Growth-Dependent Podocyte Failure Causes Glomerulosclerosisjasn.asnjournals.org/content/23/8/1351.full.pdf · Growth-Dependent Podocyte Failure Causes Glomerulosclerosis Akihiro Fukuda,*

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Growth-Dependent Podocyte Failure CausesGlomerulosclerosis

Akihiro Fukuda,* Mahboob A. Chowdhury,* Madhusudan P. Venkatareddy,* Su Q. Wang,*Ryuzoh Nishizono,* Tsukasa Suzuki,† Larysa T. Wickman,‡ Jocelyn E. Wiggins,*Timothy Muchayi,* Diane Fingar,*§ Kerby A. Shedden,| Ken Inoki,*†¶ and Roger C. Wiggins*

*Nephrology Division, Department of Internal Medicine; †Life Sciences Institute; ‡Department of Pediatrics;§Department of Cell and Developmental Biology; |Department of Statistics; and ¶Department of Molecular andIntegrative Physiology, University of Michigan, Ann Arbor, Michigan

ABSTRACTPodocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes torespond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenicFischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter;a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulatescap-dependent translation, which is a key determinant of a cell’s hypertrophic response to nutrients andgrowth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology andprotein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulo-sclerosiswere linearly related toboth increasing bodyweight and transgene dose. Uni-nephrectomy reducedthe body weight at which proteinuria first developed by 40%–50%. The initial histologic manifestation ofdisease was the appearance of bare areas of glomerular basement membrane from the pulling apart ofpodocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis con-firmed the mismatch between glomerular tuft volume and total podocyte volume (number 3 size) pertuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop ifdietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure ofpodocytes tomatch glomerular tuft growth in response to growth signaling through themTORC1 pathwaycan trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomer-ular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.

J Am Soc Nephrol 23: 1351–1363, 2012. doi: 10.1681/ASN.2012030271

A direct causative relationship exists between de-gree of podocyte depletion and the development ofproteinuria and glomerulosclerosis.1–5 Further-more, once a critical degree of podocyte depletionhas occurred, angiotensin II–dependent glomerulardestabilization supervenes, such that glomeruli con-tinue to lose podocytes in association with progres-sive glomerulosclerosis (FSGS) until glomerulibecome globally depleted of podocytes at ESRD.6

These concepts account for how progression canbe triggered and sustained after a critical degree ofpodocyte injury and loss. However, they do notaccount for why growth-associated processes, suchas obesity, large body size, glomerulomegaly, andnephronopenia, can trigger FSGS or for why FSGS

is particularly prevalent during phases of rapid bodygrowth in childhood and adolescence. If podocytedepletion is a common mechanism underlying glo-merulosclerosis, then growth itself may be able totrigger podocyte depletion, leading to progressionin susceptible individuals.

Received March 15, 2012. Accepted May 14, 2012.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Roger C. Wiggins, 1570B MSRBII, 1150West Medical Center Drive, Ann Arbor, MI 48109-5676. Email:[email protected]

Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: 1351–1363, 2012 ISSN : 1046-6673/2308-1351 1351

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mailto:[email protected]

One mechanism for how glomerular growth could resultin relative podocyte depletion would be if podocytes havelimited capacity to increase in number and size, while otherglomerular cells do not, thereby resulting in a “mismatch”between glomerular tuft volume and total podocyte comple-ment (number3 average cell volume).7,8 To test this hypoth-esis directly, we developed a transgenic rat whose podocyteshave limited capacity to respond to hypertrophic stress.

Cell growth is driven by growth factors and nutritionthrough the mammalian target of rapamycin complex 1(mTORC1) pathway and downstream via both 4E-BP1 andS6 kinase-dependent mRNA translation.9,10 The mTORC1pathway is essential for normal podocyte function.11,12 4E-BP1–regulated CAP-dependent translation can be retardedusing a dominant negative AA-4E-BP1 transgene in whichthreonine residues (37 and 46) critical for 4E-BP1 phosphor-ylation and activation are mutated to alanine.13 AA-4E-BP1transgene expression causes reduced cell size in culture.10

To impair podocyte response to hypertrophic stimuli, wetherefore expressed the AA-4E-BP1 transgene specifically inpodocytes of Fischer 344 rats under control of the podocinpromoter. Body growth on an ad libitum diet and nephrec-tomy, known to increase glomerular capillary number andsurface area,14,15 were used to stress wild-type and transgenicrats to bring out the underlying principles.

RESULTS

Glomerular Volume Is Directly Related to Body WeightGlomerular volume increases in relation to bodyweight gainin wild type Fischer 344 intact rats (Figure 1). Podocytevolume also increases but does not increase in proportion toglomerular volume (Figure 1). If the “mismatch hypothesis” iscorrect, then impairing podocyte capacity to respond to hyper-trophic stress should result in accelerated proteinuria andFSGS in relation to growth.

AA-4E-BP1 Is Expressed by Podocytes of TransgenicFischer 344 RatsTransgenic AA-4E-BP1 rats, but not wild-type Fischer 344rats, expressed human 4E-BP1 in their podocytes (Figure 2A).Western blot of isolated glomerular extracts demonstratedhypo-phosphorylated 4E-BP1 bands in both heterozygousand homozygous transgenic rats, along with expression ofthe human 4E-BP1 protein; these were not seen in wild-typerat glomerular extracts (Figure 2B). Thus, the human AA-4E-BP1 was expressed by transgenic rat podocytes.

