View Histology, Slides Were Stained by PAS

BASIC RESEARCH www.jasn.org

Increased Tubular Proliferation as an Adaptive Response to Glomerular Albuminuria

† † † † ‡ Jian-Kan Guo,* Arnaud Marlier,* Hongmei Shi,* Alan Shan,* Thomas A. Ardito, ‡ ‡| † Zhao-Peng Du,§ Michael Kashgarian, Diane S. Krause, Daniel Biemesderfer,* † and Lloyd G. Cantley*

† *Section of Nephrology and Departments of Internal Medicine, ‡Pathology, §Cellular and Molecular Physiology, and |Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specificablation of podocytes by administering diphtheria toxin. Ablation of ,20% of podocytes resulted in profound albuminuria that resolved over 1–2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increasedapoptosis,resultinginanincreaseintotal tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of .40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.

J Am Soc Nephrol 23: 429–437, 2012. doi: 10.1681/ASN.2011040396

Podocytes, mesangial cells, and glomerular endo- There have been numerous animal models de- thelial cells together contribute to the formation veloped to study the effect of glomerular injury on and maintenance of the kidney ultrafiltration kidney function.9,10 Typically, these models involve barrier that serves to retain the majority of plasma injection of general cellular toxins such as adriamycin proteins within the vascular space.1–5 This barrier or nephritic serum containing antibodies directed can be compromised by injury to any of these cells, against either the GBM or podocyte-specific anti- resulting in glomerular proteinuria that has in gens.Inmostcases,thereiswidespreaddamageto turn been associated with progressive renal dys- multiple cell types in the glomerulus with a sub- function. Of these cells, podocyte damage is the stantial inflammatory response, making it difficult most problematic because podocytes provide a critical component of the filtration barrier and seem to lack the ability to proliferate in response Received April 19, 2011. Accepted October 24, 2011. to cell loss. Multiple causes of podocyte dysfunc- Published online ahead of print. Publication date available at tion have been identified, including genetic defects www.jasn.org. in components of theslit diaphragm, toxins,depo- Correspondence: Dr. Jian-Kan Guo, Yale University School of sition of abnormal proteins in the glomerular base- Medicine, 333 Cedar Street, P.O. Box 208029, New Haven, CT ment membrane (GBM), and immune-mediated 06520. Email: [email protected] – injury.6 8 Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: 429–437, 2012 ISSN : 1046-6673/2303-429 429 BASIC RESEARCH www.jasn.org to determine which components of the subsequent kidney injury are related specifically to podocyte injury and which are related to the ensuing response. One of the key responses to glomerular damage is albuminuria, and several studies have suggested that glomerular albuminuria can lead to subsequent tubule damage.11,12 In vitro studies in which albuminuria was modeled by culturing tubular cells in the presence of albumin have led to conflicting results. Whereas some groups demonstrated that albumin can cause oxidative stress13 and programmed cell death,14,15 other studies showed that albumin is one of the major serum survival factors for renal tubular cells and can serve to scavenge reactive oxygen species.16,17 To induce selective podocyte loss via a nonimmune mechanism, we utilized mice that contained the transgene for diphtheria toxin receptor (DTR) downstream of a poly- adenylation signal flanked by loxP sites (iDTR mouse18). Be- causemicenormallylacktheDTR,expressionoftheCre recombinase in a podocyte-specific manner (Pod-Cre19) results in DTR expression selectively in podocytes, allowing subsequent podocyte ablation induced