in response to Ang IIDOCA compared to SHAM controls


Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)

Figure 5 Impact of gender on CKD induced by Ang IIDOCA salt model For all graphs lowast indicates a significant difference between twogroups where 119875 lt 005 Ang IIDOCA salt is denoted by ◼ whereas ◻ denotes SHAM operated controls ldquoMrdquo and ldquoFrdquo indicate sections frommale and female mouse kidneys respectively Influence of gender on the development of (a) proteinuria (b) albuminuria (c) protein castformation (d) apoptosis and (e) interstitial fibrosis

mimics human CKD Developing this model allows the useof genetically modified mice in order to identify gene targetsthat play a role in CKD development and progression

Similar to the description of themodel provided byKirch-hoff et al [6] we found systolic blood pressure to be elevatedadditionally we determined that diastolic blood pressure wasalso elevated We present data for the total 24-hour excretionof both protein and albumin in the urine demonstrating thatthe Ang IIDOCA salt model induced significant proteinuriaand albuminuria This finding is similar to the increase inalbuminuriacreatinine ratio described by Kirchhoff et al[6] Further we show the evolution of proteinuria in the

model at days 0 7 14 18 and 21 where we observe astatistically significant increase in proteinuria at day 18 andday 21 We also demonstrated that protein cast formationand glomerular sclerosis were significantly increased in themodel similar to findings by Kirchhoff et al [6] We furtherinvestigated renal injury by examining apoptosis throughTUNEL staining and found it to be significantly elevatedAdditionally the kidneys were found to have a significantinfiltration of macrophages and showed renal interstitialfibrosis through trichrome staining Similar to Kirchhoff et al[6] we found end-organ damage in the heart characterized bycardiac hypertrophy We extended these findings to examine


the lung where we noted an edematous effusion in airspacesFurther we characterized the model for gender differencesand found that the female gender imparted protection fromproteinuria albuminuria protein cast formation apoptosisand renal interstitial fibrosis

Hypertensive proteinuria is a common feature of CKDTherefore animal models that display this feature have beenused to investigate disease-specific mechanisms molecu-lar pathogenesis and potential therapies [1] The increasedglomerular permeability during hypertensive CKD allowsprotein hyperfiltration into the proximal tubules causingrenal tissue damage Filtered albumin and other proteins thataccumulate within intracellular compartments of proximaltubular cells perturb cell function by several mechanisms[17] Hypertension is an important contributor to ESRD[18] Proteinuria is associated with glomerular damage andpodocyte depletion [19] and can be used in the classificationof different stages of CKD clinically [20] The Ang IIDOCAsalt model displays hypertensive proteinuria that mirrorshuman CKD as demonstrated by a significant increase insystolic and diastolic blood pressure as well as proteinuriaand albuminuria Overt proteinuria evolved later in thismodel at days 18 and 21 indicating disease progression inresponse to glomerular injury The glomerulosclerosis seenin our model involves inflammation and fibrosis around thearea of the damaged glomeruli and could potentially resultin tubular atrophy and degeneration [19] Tubulointerstitialinjury caused by proteinuria includes the formation of pro-tein casts that may block the tubular lumen [19] and thisphenomenon has been shown in our model

As CKD progresses tubular cell atrophy results primarilydue to apoptosis An increase in apoptosis has been demon-strated using TUNEL staining on kidney sections fromdiabetic nephropathy patients [21] and from rats in the strep-tozotocin-induced diabetic nephropathymodel [22] In addi-tion apoptotic nuclei have been observed in polycystichuman kidneys and kidney sections from mouse models ofthe disease [23] The loss of renal tubular epithelial cellsthrough apoptosis occurs in both acute and chronic kidneydiseases [24] Apoptotic cell loss from nephron segmentsleads to tubular atrophy and their loss eventually leads to adecline in GFR Since the Ang IIDOCA salt model displaysa robust apoptotic response it could be used in identifyingtherapeutic targets against apoptosis that are able to halt CKDprogression [24]