Heterozygous AA-4E-BP1 Rats at 100-g Body WeightAre Structurally and Functionally Normal, WhereasHomozygous Rats Have Smaller Podocytes andGlomerular TuftsBy 100-g body weight, transgenic rats were normal in ap-pearance, had no increase in proteinuria (Figure 3A), were not

hypertensive, had normal histologic features of the kidney bylight microscopy (Figure 4A), and had no abnormality detect-ed by scanning electron microscopy (SEM) and transmissionelectron microscopy (TEM) (Figure 2C). Morphometric ex-amination at 100-g bodyweight showed no difference betweenwild-type and heterozygous rats, but homozygous rats had asmaller glomerular volume, a smaller total podocyte volumemade up of smaller podocyte volume, and slightly reducedpodocyte number (Table 1, left columns). Western blotshowed increased expression of ribosomal protein S6 andS6 phosphorylation (activation) in both heterozygous andhomozygous rat glomerular extracts (Figure 2D). Figure 2Eshows that phosphorylated S6 in homozygous AA-4E-BP1glomeruli was predominantly localized to podocytes. Thus,expression of the AA-4E-BP1 transgene designed to reducetraffic through the 4E-BP1 pathway resulted in a compensa-tory increase in activity in the S6 pathway. However, there wasno obvious reduction in expressed levels of podocyte proteinsin transgenic rats, including nephrin, podocin, glomerularepithelial protein 1 (GLEPP1), and vascular endothelialgrowth factor (Figure 2D). Therefore, transgene expressionappeared to be well compensated in both heterozygous andhomozygous rats at 100 g.

However, by 1 year of age, most heterozygous and homo-zygous rats (male and female) had reached ESRD (Figure 2F).Therefore, podocyte-specific expression of the AA-4E-BP1transgene under the direction of the podocin promoter thatcomes on late during the capillary-loop stage of glomerulardevelopment did not significantly alter podocyte structure or

Figure 1. Relationships between body weight and glomerulartuft volume (closed triangles) and body weight and total podo-cyte volume as measured by the GLEPP1-positive tuft volume(open squares). In wild-type Fischer 344 intact rats, glomerulartuft volume increased exponentially in relation to body weightgain in rats kept on an ad libitum diet. Podocyte volume alsoincreased in relation to body weight gain, but not at the samerate as the glomerular tuft volume, representing an apparent mis-match developing between these two variables as body weightincreases.

1352 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1351–1363, 2012


function in young rats, but conferred amarkedly increased propensity to developESRD.

Body Weight Gain as a Driver ofIncreased Proteinuria andGlomerulosclerosis in Relation to theTransgene Dose, and Acceleration byUni-nephrectomyFigure 3A shows the time course (startingat 100-g body weight and continuing for 14weeks) of proteinuria for wild-type, het-erozygous, and homozygous Fischer 344rats both in the intact state and after uni-nephrectomy performed at 100 g of bodyweight. Intact wild-type rats did not havesignificantly increased proteinuria by 14weeks. In contrast, uni-nephrectomy ofwild-type rats caused normal urine proteinexcretion for 8 weeks, followed by a smallbut significant increase in proteinuria at 9weeks that persisted until week 14. Intactheterozygous rats had no increase in pro-teinuria until week 5, after which theydeveloped proteinuria. Nephrectomy ofheterozygous rats resulted in normal pro-tein excretion for 2 weeks and then anincrease in proteinuria by week 3 after ne-phrectomy. Intact homozygous rats hadnormal proteinuria for 3 weeks and thendeveloped increased proteinuria by week 4.Nephrectomy of homozygous rats resultedin a normal level of proteinuria for 1 weekand then sharply increasing proteinuriathereafter until these animals developedESRD by 9 weeks. Thus, in each case (exceptthe intact wild-type rats) during the 14-weekperiod of observation, therewas normal pro-tein excretion for a period of time, afterwhich some threshold was crossed and urineprotein excretion then increased.

Figure 3B shows these same proteinuriadata plotted against body weight. There is aweight threshold above which rats developproteinuria (the “weight-proteinuria thresh-old”). This threshold varied depending on

Figure 2. Characterization of AA-4E-BP1 transgenic rats. (A) Expression of human4E-BP1 by immunofluorescence (red) in podocytes as identified by WT1-positive nuclearimmunofluorescence (green) in homozygous transgenic rats (upper panels) and wild-type rats (lower panels). The right two panels show DAPI-stained nuclei for the sameimages. n=3. Ab, antibody. Original magnification, 3160 (B) Western blots of proteinextracts from isolated glomeruli of wild-type (WT), heterozygous (Het), and homozy-gous (Homo) rats. Note that the heterozygous and homozygous glomeruli containhypophosphorylated lower-molecular-weight bands not seen in wild-type glomeruli(upper blot), and antihuman 4E-BP1 antibodies recognize bands in the heterozygousand homozygous rat glomerular extracts not present in wild-type extracts (middleblot). b-actin is shown as a reference loading marker. n=2 per experiment, with twoexperiments performed. (C) SEM (top panel) and TEM (bottom panel) images ofhomozygous AA-4E-BP1 rats, illustrating normal podocyte structure at 100-g bodyweight. n=4 for SEM and n=2 for TEM. Original magnification 32500. (D) Westernblots of wild-type, heterozygous, and homozygous isolated glomerular extractsdemonstrating increased S6 protein and phospho-S6 in heterozygous and homozy-gous rats compared with wild-type rats; there is no obvious difference in expressionof podocyte products (nephrin, podocin, GLEPP1, and vascular endothelial growthfactor [VEGF]). b-actin is shown as a reference loading marker. n=2 per experiment,with two experiments performed. (E) Glomerulus from a 100-g homozygous AA-4EBP1 rat developed for phospho-S6 immunofluorescence demonstrating pre-dominant expression of phospho-S6 in podocytes as confirmed by WT1-positive(green) nuclei. The right panel shows DAPI-stained nuclei in the same image. n=3.

Original magnification 3160. (F) Hematoxylinand eosin (H and E)–stained sections of ratrenal cortex from an 11-month-old heterozy-gous AA-4EBP1 rat (left panel) versus the cor-tex from an 11-month-old wild-type Fischer344 rat (right panel), demonstrating that ESRDhad developed in the transgenic rat. n=7.Original magnification 350.