by diphtheria toxin (DT) Figure 1. DTR expression in podocytes. Immunofluorescent to be performed in a dose-dependent manner without con- staining with anti-DTR (green) and anti-Wt1 (pink) was performed to comitant injury to other glomerular components. A similar evaluate kidney sections. (A) Podocytes of the single transgenic 2 approach has been successfully used for selective podocyte ab- iDTR+/ mouse do not express DTR (arrow, podocyte expressing 2 2 lation in the rat.20 In these studies, we demonstrate that struc- Wt1). (B) Podocytes from iDTR+/ ;Pod-Cre+/ express DTR only in tural tubular injury occurs only when podocyte loss is sufficient podocytes (arrow, podocyte double positive for DTR and Wt1). (C) to cause glomerular damage, and is not seen in the setting of After 25 ng DT, podocytes numbers are reduced. The white arrow + + isolated glomerular albuminuria. In fact, modest podocyte loss indicates a DTR /Wt1 podocyte that survived the initial ablation results in marked albuminuria that is accompanied by a pro- by DT. (D) Podocytes are undetectable after treatment with 200 ng fi m fi 3 liferative response of proximal tubule cells without evident tu- DT. Paraf nsectionsat3 m. Original magni cation, 400. bular injury, leading to an increase in total proximal tubule cell numbers. We propose that the proteinuria that accompanies selective podocyte loss, in the absence of more generalized glo- DT Exposure Selectively Ablates Podocytes +/2 +/2 merular injury, leads to an increase in proximal tubule cell in iDTR ;Pod-Cre Mice numbers that may provide an adaptive response to limit the It has been demonstrated that binding of one molecule of loss of filtered proteins in the urine. DT to the DTR will trigger cell death.21,22 Increasing the dose of DT injected should therefore result in progressively greater podocyte loss. The rate of podocyte ablation was tested in RESULTS 4- to 5-week-old mice (18–20 g body wt) exposed to DT doses ranging from 25 to 200 ng. Double immunostaining using +/2 +/2 iDTR ;Pod-Cre Mice Express DTR in Podocytes anti-Wt1 and anti-DTR antibodies was performed to calculate 2 2 iDTR+/ ;Pod-Cre+/ mice were found to be viable and fertile. the ablation rate. One week after a single dose of 25 ng DT Toexamine the expression pattern of DTR, multiple organs were there was a 19.6% decrease in podocyte numbers (from 2 2 isolated from double transgenic (iDTR+/ ;Pod-Cre+/ )18,19 and 11.663.2 per glomerular cross-section to 9.362.4, n=11 2 2 control mice (iDTR+/ ,Pod-Cre+/ ,andwildtype).These mice, P,0.001), whereas 50 ng DT led to 46.1% podocyte included heart, lung, liver, pancreas, small intestine, spleen, loss (6.861.1 podocytes per glomerular cross-section, n=7 kidney, and skeletal muscle. Immunofluorescence microscopy mice, P,0.001) and 200 ng DT resulted in complete loss of demonstrated that DTR expression was found only in the Wt1-expressing cells (n=3 mice, P,0.001). 2 2 glomeruli of the iDTR+/ ;Pod-Cre+/ mice, with anti-Wt1 and antinephrin staining confirming co-localization restricted Podocytes Fail to Proliferate after Partial Ablation to podocytes (Figure 1, A and B and data not shown). No Semi-quantitative analysis of Wt1-expressing cells in the 2 DTR expression was detected in any other organ of iDTR+/ ; glomeruli of mice treated with 200 ng DTreveals a progressive 2 2 Pod-Cre+/ mice or in any organ of iDTR+/- or Pod-Cre+/ decline in podocyte numbers beginning after the first day mice. Analysis of DTR immunostaining of multiple kidney (Figure 2A). Transmission electron microscopy (EM) of these 2 2 sections from iDTR+/ ;Pod-Cre+/ mice demonstrated that glomeruli revealed that there are no detectable changes 24 99.5% of Wt1+ podocytes co-expressed the DTR. hours after DT injection (Figure 2B), whereas effacement of