Macrophage infiltration has been studied as a universalfeature of tubulointerstitial damage regardless of diseaseorigin [25 26] and is a key feature of experimental andhuman kidney disease models [26 27] Macrophages havebeen shown to release cytotoxic moieties such as proteolyticenzymes reactive oxygen and nitrogen species as well asproinflammatory cytokines and chemokines [28] By express-ing cytokines such as TGF-120573 and connective tissue growthfactor macrophages are able to induce myofibroblast differ-entiation and extra cellular matrix deposition key processesin renal interstitial fibrosis [29 30] Increased macrophageinfiltration is associated with glomerulosclerosis and tubu-lointerstitial fibrosis [27 31 32] Bothmacrophage infiltrationand renal interstitial fibrosis have been demonstrated in the

Ang IIDOCA salt model of CKD Further studies wouldbe focused on the time course of the development of thesephenomena in order to establish pathways that lead to CKDprogression

CKD through its effects on volume overload plays animportant pathophysiological role in multiple organ failureThe effect of volume overload on the heart is to developcardiac hypertrophy through myocardial stretch This effectis reflected in our model As hypertrophy progresses fromcompensated to decompensated heart failure volume over-load affects the lungs and results in pulmonary edema [33]This phenomenon is also reflected in our model

The findings that Ang IIDOCA salt mice show a gender-based difference in severity of CKD are consistent withfindings in the human population [34] and in agreementwith previous clinical and experimental studies on the pro-tective effect of female gender on the development of renaldiseases [35ndash38] Female mice subjected to this model showsignificantly lower levels of proteinuria Ang IIDOCA saltfemale mice also experienced a trend for decreased proteincast formation and collagen deposition thereby indicating alower level of renal tissue damage compared toAng IIDOCAsalt male mice A recent study identified gender differenceas a modifier of susceptibility to ER stress-induced injury intunicamycin-treated mice a model of acute kidney injuryshowing reduced renal pathology in female mice [39] Thereduced induction of apoptosis in female Ang IIDOCA saltmice in our CKDmodel is therefore in agreement with in vivoexperiments of endoplasmic reticulum stress-induced acutekidney injury in the tunicamycin mouse models [39]

Clinical studies have demonstrated an associationbetween male gender and a faster rate of CKD progression[36] Interestingly the impact of gender is restricted to pre-menopausal women and is therefore thought to be anestradiol-mediated protective effect [35] This finding issupported by experimental studies Aged male rats havebeen shown to develop decreased GFR increased glomerularinjury and increased proteinuria earlier than female rats[37] In addition estradiol treatment caused a reduction inglomerulosclerosis expression of adhesion and extracellularmatrix molecules and prevented tubular damage in animalmodels of unilateral nephrectomy and chronic renal allograftrejection [38 40]These studies indicate a renoprotective rolefor estradiol and although our experiments show that femalegender imparts a protective effect on CKD development thepotential role of estradiol is still to be determined

In conclusion the Ang IIDOCA salt model produces asignificant degree of CKD that mimics progressive humanCKD in theC57BL6mouseThis pathology includes protein-uria intertubular protein casts renal interstitial fibrosis andtubular epithelial cell apoptosis As this model recapitulatesmany of the aspects of pathology found in human CKD [41]it will be of great utility to determine the specific effect of var-ious candidate genes on CKD severity and progression Fur-ther a gender effect was observed where female mice showedlower proteinuria and apoptosis in the CKD model Futurework in this model may allow the precise molecular nature ofthe protective effect of the female gender to be determined


Conflict of Interests

The authors declare that there is no conflict of interestsregarding publication of this paper

Acknowledgments

This work was supported by research grants to Jeffrey GDickhout from the Canadian Institutes of Health Research(OSO-115895 and MOP-133484) Financial support from StJosephrsquos HealthcareHamilton is also acknowledged JeffreyGDickhout also acknowledges salary support from St JosephrsquosHealthcare Hamilton and holds a McMaster UniversityDepartment of Medicine Internal Career Research AwardSupport from the Division of Nephrology at the Departmentof Medicine at McMaster University is also acknowledgedDr Jeffrey G Dickhout also holds a Kidney Foundation ofCanada Krescent New Investigator Award