J Am Soc Nephrol 23: 1351–1363, 2012 Growth and Hypertrophic Podocyte Stress 1353

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the dose of the transgene and on whetherthe rats had been nephrectomized. For ex-ample, we previously reported that wild-type intact Fischer 344 male rats developedproteinuria at about 400 g.8 Nephrecto-mized wild-type rats developed proteinuriaat about 250-g body weight (a 40% lowerweight-proteinuria threshold caused bynephrectomy). Similarly, nephrectomy re-duced the weight-proteinuria threshold ofheterozygous AA-4E-BP1 rats from about210 to 140 g (a 35% reduction), and forAA-4E-BP homozygous rats from 190 to100 g (a 47% reduction). Each rat straintherefore had its own weight-proteinuriathreshold for the intact animal and anapproximately 40%–50% lower weight-proteinuria threshold if one kidney hadbeen removed.

Figure 3C emphasizes the linear rela-tionship between weight gain and protein-uria, which is particularly well illustratedby comparing the nephrectomized homo-zygous and heterozygous transgenic rats.The slope for nephrectomized homozy-gous rats was twice that for heterozygousrats, thereby demonstrating a proportionalgene dose effect not only on the weight-proteinuria threshold but also on theweight-proteinuria slope. These data dem-onstrate that genes expressed only by thepodocyte can have a remarkable effect bothin lowering the weight threshold at whichproteinuria and FSGS develop as well asthe slope of the relationship between weightgain and proteinuria. Furthermore, reduc-tion in nephron mass caused by nephrec-tomy lowers this weight-proteinuriathreshold by about 40%–50% and increasesthe weight-proteinuria slope two-fold in apredictable way that depends on the podo-cyte’s genetic profile.

Figure 4, A and B, shows Massontrichrome–stained histologic features at 14weeks, together with quantitation of ad-hesions per glomerular tuft. Wild-typerats showed no detectable adhesions tothe Bowman capsule by 7 weeks, but afternephrectomy 5%of glomerular tufts had ad-hesions by 14weeks. Adhesions were presentin increasing numbers in linear proportionto proteinuria in all groups (Figure 4C).Nephrectomized homozygous rats devel-oped polyuria associated with the ruffledfur and reduced intake of food typical of

Figure 3. Relationship between proteinuria, age, and weight. (A) Time course (startingat 100-g body weight) for development of proteinuria of wild-type, wild-type uni-nephrectomized (NX), heterozygous, heterozygous uni-nephrectomized, homozygous,and homozygous uni-nephrectomized rats. Note that the time course of proteinuria varieddepending on transgene dose and whether the rats had been uni-nephrectomized.(B) Same data plotted against body weight on the x-axis, demonstrating that as weightincreases a weight proteinuria threshold is crossed; beyond this weight threshold, theproteinuria increases linearly in relation to weight gain. Nephrectomy reduced thisthreshold by 40%–50% for each rat strain (wild-type, heterozygous, or homozygous).(C) For the uni-nephrectomized rat strains, the slope of proteinuria versus weight gainin homozygous rats is approximately twice that for heterozygous rats under bothnephrectomy and intact conditions, thereby demonstrating that the gene dose playsa critical role in determining both the weight proteinuria threshold and the relation-ship between weight gain and proteinuria. Data are shown as the mean 6 SEM.



ESRD; they were euthanized at 9 weeks, by which time almostevery glomerulus had adhesions to the Bowman capsule.Heterozygous transgenic rats developed adhesions in 35%of glomeruli by 7 weeks and 60% of glomeruli by 14 weeks afteruni-nephrectomy (Figure 4B).

Table 1 shows morphometric data documenting how glo-merular tuft volume, the GLEPP1-positive (podocyte) area ofthe tuft, podocyte number, total podocyte volume, the glo-merular volume per podocyte, and the tuft nonpodocyte vol-ume change with normal growth in the intact rat on an adlibitum diet, and how this can be accelerated by nephrectomy.In wild-type intact rats, tuft volume increased 2.2-fold duringthe 14 weeks of growth, and nephrectomy caused a furtherincrease in tuft volume to 3-fold above baseline. In rats whosepodocytes carried the AA-4E-BP1 transgene, glomerular tuftvolume increased to a greater extent than in wild-type rats,particularly after nephrectomy, to 4.8-fold and 8.1-fold inheterozygous and homozygous rats, respectively. The totalpodocyte volume (number 3 size) increased only 2.2-fold,2.0-fold, and 1.4-fold in nephrectomized wild-type, heterozy-gous, and homozygous rats, respectively. Therefore, althoughthe glomerular tuft volume that was not GLEPP1 positive (i.e.,nonpodocyte tuft volume) increased 3.6-fold in wild-typenephrectomized rats (which was appropriate for the 3-foldincrease in glomerular tuft volume), in heterozygous andhomozygous transgenic rats the nonpodocyte glomerular vol-ume increased 7-fold and 12.2-fold, respectively. Thus, theglomerular volume per podocyte increased 2.9-fold in thewild-type rats during the 14-week period but 16-fold inthe heterozygous and 19.2-fold in the homozygous transgenicrats. Therefore, the total podocyte complement (number 3volume) did not keep up with glomerular tuft enlargement,particularly after the added hypertrophic stimulus of ne-phrectomy. Collectively, these data strongly support the con-cept that in the presence of defective podocyte function,glomerular tufts have an enhanced capacity to enlarge in thepresence of normal growth environment and after nephrec-tomy, whereas the podocyte number3 size has a much morelimited capacity for growth. This creates a “mismatch” be-tween tuft volume and the podocyte complement, whichwas associated with development of proteinuria, adhesionsto the Bowman capsule, and glomerulosclerosis.