430 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 429–437, 2012 www.jasn.org BASIC RESEARCH

podocyte foot processes was obvious by day 3 (when total podocyte numbers are reduced by 75%). On day 5, retraction of foot processes with microvillous transformation of the podocytes had occurred, leading to podocyte detachment and complete denudation of the GBM by day 7 at which time ,1 podocyte/glomerulus was detectable by Wt1 or DTR staining (Figure 2, A and B). Transmission electron microscopy performed after 25-ng DT injection revealed focal podocyte effacement in .80% of the glomeruli examined on day 7, but surviving podocytes remained at- tached and foot process morphology returned to normal by 4 weeks (Figure 2C). Consistent with the initial loss of foot process integrity after administration of either 25 ng or 200 ng DT,the concentration of albumin in the urine increased to a similar degree in both groups beginning 3– 5 days after DT treatment (Figure 2D). In the mice treated with 200 ng DT, albuminuria persisted until their death on day 9–14, whereas those treated with 25 ng DT exhibited a peak of albuminuria between days 5 and 7 with resolution by days 9 and 10. Normalization of albumin concentration to creatinine revealed equal proteinuria on day 5 with an increase in the normalized urine albumin value on day 7 in the group treated with 200 ng DT due to a marked decrease in urinary creatinine concentration that correlated with the acute renal failure and oliguria present at that time point (Supplemental Figure 1). To determine if resolution of foot process effacement in the group treated with 25 ng DT was due to proliferation of surviving podocytes, mice were given bromo- Figure 2. Reduction of podocyte numbers after DT treatment. (A) Podocyte numbers deoxyuridine (BrdU) daily in their drinking were estimated after Wt1 immunostaining. Treatment with 200 ng DT resulted in a time- water for up to 28 days after DT injection, + , dependent loss of Wt1 podocytes. ***P 0.005 compared with control. n=3 mice for and then sections were stained for BrdU, each group. (B) EM analysis after 200 ng DT injection. (a) Untreated control glomeruli Wt1, and DTR. Neither BrdU+Wt1+ nor demonstrate normal foot processes (arrow). (b) Podocytes retain normal foot processes 1 + + day after DT injection (arrow). (c) Foot process effacement is evident 3 days after DT BrdU DTR cells were detected along the injection (arrow), along with microvillous transformation (arrowhead). (d) GBM is de- GBM (data not shown). Staining for Ki-67 nuded 5 days after injection (arrow) with microvillous transformation of the podocytes also failed to detect any podocyte prolifer- (arrowhead). Original magniﬁcation, 310,000. CL, capillary lumen. (C) (a) EM analysis of ation after DT treatment. Consistent with a control glomerulus showing normal ultrastructure of the foot processes (arrowhead). the absence of detectable podocyte prolif- (b) Focal areas of foot process effacement are evident 7 days after 25 ng DT treatment eration, podocyte numbers fell to a nadir of (arrowhead). (c) Foot process morphology is normal 30 days after 25 ng DT treatment 9.362.4 per glomerular cross-section by (arrowhead). (D) ELISA quantiﬁcation of albumin loss in the urine. *P,0.05, ***P,0.005, 7 days after 25-ng DT injection and then # , and P 0.0001 versus control. (E) Wt1 positive cell numbers were counted per glo- remained constant for the following merular cross-section at the indicated times after 25 ng DT injection. n=44, 7, and 11 for 3 weeks (Figure 2E). The absence of detect- control, day 7, and day 30, respectively. P,0.0001 by one-way ANOVA. able podocyte proliferation or restoration

J Am Soc Nephrol 23: 429–437, 2012 Tubular Proliferation after Podocyte Damage 431 BASIC RESEARCH www.jasn.org of podocyte numbers in adult mice during the 30 days after markedly increased BUN values and animal death by day treatment with 25 ng DTsuggests that podocyte hypertrophy is 10–14 days after treatment (Figure 3B). Mice receiving 50– the primary feature that accounts for the reconstitution of 100 ng DTexhibited an intermediate phenotype with the pres- normal podocyte morphology and foot process function in ence of glomerulosclerosis beginning at 1 week after treatment these animals. and progressively worsening by 4 weeks (Supplemental Figure 2A). Interestingly, DTR+/a-smooth muscle actin+ cells were Tubular Injury and Decline of Renal Function Correlates occasionally found in these crescents (Supplemental Figure with Glomerular Damage 2B). To note, administration of as much as 400 ng/d DT for Light microscopic and functional changes closely mirrored 6 consecutive days had no detectable adverse effect on control 2 these podocyte events. After treatment with 25 ng DT, most mice (wild-type, iDTR+/-,orPod-Cre+/ single transgenic glomeruli were normal at the light microscopic level with mice) as indicated by normal survival, normal histology of rare glomeruli showing focal areas of glomerulosclerosis various organs, absence of albuminuria, and normal renal (Figure 3A and data not shown), and BUN values remained function (data not shown). normal (Figure 3B). However, 200 ng DT led to development Although the concentration of urinary albumin on days 5 of glomerular pseudocysts, crescent formation, collapse of the and 7 was comparable between mice receiving 25 and 200 ng glomerular tuft, and glomerulosclerosis (Figure 3A), with DT (Figure 2D), histologic changes in the tubulointerstitial compartment were markedly different and strongly correlated with those in the glomerulus. The tubules of mice treated with 25 ng DT had preserved cellular archi- tecture with no interstitial changes observed and only occasional proteinaceous casts on day 7 (Figure 3C), with no detectable casts or tubular changes present on day 14. In contrast, tubular damage was extensive on day 7 in those mice treated with 200 ng DT, consisting of marked cast formation, tubular cell flattening. and interstitial damage (Figure 3C). Again, animals receiving a 50-ng dose of DT exhibited a less rapid progression of the tubular changes, but had obvious cast formation, tubular simplification, and interstitial changes that progressively worsened over the 4 weeks after DT (Supplemental Figure 2A). These findings demonstrate that the degree of tubular injury and renal failure correlates closely with the severity of glomerular damage but not with the level of proteinuria.