References

[1] H-C Yang Y Zuo and A B Fogo ldquoModels of chronic kidneydiseaserdquo Drug Discovery Today Disease Models vol 7 no 1-2pp 13ndash19 2010

[2] W C Skarnes B Rosen A P West et al ldquoA conditional knock-out resource for the genome-wide study of mouse gene func-tionrdquo Nature vol 474 no 7351 pp 337ndash344 2011

[3] F Zheng G E Striker C Esposito E Lupia and L J StrikerldquoStrain differences rather than hyperglycemia determine theseverity of glomerulosclerosis in micerdquo Kidney Internationalvol 54 no 6 pp 1999ndash2007 1998

[4] N Kato YWatanabe Y Ohno et al ldquoMapping quantitative traitloci for proteinuria-induced renal collagen depositionrdquo KidneyInternational vol 73 no 9 pp 1017ndash1023 2008

[5] A El-Meanawy J R Schelling S K Iyengar et al ldquoIdentifica-tion of nephropathy candidate genes by comparing sclerosis-prone and sclerosis-resistant mouse strain kidney transcrip-tomesrdquo BMC Nephrology vol 13 no 1 article 61 2012

[6] F Kirchhoff C Krebs U N Abdulhag et al ldquoRapid develop-ment of severe end-organ damage in C57BL6 mice by combin-ing DOCA salt and angiotensin IIrdquo Kidney International vol73 no 5 pp 643ndash650 2008

[7] D L Longo D L Kasper J L Jameson A S Fauci S L Haus-er and J Loscalzo ldquoClinical and laboratory manifestations ofchronic kidney disease and uremiardquo in Harrisonrsquos Principles ofInternal Medicine p 1410 McGraw-Hill New York NY USA18th edition 2012

[8] G Remuzzi and T Bertani ldquoPathophysiology of progressivenephropathiesrdquoThe New England Journal of Medicine vol 339no 20 pp 1448ndash1456 1998

[9] S A Atlas ldquoThe renin-angiotensin aldosterone system patho-physiological role and pharmacologic inhibitionrdquo Journal ofManaged Care Pharmacy vol 13 no 8 supplement B pp S9ndashS20 2007

[10] A H Gradman J N Basile B L Carter et al ldquoCombinationtherapy in hypertensionrdquo Journal of the American Society ofHypertension vol 4 no 2 pp 90ndash98 2010

[11] Y Gavrieli Y Sherman and S A Ben-Sasson ldquoIdentificationof programmed cell death in situ via specific labeling of nuclearDNA fragmentationrdquo Journal of Cell Biology vol 119 no 3 pp493ndash501 1992

[12] L Raij S Azar and W Keane ldquoMesangial immune injuryhypertension and progressive glomerular damage inDahl ratsrdquoKidney International vol 26 no 2 pp 137ndash143 1984

[13] J M Austyn and S Gordon ldquoF480 a monoclonal antibodydirected specifically against the mouse macrophagerdquo EuropeanJournal of Immunology vol 11 no 10 pp 805ndash815 1981

[14] A S Levey K-U Eckardt Y Tsukamoto et al ldquoDefinition andclassification of chronic kidney disease a position statementfrom kidney disease improving global outcomes (KDIGO)rdquoKidney International vol 67 no 6 pp 2089ndash2100 2005

[15] M Kerr B Bray JMedcalf D J OrsquoDonoghue and BMatthewsldquoEstimating the financial cost of chronic kidney disease to theNHS in Englandrdquo Nephrology Dialysis Transplantation vol 27supplement 3 pp iii73ndashiii80 2012

[16] V Jha G Garcia-Garcia K Iseki et al ldquoChronic kidney diseaseglobal dimension and perspectivesrdquo The Lancet vol 382 no9888 pp 260ndash272 2013

[17] G Remuzzi N Perico M Macia and P Ruggenenti ldquoThe roleof renin-angiotensin-aldosterone system in the progression ofchronic kidney diseaserdquo Kidney International no 99 pp S57ndashS65 2005