SEM of glomeruli from both heterozygous and homozy-gous transgenic rats in this study showedareasof patchy absenceof podocytes from the capillary loop surface (Figure 4D). Atlater time points, TEM showed that adhesions to the Bowmancapsule had developed (Figure 4E, upper panel). By SEM,glomerular tuft could not be visualized because all capsulesremained adherent to the tuft (Figure 4E, lower panel), pre-sumably because of adhesions. Of note, podocyte foot pro-cesses were not diffusely effaced, although there were localizedareas of foot process effacement (not shown) and cyst forma-tion within podocytes (Figure 4E, upper panel). This result iscompatible with the hypothesis that as glomerular tufts enlargethe capacity of transgenic podocytes to hypertrophy and cover

the glomerular capillary surface area becomes locally inade-quate, so that podocytes pull apart to reveal bare areas of glo-merular basementmembrane (GBM).These lesions are the likelysource of proteinuria and give rise to the adhesions to the Bow-man capsule seen by light microscopy and TEM, as describedby Kriz and LeHir.16 This finding would also be consistent withthe linear relationship between the proportion of glomerulartufts with adhesions and the degree of proteinuria (Figure 4C).

Urine podocyte biomarker analysis provides real-timeinformation about podocyte stress and rate of loss.6,17 Asshown in Figure 4F, the urine podocin-to-nephrin mRNAratio (a measure of podocyte stress) was not statistically sig-nificantly upregulated after nephrectomy in wild-type rats butwas significantly increased after nephrectomy in both heterozy-gous and homozygous rats. The urine podocin-to-aquaporin2ratio (a measure of the rate of podocyte loss) was not detectablyupregulated in nephrectomized wild-type rats but was in-creased by 3 weeks after nephrectomy to a greater extent inhomozygous than heterozygous rats. We interpret this result todemonstrate that although podocyte number per tuft was notdetectably reduced in glomeruli of heterozygous nephrecto-mized transgenic rats by 14 weeks (Table 1), the rate of podo-cyte loss was increased, reflecting increased podocyte turnoverin these animals where podocyte replacement was able toapproximately keep up with the rate of loss. In contrast, in ho-mozygous nephrectomized transgenic rats the higher rate ofpodocyte loss could not be compensated for and was thereforeassociated with a significant reduction in the number of glomer-ular tuft podocytes by 14 weeks (Table 1).

Calorie Restriction Prevents Weight Gain, GlomerularEnlargement, Proteinuria, and FSGSIf glomerular enlargement is driving the development ofFSGS, we would hypothesize that prevention of glomerulargrowth would prevent development of proteinuria and FSGSin AA-4E-BP1 transgenic rats. To test this hypothesis, we used300-g male heterozygous AA-4E-BP1 rats (to avoid exposinggrowing rats to the 40% reduced calorie diet). As shown inFigure 5A, 40% calorie restriction resulted in 20% weightloss to a weight of about 240 g (the average weight of malerats in the wild).18 Rats fed ad libitum continued to gainweight. After nephrectomy, calorie-restricted rats did not de-velop proteinuria, adhesions to the Bowman capsule, or glomer-ulosclerosis (Figure 5, B–D). Morphometric analysis (Table 2)showed that in calorie-restricted rats, glomerular tuft volumedid not increase, the podocyte complement as a proportion ofthe tuft volume did not change, and urine podocyte biomarkersdid not show an increase in the rate of podocyte loss fromglomeruli (Figure 5E). In contrast, ad libitum–fed transgenicrats showed increased volume of glomerular tufts, a decrease inthe podocyte complement (number 3 volume) of glomerulartufts in proportion to glomerular volume, development of pro-gressive proteinuria in relation to weight gain, increase in urinepodocyte biomarkers, and FSGS. This result demonstrates thatreduced nutritional signaling in the glomerulus has a powerful



Figure 4. Relationships between histologic features, proteinuria, and urine podocyte biomarkers in wild-type, heterozygous, and ho-mozygous nephrectomized AA-4E-BP1 rats. Original magnification360. (A) Masson trichrome–stained histologic sections at 7 weeks and 14weeks after nephrectomy (at 100-g body weight). Homozygous rats were euthanized at 9 weeks because they developed uremic symp-toms. (B) Proportion of glomeruli that contained adhesions at 7 and 14 weeks (9 weeks for homozygous nephrectomized rats). Het,



determinative effect on whether glomerular enlargement oc-curs together with its downstream consequences in terms ofpodocyte stress and loss, development of proteinuria, adhe-sions, and FSGS. This result also strongly supports the mis-match hypothesis.

DISCUSSION

Increase in glomerular tuft volume is remarkably related tobody weight gain. As previously demonstrated by Nyengaardand colleagues, this increase in glomerular tuft volume resultsfrom increased glomerular capillary number and surfacearea.14,15 At the same time, podocyte number per tuft didnot increase in proportion to glomerular volume. This meansthat compensation for glomerular tuft enlargement mustoccur largely through podocyte hypertrophy, as previouslynoted for aging ad libitum–fed rats.8 This finding raises thepossibility that failure of podocyte hypertrophy to matchglomerular enlargement could potentially drive developmentof glomerulosclerosis. To test this concept, we engineeredtransgenic rats to selectively compromise the capacity of theirpodocytes to respond to hypertrophic stress. The resultingenhanced sensitivity of these transgenic rats to develop pro-teinuria and FSGS in response to both body growth (weightgain) and nephrectomy, and the prevention of these events byreducing nutritional signaling by calorie restriction, stronglysupports the “mismatch” hypothesis.