Podocyte Damage Stimulates Proliferation of Proximal Tubular Cells Although no BrdU+ or Ki67+ podocytes were detected in the glomerular tufts of the DT-treated mice, large numbers of tubular cells were found to be positive for proliferation markers in mice treated with Figure 3. Extensive podocyte ablation leads to glomerulosclerosis and tubular injury. either 25 or 200 ng DT (Figure 4A and data (A) Representative glomerular histology 7 days after control, 25 ng DT injection, or ﬁ 3 not shown). In addition, tubular cells at the 200 ng DT injection. Arrow, crescent formation. Original magni cation, 400. (B) BUN ’ values 1 week after DT treatment. n=13, 7, and 15 for control, 25 ng group, and 200 urinary pole of Bowman s capsule were also ng group, respectively. *P,0.001 versus control. (C) Representative tubular histology found to be proliferating (Figure 4, A, C, 7 days after control, 25 ng DT injection, or 200 ng DT injection. In the 200 ng–treated and D). To further characterize the tubular animals, there is extensive cast formation (asterisk) and epithelial simpliﬁcation proliferation in response to podocyte (arrowhead). ablation, a time course of the proliferative

432 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 429–437, 2012 www.jasn.org BASIC RESEARCH

and resolution (day 8) of albuminuria seen with this dose of DT (Figure 2D). Approx- imately 50% of the proliferating cells 1 week after partial podocyte ablation were in the proximal tubule based on co-staining with megalin, with the remaining proliferating cells found in the interstitial compartment, other tubular segments, and rarely in glomeruli (nonpodocytes) (Figure 4C). Addition of albumin has been shown to induce apoptosis of cultured tubular cells.23 Thus, it was possible that the tubular proliferation occurred in response to albuminuria-induced apoptosis and reduced tubular cell numbers. However, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining at 3, 5, and 7 days after podocyte ablation with 25 ng DT revealed that there was no increase in tubular cell apoptotic rates on days 3 and 5 (i.e., preceding the proliferation observed on days 6–7) and only a modest increase in apoptotic rates on day 7 (Figure 4, D and E). Furthermore, counting the nuclei of brush border positive tubular cells revealed a significant increase of PTC by day 7 (P,0.001) and day 30 (P,0.005) after 25 ng DT compared with baseline (Figure 4F). At 3 months after 25- ng DT injection, a time when albuminuria has completely resolved, tubular cell numbers had returned to normal. Figure 4. Tubular proliferation after low-level podocyte ablation. (A) Ki-67 immunostaining to identify cell proliferation (a-d) double immunostaining (anti-Ki-67 [red] and anti-DTR Activation of Tubular Cell Axl + [green]) to identify proliferating cells after DT treatment. (a) Control (arrows indicate Ki-67 Coincides with Proteinuria-Induced tubular cells). (b) Multiple tubular cells are Ki-67+ 1 week after 25 ng DT treatment. (c and Proliferation d) Two and 4 weeks after 25 ng DT, proliferating tubular cells are still detected in the To determine if glomerular proteinuria re- urinary pole of Bowman’s capsule (arrows). Original magnification, 3400. (B) Quantifica- 2 2 sults in exposure of tubular cells to growth tion of Ki-67+ tubular cells after administration of 25 ng DT to DTR+/ ;Pod-Cre+/ mice. n=4, 3, 6, and 3 for control, day 7, day 14, and day 30, respectively. &P,0.001 versus factors present in the serum, we examined control. (C) Quantification of BrdU-positive cells as well as BrdU+Megalin+ cells after 25 the effect of proteinuric urine on tubular cell ng DT injection. n=4 for no DT group, n=3fordays2,4,6,and8.*P,0.05 versus control. proliferation. Urine collected from mice (D) Terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end 7 days after treatment with 25 ng DT labeling+ (TUNEL) cells from (a) control, (b) 3 days after DT treatment, (c) 5 days after DT significantly stimulated cell proliferation of treatment, and (d) 7 days after 25 ng DT treatment. (E) Quantification of TUNEL+ cells cultured proximal tubular cells, whereas counted from randomly chosen 3400 fields in renal cortex. n=5 each. *P,0.05 versus control urine had no effect (Figure 5A). control. Panels B, C, and E are plotted with the same y-axis scale to present the relative To examine the mechanism of this effect, 3 levels of proliferation versus apoptosis. (F) Counting of proximal tubular cells per 400 we isolated membrane microsomes from fi eld reveals an increase in proximal tubule cells at 7 and 30 days after DT treatment. By day total kidney homogenates of both controls 90, however, proximal tubular cells returned to baseline. n=4,6,4,and5forcontrol,day7, and mice treated with 25 ng DT to deter- day 30, and day 90, respectively. &P,0.001 versus control, ***P,0.005 versus control. mine if they exhibited differential tyrosine kinase receptor activation. Incubation of response was established in mice treated with 25 ng DT. The the microsomes on an array that can detect phosphorylation rate of tubular proliferation was found to increase after day 5, of 39 different receptor tyrosine kinases (RTKs) (mouse RTK peak on days 6–7, and return to baseline after day 8 (Figure 4, B array; R&D Systems, Minneapolis, MN) revealed increased and C). This timing correlates closely with the onset (days 4–5) phosphorylation of the PDGF receptor-a (PDGFR-a) and the