[18] J M Turner C Bauer M K Abramowitz M L Melamed andT H Hostetter ldquoTreatment of chronic kidney diseaserdquo KidneyInternational vol 81 no 4 pp 351ndash362 2012

[19] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo Pediatric Neph-rology vol 27 no 6 pp 901ndash909 2012

[20] A S Levey and J Coresh ldquoChronic kidney diseaserdquoThe Lancetvol 379 no 9811 pp 165ndash180 2012

[21] D Kumar S Robertson and K D Burns ldquoEvidence of apopto-sis in human diabetic kidneyrdquoMolecular and Cellular Biochem-istry vol 259 no 1-2 pp 67ndash70 2004

[22] G Liu Y Sun Z Li et al ldquoApoptosis induced by endoplasmicreticulum stress involved in diabetic kidney diseaserdquo Biochem-ical and Biophysical Research Communications vol 370 no 4pp 651ndash656 2008

[23] D Woo ldquoApoptosis and loss of renal tissue in polycystic kidneydiseasesrdquo The New England Journal of Medicine vol 333 no 1pp 18ndash25 1995

[24] A B Sanz B Santamarıa M Ruiz-Ortega J Egido and AOrtiz ldquoMechanisms of renal apoptosis in health and diseaserdquoJournal of the American Society of Nephrology vol 19 no 9 pp1634ndash1642 2008

[25] K A Nath ldquoThe tubulointerstitium in progressive renal dis-easerdquo Kidney International vol 54 no 3 pp 992ndash994 1998

[26] K S Eardley and P Cockwell ldquoMacrophages and progressivetubulointerstitial diseaserdquo Kidney International vol 68 no 2pp 437ndash455 2005

[27] Y Wang and D C H Harris ldquoMacrophages in renal diseaserdquoJournal of the American Society of Nephrology vol 22 no 1 pp21ndash27 2011

[28] B Rodrıguez-Iturbe H Pons J Herrera-Acosta and R J John-son ldquoRole of immunocompetent cells in nonimmune renaldiseasesrdquo Kidney International vol 59 no 5 pp 1626ndash16402001

[29] Y Liu ldquoRenal fibrosis new insights into the pathogenesis andtherapeuticsrdquo Kidney International vol 69 no 2 pp 213ndash2172006

[30] F M Strutz ldquoEMT and proteinuria as progression factorsrdquoKidney International vol 75 no 5 pp 475ndash481 2009


[31] J S Duffield ldquoMacrophages and immunologic inflammation ofthe kidneyrdquo Seminars in Nephrology vol 30 no 3 pp 234ndash2542010

[32] A B Fogo ldquoMechanisms of progression of chronic kidneydiseaserdquo Pediatric Nephrology vol 22 no 12 pp 2011ndash20222007

[33] N Aspromonte D N Cruz C Ronco and R Valle ldquoRoleof bioimpedance vectorial analysis in cardio-renal syndromesrdquoSeminars in Nephrology vol 32 no 1 pp 93ndash99 2012

[34] W L LeeMH Cheng D C TarngWC Yang F K Lee and PH Wang ldquoThe benefits of estrogen or selective estrogen recep-tor modulator on kidney and its related disease-chronic kidneydisease-mineral and bone disorder osteoporosisrdquo Journal of theChinese Medical Association vol 76 no 7 pp 365ndash371 2013

[35] R K Dubey S Oparil B Imthurn and E K Jackson ldquoSexhormones and hypertensionrdquo Cardiovascular Research vol 53no 3 pp 688ndash708 2002

[36] S R Silbiger and J Neugarten ldquoThe impact of gender onthe progression of chronic renal diseaserdquo American Journal ofKidney Diseases vol 25 no 4 pp 515ndash533 1995

[37] C Baylis ldquoAge-dependent glomerular damage in the rat Dis-sociation between glomerular injury and both glomerularhypertension and hypertrophy Male gender as a primary riskfactorrdquo The Journal of Clinical Investigation vol 94 no 5 pp1823ndash1829 1994