Themechanism by which proteinuria occurs in the AA-4E-BP1model appears to be bymechanical failure of the podocyteepithelial layer. The integrity of the glomerular filtrationsurface requires exact coverage of theGBMsurface by podocytefoot processes. As tufts hypertrophy in response to growth,the filtration surface area increases; thus, in the setting ofreduced capacity to hypertrophy, the podocyte interdigitatingfoot processes eventually pull apart at certain sites to exposebare areas of GBM. These bare areas of glomerular capillarysurface facilitate protein leak and are where adhesions to theBowman capsule then develop (as demonstrated by Massontrichrome staining at the light microscopic level and by TEM).

These events lead to development of FSGS lesions and pro-gression to glomerulosclerosis in the sequence described by Krizand LeHir.16 The linear relationship between the adhesions andproteinuria is compatible with a direct causative relationship.

Morphometry data emphasize that glomerular tuft vol-ume increases more in transgenic rats than in wild-type rats,and in homozygous transgenic rats versus heterozygous trans-genic rats. Therefore, wild-type rats were better able to controlglomerular tuft volume in the presence or absence of hyper-trophic stress compared with transgenic rats, and heterozy-gous rats were better able to control glomerular tuft volumecompared with homozygous rats. There was therefore a trans-gene dose-dependent effect. This must mean that one job ofthe normal podocyte is to control tuft volume, and that itrequires active podocyte function by an unidentified mech-anism to achieve this important regulatory process. Thus, themismatch resulting from podocyte loss or dysfunction is acompounded result of both a reduction in effective podocytevolume (size 3 number) and an increase in glomerular tuftvolume. The mechanism by which this loss of tuft volumecontrol occurs is not yet identified but could provide animportant target for intervention to prevent progression.

We demonstrate a remarkable direct linear relationshipbetween body weight gain and development of proteinuriaand glomerulosclerosis, and how this relationship is affectedby podocyte-specific expression of gene variants, which com-promise podocyte capacity to adapt to hypertrophic stress.This result is compatible with the general concept that everyindividual has a body weight above which proteinuria willdevelop (the weight-proteinuria threshold). The developmentof proteinuria and FSGS for any individual will thereforedepend on several variables: (1) body weight and closely asso-ciated glomerular tuft volume (governed by both genetic andenvironmental factors); (2) nephron number (influenced bybirthweight andmaternal and genetic factors, as well as by lossof renal mass caused by nephrectomy [such as occurs in kidneydonation, transplantation, and cancer surgery]); (3) the geneti-cally endowed capacity of their podocytes to adapt to hypertro-phic stress; and (4) accelerated loss of nephrons or podocytescaused by diverse disease mechanisms (both genetic and

heterozygous; Homo, homozygous; Wt, wild-type. *P,0.05 and **P,0.01, as assessed by Kruskal-Wallis test and then Scheffe’s test. (C)There was a direct relationship between the urine protein-to-creatinine ratio and the proportion of glomeruli that contained adhesions.(D) SEM images from heterozygous nephrectomized rats demonstrating that foot processes were mostly intact (upper left panel) butthat glomeruli contained patches of bare GBM where the foot processes had apparently been pulled apart (three examples shown).Foot processes abutting these areas remained relatively intact. Original magnification 34000. (E) Upper panel shows a TEM imagedemonstrating two adhesions (arrows) distant from the pole of the glomerulus (P), and podocytes adjacent to these areas containedgranules and cysts (arrowheads). Most capillary loops were lined by intact foot processes, although occasional areas of effaced footprocesses were present (not shown). Also shown are plump parietal epithelial cells adjacent to the adhesions. Original magnification 3260.Glomerular tufts could not bevisualizedbySEMbecause capsules remainedattached to the tuft, presumablybecauseof adhesions (lowerpanel).Originalmagnification370. (F) Time course of urine protein-to-creatinine ratio (upper panel), urine podocin-to-nephrinmRNA ratio as ameasureofpodocyte stress (middlepanel), andurinepodocin-to-aquaporin2mRNA ratioper as ameasureof the rateofpodocyte loss into theurine.Notethat there was no measurable increase in the rate of podocyte loss in wild-type rats, although podocyte stress was increased throughout the14-week observation period. In contrast, podocyte stress was markedly increased in both heterozygous and homozygous rats, and the rate ofpodocyte loss was higher in homozygous than in heterozygous rats. As homozygous rats reached ESRD, glomeruli became depleted ofpodocytes, as reflected by a reduced rate of podocyte loss. Data are shown as the mean6 SEM.



Table

1.Morpho

metricva

riab

lesmea

suredin

wild

-typ

e,he

terozygou

s,an

dho

moz

ygou

sAA-4E-BP1

Fische

r34

4rats

withan

dwitho

utprior

nephrec

tomy

Variable

Wild

Type

Heterozy

gous

Homozygous

Wk0,

100g

(n=5)

NoNX

NX

Wk0,

100g

(n=10

)

NoNX

NX

Wk0,

100g

(n=5)

NoNX

NX

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=10

)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk9

(n=5)

Fold

Cha

nge

ove

rWk0

Glomerular

tuft

volume(mm

3)

mea

n46

9,01

21,04

0,08

12.2

1,39

4,31

93.0

465,42

91,04

9,94

22.3

2,22

0,89

34.8

332,47

31,28

5,27

13.9

2,68

8,46

48.1

SD84

,100

54,891

189,86

592

,952

212,90

672

9,85

954

,000

295,20

230

3,84

6Pvaluewith

inWT,

heterozygou

s,or

homoz

ygou

sgroup

,0.01

,0.01

,0.01

0.01

,0.01

,0.01

,0.01

,0.01

,0.01

Pvaluebetwee

nWT,

heterozygou

s,an

dho

moz

ygou

sgroup

s

NS

NS

0.03

0.01

NS

NS

0.02

NS

,0.01

GLP

P1-positive

tuft

area

(%)

mea

n47

400.9

360.8

4441

0.9

220.5

3536

1.1

60.3

SD6

44

66

182

63

Pvaluewith

inWT,

heterozygou

s,or

homoz

ygou

sgroup

0.02

NS

,0.01

NS

,0.01

,0.01

NS

,0.01

,0.01

Pvaluebetwee

nWT,

heterozygou

s,an

dho

moz

ygou

sgroup

s

NS

NS

NS

,0.01

NS

NS

,0.01

NS

,0.01

GLP

P1-positive

volume(mm

3)

mea

n22

1,74

741

5,21

71.9

495,05

12.2

203,67

642

6,97

12.1

399,11

82.0

117,25

946

8,37

93.9

158,36

51.4

SD61

,861

47,401

58,717

42,568

53,570

270,77

319

,676

128.70

389

,343

Pvaluewith

inWT,

heterozygou

s,or

homoz

ygou

sgroup

,0.01

,0.01

,0.01

0.01

0.02

0.02

,0.01

,0.01

NS

Pvaluebetwee

nWT,

heterozygou

s,an

dho

moz

ygou

sgroup

s

NS

NS

NS

,0.01

NS

NS

,0.01

NS

,0.01

Podocytesper

tuft(n)

mea

n12

013

01.1

122

1.0

118

125

1.1

930.8

103

137

1.3

530.5

SD9

119

1317

5511

2826

Pvaluewith

inWT,

heterozygou

s,or

homoz

ygou

sgroup

NS

NS

NS

NS

NS

NS

,0.01

,0.01

,0.01



Table

1.Con

tinu

ed

Variable

Wild

Type

Heterozy

gous

Homozygous

Wk0,

100g

(n=5)

NoNX

NX

Wk0,

100g

(n=10

)

NoNX

NX

Wk0,

100g

(n=5)

NoNX

NX

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk14

(n=10

)

Fold

Cha

nge

ove

rWk0

Wk14

(n=5)

Fold

Cha

nge

ove

rWk0

Wk9

(n=5)

Fold

Cha

nge

ove

rWk0

Pvaluebetwee

nWT,

heterozygou

s,an

dho

mozygous

group

s

NS

NS

NS

0.04

NS

NS

0.03

NS

,0.01

GV/P

(mm

3)

mea

n39

0780

702.2

1137

92.9

3939

8391

2.1

52,853

16.0

3,22

59,62

13.0

62,044

19.2

SD66

310

6012

1555

511

0664

,753

428

2,74

434

,002

Pvaluewith

inWT,

heterozygou

s,or

homozygous

group

,0.01

,0.01

,0.01

,0.01

0.01

0.01

NS

,0.01

,0.01

Pvaluebetwee

nWT,

heterozygou

s,an

dho

mozygous

group

s

NS

NS

NS

0.01

NS

NS

NS

NS

0.01

Mea

npod

ocyte

volume(mm

3)

mea

n18

3332

291.8

4059

2.2

1734

3440

2.0

4176

2.4

1133

3422

3.0

2945

2.6

SD40

359

253

433

237

715

5211

666

012

17Pvaluewith

inWT,

heterozygou

s,or

homozygous

group

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

NS

,0.01

Pvaluebetwee

nWT,

heterozygou

s,an

dho

mozygous

group

s

NS

NS

NS

,0.01

NS

NS

0.01

NS

NS

GLE

PP1-ne

gativetuft

volume(mm

3)

mea

n24

7,26

462

4,86

32.3

899,26

93.6

261,75

462

2,97

12.4

1,82

1,77

57.0

215,21

481

6,89

23.8

2,53

0,09

912

.2SD

42,767

43,760

152,52

467

,173

176,02

389

7,84

835

,969

195,80

836

3,27

9Pvaluewith

inWT,

heterozygou

s,or

homozygous

group

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

,0.01

Pvaluebetwee

nWT,

heterozygou

s,an

dho

mozygous

group

s

NS

NS

0.04

NS

NS

NS

NS

0.03

,0.01

Maleratsat

100geither

nephrec

tomyor

remaine

dintact.T

hekidne

ysob

tained

byne

phrec

tomyat

100gwereused

asabaseline.

After

14wee

kson

anad

libitum

diet,ratswereeu

than

ized

(with

theex

ceptio

nof

theho

moz

ygous

nephrec

tomygroup

,which

reac

hedES

RDan

dwereeu

than

ized

at9wee

ksaftern

ephrec

tomy).M

orpho

metric

varia

blesweremea

suredas

show

ninthetable.T

hePvalues

fore

achdatasetinthe

upper

lineco

mpareva

lues

with

inea

chgroup

.Pvalues

areshowninthreeco

lumns.T

heleftco

lumnco

mpares

100-gratsto

theintactrat1

4wee

kslater.Th

emiddleco

lumnco

mpares

theintactrata

t14wee

ksto

the

nephrec

tomized

rata

t9wee

ks.T

herig

htco

lumnco

mpares

thene

phrec

tomized

rata

t9wee

ksto

the10

0-grata

t0wee

ks.T

hePvalues

shownfore

achdatasetinthelower

lineco

mparedataforthe

samevaria

bles

betwee

nwild

-typ

e,he

terozygou

s,an

dho

mozyg

ousg

roup

s.Th

ePva

lues

inthewild

-typ

eco

lumnshowva

lues

comparingwild

-typ

eto

heterozygou

srats.T

hedatainthemiddleco

lumns

comparehe

terozygou

srats

toho

mozyg

ousrats.T

hevalues

intheho

mozyg

ousco

lumns

compareho

mozyg

ousrats

towild

-typ

erats.N

X,n

ephrec

tomy;

WT,

wild

-typ

e;GV/P,g

lomerular

volumeper

pod

ocyte.



acquired). The well established relation-ships between nephron mass, body size,glomerular volume, and hypertensionwere recently reviewed by Luyckx andBrenner,19 although the key role of thepodocyte in these relationships is not wellrecognized. Understanding the biologybehind these relationships can provideimproved opportunities for targeted thera-peutic intervention to prevent progression.