[38] S E Mulroney C Woda M Johnson and C Pesce ldquoGenderdifferences in renal growth and function after uninephrectomyin adult ratsrdquo Kidney International vol 56 no 3 pp 944ndash9531999

[39] R Hodeify J Megyesi A Tarcsafalvi H I Mustafa N S HtiLar Seng and P M Price ldquoGender differences control thesusceptibility to ER stress-induced acute kidney injuryrdquo TheAmerican Journal of PhysiologymdashRenal Physiology vol 304 no7 pp F875ndashF882 2013

[40] V Muller A Szabo O Viklicky et al ldquoSex hormones andgender-related differences their influence on chronic renalallograft rejectionrdquoKidney International vol 55 no 5 pp 2011ndash2020 1999

[41] W Metcalfe ldquoHow does early chronic kidney disease progressA background paper prepared for the UK consensus conferenceon early chronic kidney diseaserdquoNephrologyDialysis Transplan-tation vol 22 supplement 9 pp ix26ndashix30 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


as first line therapies in the treatment of hypertension andkidney disease patients [9 10] As previously demonstratedby Kirchhoff et al (2008) this mouse model of CKD inducesproteinuria and renal injury and increased systolic bloodpressure [6] In this paper we will further characterizethis model by quantifying the apoptotic responses in renaltubular epithelium as well as renal interstitial fibrosis andthe inflammatory response Further we sought to determinewhether gender alters the severity of the development of thesefeatures of CKD in this model

2 Methods and Materials

21 Model of CKD in C57BL6 Mice Ten-week-old mice(gender-balanced groups) underwent uninephrectomy (Unx)or a sham uninephrectomy under isofluraneoxygen anaes-thesia 2 weeks before the start of the experiment and wereallowed to recover (Figure 1) The Unx mice were then given1 sodium chloride in the drinking water and receivedDOCA pellet implants and Ang II infusion using osmoticminipumps Model 1004 ALZET osmotic infusion pumps(Durect) containing Ang II in sterile water were subcuta-neously implanted in the back of the necks of mice underisofluraneoxygen anaesthesia to deliver a dose of 15 ng AngII (Sigma) per minute per gram body weight At this timea 50mg 21-day release DOCA pellet (Innovative Research ofAmerica M-121) was also implanted subcutaneously Micethat underwent the sham Unx were also treated with asham procedure for subcutaneous implantation All micewere sacrificed on day 21 after implantation This animalutilization and the described procedures were approved bythe McMaster University Research Ethics Board

22 Blood Pressure Measurements Blood pressure measure-ments were obtained with tail cuff plethysmography using aCODA (Kent Scientific) blood pressure analyzer before AngIIDOCA implantation and also before sacrifice (Figure 1)Briefly animals were placed in restraint and positioned ona heating pad with a tail cuff attached to the machine Thecuff then measured systolic blood pressure diastolic bloodpressure and heart rate

23 Urinalysis Metabolic Cages and Microalbumin ELISABefore the surgical procedure and after 3 weeks on treatmentwith AngIIDOCA salt mice were placed in metabolic cagesfor 24 h urine collection (Figure 1) Urine samples were sentto our in-house laboratory to evaluate total protein concen-trations and an ELISA was used to measure mouse urinealbumin concentration (BETHYLLaboratories) to determinehypertension-induced proteinuria

24 Tissue Preparation for Histological Assessment andImmunohistochemical Analysis of Protein Cast FormationRenal Interstitial Fibrosis Apoptosis and Glomerular SclerosisRenal tissue was prepared for histological analysis The tissuewas fixed in 4 paraformaldehyde upon sacrificing theanimal The tissue was then embedded in paraffin blocks andsectioned (4 120583m) using a microtome To assess protein cast

Day 1 Day 21

CKD

dev

elopm

ent

Uninephrectomy

Ang II + DOCA

pellet implantation1 Na+ in drinkingwater

Sacrifice



Day 14

Figure 1 Ang IIDOCA salt model of chronic kidney disease inthe C57BL6 mouse Graph describing the time course of CKDdevelopment

formation and glomerular injury score these tissues werestained with Periodic Acid Schiff (PAS) stain and imagedusing a light microscope (Olympus)