The mechanism of podocyte depletiondemonstrated by this model system isdifferent from direct podocyte damageand death causing reduced podocyte num-ber, leading to podocyte depletion andglomerulosclerosis.2,4,6 Growth-associatedpodocyte failure provides a mechanisticexplanation for a separate group of diseasesleading to FSGS or global glomerulosclero-sis. These include (1) obesity and largebody size, associated with proteinuria,glomerulomegaly FSGS, and progressionto ESRD for all causes;20–23 (2) idiopathicFSGS and FSGS due to known geneticcauses that are most prevalent during pe-riods of rapid growth in childhood andadolescence; (3) nephronopenic and glomer-ulomegalic conditions (congenital andacquired);24 and (4) diabetic glomeruloscle-rosis, in which glomerulomegaly occurs inthe setting of diabetes-associated renal hyper-trophy, leading to reduced podocyte densityand subsequent podocyte loss.25–29

The remarkable enhancement of FSGScaused by increased body growth in rats fedan ad libitum diet (and the even more re-markable total protection against FSGScaused by calorie restriction) could helpexplain the increased prevalence of FSGSreported in diverse ethnic populations30–39

in the setting of a worldwide increase inbody mass index.40 The data are compat-ible with the concept that dietary modifi-cation and therapeutic modulation ofgrowth/nutrition signaling pathways couldbe powerful adjunctive strategies to preventprogression of kidney diseases.

CONCISE METHODS

All animal studies were approved by the Uni-

versity of Michigan Committee on Use and

Care of Animals. Male Fischer 344 rats (Harlan,

Indianapolis, IN) fed an ad libitum diet were

Figure 5. Effect of calorie restriction in preventing podocyte stress, podocyte loss, andadhesion formation in 300-g nephrectomized heterozygous AA-4EBP1 rats. (A) Fortypercent calorie restriction resulted in loss of about 70 g in weight, whereas ad libitum–fedrats continued to gain weight over the 16-week period of observation. (B) Urine protein-to-creatinine ratio remained at baseline in calorie-restricted rats and increased in adlibitum–fed rats. (C) Representative Masson trichrome–stained sections illustrating thedevelopment of adhesions present in ad libitum–fed rats, which were not present incalorie-restricted rats. Original magnification 3110. (D) Quantitation of the adhesions.*P,0.05, as assessed by t test. (E) Both the urine podocin-to-nephrin mRNA ratio asa measure of podocyte stress and the urine podocin-to-aquaporin2 ratio as a measure ofthe rate of podocyte loss remained at baseline in calorie-restricted rats but increased inad libitum–fed rats. Data are shown as the mean 6 SEM.



used for these experiments because they do not spontaneously

develop diabetes or hypertension. Calorie restriction was as previ-

ously described using the National Institute on Aging rat protocol

and pellets purchased from this agency for this purpose as previously

described.8 Urine was collected weekly using metabolic cages. Sys-

tolic BP; serum and urine creatinine, urine protein, and urinemRNA

assays; and Western blots of glomerular extracts under reducing

conditions were as previously described.2,4,6,8

AA-4E-BP1 Fischer 344 Rat ModelThe AA-EFI4EBP1 cDNA, a double point mutant of EFI4EBP1 at

phosphorylation sites A37 and A46, was isolated from a pACTAG-

2-h4E-BP1 plasmid kindly provided by Dr. Anne-Claude Gingras.13

The AA-EFI4EBP1 cDNAwas inserted at the 39 position of the 2.5-kb

human podocin (Nphs2) promoter at theNcoI site of plasmid p2.5P-

nlacF, which we previously showed drives podocyte-specific LacZ

expression in mice and human diphtheria toxin receptor expression

in rats.4,41 The podocin promoter/AA-EFI4EBP1 Tg construct was iso-

lated from plasmid (p2.5PnLacF_EFI4EBP1 Thr37Ala/Thr46Ala) by

XbaI/HindIII restriction enzyme digestion and injected into the pro-

nuclei of fertilized Fischer344 rat ova using standard techniques.

Three Tg founders were obtained, only one of which (#110) produced

Tg offspring. Transgenic rats were identified by PCR analysis of tail

DNA using the primer pair: p2.5_EGFdetect.fwd (59-ACCCGACGG-

TCTTTAGGG-39); h4E-BP1_FixB.rev (5-ATCCCCCATGGATTCCTTG

AGCACA AGTCTCT CTTAAATGTCC ATCTCAAA CTGTGACTC-3),

Table 2. Morphometric variables for ad libitum–fed and calorie-restricted heterozygous AA-4E-BP1 rats nephrectomizedat 300-g body weight

Variable

Ad Libitum Diet Calorie-Restricted Diet Fold Change: AdLibitum versus

Calorie-Restricted

Diets

Control(n=5)

8 wk(n=5)

Fold Changeversus Control

Control(n=5)

8 wk(n=5)

Fold Changeversus Control

Glomerular tuft volume (mm3)mean 988,394 1,537,509 1.6 988,509 829,346 0.8 1.9SD 178,504 331,376 189,784 149,557P value within each group ,0.01 NSP value between groups NS ,0.01

GLPP1-positive tuft area (%)mean 57 43 0.8 55 53 1.0 0.8SD 6 9 4 4P value within each group ,0.01 NSP value between groups NS NS

GLPP1-positive volume (mm3)mean 557,405 640,941 1.1 538,970 435,234 0.8 1.5SD 101,091 41,783 104,964 75,625P value within each group 0.03 NSP value between groups NS ,0.01

Podocytes per tuft (n)mean 116 132 1.1 106 128 1.2 1.0SD 11 41 8 26P value within each group NS NSP value between groups NS NS

GV/P (mm3)mean 8552 12453 1.5 9253 6518 0.7 1.9SD 1676 3927 1302 696P value within each group ,0.01 ,0.01P value between groups NS 0.01

Mean podocyte volume (mm3)mean 4826 5221 1.1 5032 3436 0.7 1.5SD 970 1627 637 466P value within each group NS ,0.01P value between groups NS 0.05

GLEPP1-negative tuft volume (mm3)mean 430,989 896,567 2.1 449,538 394,112 0.9 2.3SD 102,443 332,279 99,656 83,445P value within each group ,0.01 NSP value between groups NS 0.01

Fold differences are used to summarize the comparison between the ad libitum and calorie-restricted animals after 16 weeks of diet. Note that calorie restrictionprevented glomerular enlargement, podocyte hypertrophy, and nonpodocyte glomerular volume expansion. GV/P, glomerular volume per podocyte.



which produced a 550-bp amplicon. Crossing of heterozygous AA-4E-

BP1 transgenic rats resulted in the expected ratio of 1:2:1 wild-type:

heterozygous:homozygous offspring ratios (data not shown).

Reagents and AntibodiesThe following primary antibodies were used for immunofluorescence

and Western blot. Nephrin and podocin (rabbit polyclonal) were

supplied by Dr. Lawrence Holzman. GLEPP1 (also more recently

designated in the tyrosine phosphatase literature as PTPro [protein

tyrosine phosphatase ro]) mouse monoclonal antibody was raised

against the recombinant rat GLEPP1 extracellular domain and used

as described previously.2 Vascular endothelial growth factor (no.

ab46154) and human 4E-BP1 (no. ab32130) antibodies were ob-

tained from Abcam (Cambridge, MA). Phospho-S6 (no. 2215), S6

(no. 2217), and 4E-BP1 (no. 9644) antibodies were obtained from

Cell Signaling Technology (Beverley, MA). b-actin antibody (no.

A5441) was obtained from Sigma-Aldrich (St. Louis, MO).

HistomorphometryImmunofluorescence and immunoperoxidase staining were per-

formed as previously described.2,4,6,8 All histologic testing was per-

formed on paraformaldehyde-lysine-periodate–perfused and fixed

paraffin-embedded tissue sectioned on a Reichert-Jung 820-II micro-

tome (Cambridge Instruments, Nussloch, Germany). For quantifica-

tion of glomerular tuft adhesions, 3-mm sections were stained with

Masson trichrome and read by a blinded observer. The proportion of

glomeruli demonstrating adhesions in 100 consecutive glomeruli was

measured. Glomerular volume was estimated by measuring the

mean glomerular radius (r) using the Metamorph Imaging program,

and then using the Weibel formula to calculate the mean maximal

tuft radius (R) and hence the mean glomerular volume (4/3pR3) as

previously described.42 For measurement of GLEPP1-positive area,

50 consecutive glomerular tuft areas were measured and the per-

centage of each glomerular tuft that stained positive using GLEPP1

immunoperoxidase was measured using the Metamorph Imaging

program as previously described.6 The mean GLEPP1-positive (po-

docyte) volume per glomerulus was estimated by multiplying

the mean glomerular volume by the mean percentage of GLEPP1-

positive area. The number of podocytes per glomerular tuft was

measured using WT1 immunofluorescence, as previously de-

scribed.6 The mean individual podocyte volume was estimated

by dividing the mean GLEPP1-positive tuft volume (estimated

as outlined above) by the mean podocyte number per tuft. The

nonpodocyte volume was estimated by subtracting the mean

podocyte volume from the glomerular volume.

Transmission and SEM AnalysisThe kidney samples were fixed with Sorensen phosphate buffer con-

taining 4% paraformaldehyde and 2.5% glutaraldehyde. The processed

samples were analyzed by Philips CM100 TEM and AMRAY 1910 field

emission SEM.

Western Blot and AntibodiesGlomeruli were isolated as previously described.2,4 Purified glomer-

uli from one rat was lysed in lysis buffer (10 mMTris-HCl [pH, 7.5],

100 mMNaCl, 1%NP-40, 50 mMNaF, 20 mM b-glycerophosphate,

2 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF, 10 mg/ml leupeptin,

and 10 mg/ml aprotinin) as a lysate sample. Lysates were then boiled in

SDS sample buffer (20 mM Tris [pH, 6.8], 8% SDS, 0.05% bromophe-

nol blue, 40% glycerol, 20% 2-mercaptoethanol) and subjected to SDS-

PAGE and Western blotting according to standard techniques.

Urine mRNA AnalysisUrine was collected in metabolic cages and the overnight urine

pellet was used for purification of RNA; production of cDNA; and

quantitation of podocin, nephrin, and aquaporin2 cDNAs as pre-

viously described.6,17

Statistical AnalysesAll results were presented as mean 6 SEM except where otherwise

noted. Differences among two groups was tested by a t test and,

amongmore than two groups, by Kruskal-Wallis test.When the result

of the Kruskal-Wallis test was significant, a Scheffe test was carried

out for post hoc analysis. For data shown in the tables, an unpaired

t test was used for most comparisons; for comparisons of data from

the same animals, a paired t test was used.

ACKNOWLEDGMENTS

We are grateful to Bryan Wharram for his help in producing the

AA-4E-BP1 transgenic rat.

This work was supported by the National Institutes of Diabetes

and Digestive and Kidney Diseases, National Institutes of Health

(grants DK RO1 46073 and P30 DK081943).

DISCLOSURESNone.

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Growth-Dependent Podocyte Failure Causes Glomerulosclerosisjasn.asnjournals.org/content/23/8/1351.full.pdf · Growth-Dependent Podocyte Failure Causes Glomerulosclerosis Akihiro Fukuda,*