Collagen deposition indicating extracellular matrix accu-mulation and renal interstitial fibrosis was evaluated usingMassonrsquos trichrome stain (Sigma-Aldrich)

To assess apoptosis kidney sections were stained usingthe protocol and reagents provided by the TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit (Trevigen Cat 4812-30-K) This method is based on specific binding of TdTto 31015840-OH ends of DNA and the incorporation of biotiny-lated deoxyuridine at sites of DNA breaks This signal isthen amplified by avidin-peroxidase allowing apoptotic cellswhere DNA fragmentation has occurred to be visualizedwith light microscopy [11]

The sections were analyzed using an Olympus BX41microscope Immunohistochemistry sections were imagedwith 20x and 40x objective lens For the quantification ofprotein cast formation apoptosis and F480 staining tenmicroscopic fields were randomly sampled in each of thecortex and the medulla To score glomeruli ten microscopicfields were randomly sampled from the cortex allowing thescoring of approximately 50 glomeruli per animal Imageswere analyzed for protein cast formation using the Meta-Morph program to select and quantify PAS-stained areas asa percentage of the total area of each image The average ofprotein cast area density was then calculated for each animalTUNEL-stained sections were processed using the cell counttool in Image J software Glomerular sclerosis was assessedbased on the scale and method used in a previous study [12]

Lungs from AngIIDOCA salt and sham mice wereextracted without performing a bronchoalveolar lavage pro-cedure and fixed in 4 paraformaldehyde for 24 hoursAfter standard paraffin embedding 4 120583M thick specimenswere sectioned and stained with HampE stain to visualizelung damage Mouse hearts were weighed upon sacrifice andthis parameter was normalized to body weight in grams toprovide a measure of increase in cardiac mass The heartswere then fixed in 4 paraformaldehyde for 24 hoursembedded in paraffin sectioned and stained with PAS toevaluate areas of hypertrophy



3 Results








Ang IIDOCA Sham120

140

160

180

200

Systo

lic b

lood

pre

ssur

e (m

mH

g) lowast

lowast

(a)

Ang IIDOCA Sham100

120

140

160

Dia

stolic

blo

od p

ress

ure (

mm

Hg)

lowast

lowast

(b)

Ang IIDOCA Sham0

10

20

30

40

Tota

l pro

tein

24

h (m

g)

lowast

lowast

(c)

Ang IIDOCA Sham0

10

20

30

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(d)

0 7 14 18 210

20

40

60

Ang IIDOCASham


Tota

l pro

tein

24

h (m

g) lowast

lowast

(e)



4 Discussion




Cortex MedullaA

ng II

DO

CASh

am

(a)

Cortex Medulla0

2

4

6

8

Ang IIDOCA Sham

Prot

ein

cast

form

atio

n (

) lowast

lowast

(b)

Ang IIDOCA Sham

(c)

Ang IIDOCA Sham0

1

2

3

4

Glo

mer

ulos

clero

sis (m

ean

scor

e)lowast

(d)

Ang IIDOCA Sham

(e)

Ang IIDOCA Sham0

5

10

15

20

TUN

EL p

ositi

ve ce

llsm

m2

lowast

(f)

(g)

Ang IIDOCA Sham00

05

10

15

20

F48

0 sta

inin

g in

kid

ney

(are

a ) lowast

(h)

Figure 3 Continued


40x 40x

20x 20x

(i)


LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)



Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results








Ang IIDOCA Sham120

140

160

180

200

Systo

lic b

lood

pre

ssur

e (m

mH

g) lowast

lowast

(a)

Ang IIDOCA Sham100

120

140

160

Dia

stolic

blo

od p

ress

ure (

mm

Hg)

lowast

lowast

(b)

Ang IIDOCA Sham0

10

20

30

40

Tota

l pro

tein

24

h (m

g)

lowast

lowast

(c)

Ang IIDOCA Sham0

10

20

30

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(d)

0 7 14 18 210

20

40

60

Ang IIDOCASham


Tota

l pro

tein

24

h (m

g) lowast

lowast

(e)



4 Discussion




Cortex MedullaA

ng II

DO

CASh

am

(a)

Cortex Medulla0

2

4

6

8

Ang IIDOCA Sham

Prot

ein

cast

form

atio

n (

) lowast

lowast

(b)

Ang IIDOCA Sham

(c)

Ang IIDOCA Sham0

1

2

3

4

Glo

mer

ulos

clero

sis (m

ean

scor

e)lowast

(d)

Ang IIDOCA Sham

(e)

Ang IIDOCA Sham0

5

10

15

20

TUN

EL p

ositi

ve ce

llsm

m2

lowast

(f)

(g)

Ang IIDOCA Sham00

05

10

15

20

F48

0 sta

inin

g in

kid

ney

(are

a ) lowast

(h)

Figure 3 Continued


40x 40x

20x 20x

(i)


LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)



Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ang IIDOCA Sham120

140

160

180

200

Systo

lic b

lood

pre

ssur

e (m

mH

g) lowast

lowast

(a)

Ang IIDOCA Sham100

120

140

160

Dia

stolic

blo

od p

ress

ure (

mm

Hg)

lowast

lowast

(b)

Ang IIDOCA Sham0

10

20

30

40

Tota

l pro

tein

24

h (m

g)

lowast

lowast

(c)

Ang IIDOCA Sham0

10

20

30

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(d)

0 7 14 18 210

20

40

60

Ang IIDOCASham


Tota

l pro

tein

24

h (m

g) lowast

lowast

(e)



4 Discussion




Cortex MedullaA

ng II

DO

CASh

am

(a)

Cortex Medulla0

2

4

6

8

Ang IIDOCA Sham

Prot

ein

cast

form

atio

n (

) lowast

lowast

(b)

Ang IIDOCA Sham

(c)

Ang IIDOCA Sham0

1

2

3

4

Glo

mer

ulos

clero

sis (m

ean

scor

e)lowast

(d)

Ang IIDOCA Sham

(e)

Ang IIDOCA Sham0

5

10

15

20

TUN

EL p

ositi

ve ce

llsm

m2

lowast

(f)

(g)

Ang IIDOCA Sham00

05

10

15

20

F48

0 sta

inin

g in

kid

ney

(are

a ) lowast

(h)

Figure 3 Continued


40x 40x

20x 20x

(i)


LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)



Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cortex MedullaA

ng II

DO

CASh

am

(a)

Cortex Medulla0

2

4

6

8

Ang IIDOCA Sham

Prot

ein

cast

form

atio

n (

) lowast

lowast

(b)

Ang IIDOCA Sham

(c)

Ang IIDOCA Sham0

1

2

3

4

Glo

mer

ulos

clero

sis (m

ean

scor

e)lowast

(d)

Ang IIDOCA Sham

(e)

Ang IIDOCA Sham0

5

10

15

20

TUN

EL p

ositi

ve ce

llsm

m2

lowast

(f)

(g)

Ang IIDOCA Sham00

05

10

15

20

F48

0 sta

inin

g in

kid

ney

(are

a ) lowast

(h)

Figure 3 Continued


40x 40x

20x 20x

(i)


LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)



Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















40x 40x

20x 20x

(i)


LV

RV

RV

LV

Ang IIDOCA Sham

15 mm 15 mm

(a)

Ang IIDOCA Sham0

5

10H

eart

wei

ght (

mg

g bo

dy w

eigh

t)lowast

(b)

Ang IIDOCA Sham

20x 20x

40x 40x

(c)



Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Males Females0

20

40

60

Tota

l pro

tein

24

h (m

g)

lowast

lowast

lowast

(a)

Males Females0

10

20

30

40

Tota

l alb

umin

24

h (m

g)

lowast

lowast

(b)

Ang

IID

OCA

Sham

Males Females

(c)

Ang IIDOCA Sham

M

F

(d)

Ang IIDOCA Sham

M

F

(e)


















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Development of a Model of Chronic Kidney …downloads.hindawi.com/journals/bmri/2015/172302.pdf · 2019-07-31 · kidney disease patients [ , ]. As previously demonstrated