J Am Soc Nephrol 23: 429–437, 2012 Tubular Proliferation after Podocyte Damage 433 BASIC RESEARCH www.jasn.org

(data not shown). However, phospho- Axl levels were reproducibly increased in microsomes isolated from DT-treated kidneys 7 days after DT injection (Figure 5B, quantified in Figure 5C), correlating with the peak of cell proliferation at this time. Immunofluorescence staining of kidney sections from control and DT- treated mice revealed that Axl phosphorylation occurs primarily in tubular cells, with detection of the activated receptor on both the apical and basolateral membranes (Fig- ure 5, D and E). Western blotting of urine samples collected from DT-treated mice detected the presence of the Axl ligand, Gas6, during the proteinuric period between days 4 and 8, but not in control urine (Figure 5F). Con- sistent with the possibility that Gas6 in the proteinuric urine can activate tubular cell Axl, incubation of murine proximal tubular cells (MPT cells) with urine from DT-treated mice induced a 1.8-fold increase in Axl phosphorylation as compared with control, nonproteinuric urine (Figure 5G, quantified in Figure 5H).

DISCUSSION

The experiments performed in this study were designed to determine the glomerular and tubular response to selective podocyte ablation. Analysis of the data led us to three Figure 5. Increased Axl phosphorylation after podocyte damage. (A) MTT assay for conclusions that we believe are pertinent cell proliferation. n=3 each. *P,0.05 versus No FBS control. (B) Western blotting of to our understanding of the pathogenesis microsomal proteins shows increased Axl phosphorylation in DT-treated kidneys. Blot- of CKD after glomerular injury in humans. ting against E-cadherin and Na,K-ATPase was performed to confirm equal loading. First, the glomerulus and the tubules seem (C) Quantification of microsomal blotting results as shown in panel A (pAxl levels to have the ability to adapt to a modest normalized to E-cadherin levels). n=3. *P,0.05. (D) Immunofluorescence staining of , +/2 +/2 loss of podocytes ( 20%) that allows res- kidney sections for pAxl in iDTR ;Pod-Cre under control conditions (con) and 7 olution of the proteinuria without a sub- days after 25 ng DT. Both slides were processed identically. (E) Counting of all pAxl+ sequent loss of surviving podocytes or tubules per cross-section reveals a significant increase after DT treatment compared with untreated control mice. **P,0.01. HPF, high power field using 340 objective progressive glomerular or tubular injury. 2 2 lens. (F) urine was obtained from iDTR+/ ;Pod-Cre+/ mice under control conditions Second, the loss of large numbers of podo- . and 4 and 8 days after treatment with 25 ng DT, and equal protein (5 mg) loaded and cytes ( 40%) exceeds these adaptive re- 2 immunoblotted for Gas6 (predicted molecular mass is 85 kD). (G) Urine from iDTR+/ ; sponses and leads to sustained proteinuria 2 Pod-Cre+/ mice 7 days after DT treatment stimulates Axl phosphorylation. *P,0.05. and progressive glomerulosclerosis. (H) Quantification of Western blot results as shown in G. n=3. **P,0.01. Finally, tubular cell injury in response to podocyte loss correlates with the degree of secondary glomerular injury (crescent Gas6 receptor (Axl) in the microsomes from DT-treated mice formation and glomerulosclerosis), rather than the degree (Supplemental Figure 3). Western blot analysis of microsome of proteinuria. fractions with two separate phospho-specific antibodies In agreement with Wharram et al.,20 who reported a sim- (site 754 and 742) against PDGFR-a failed to confirm acti- ilar model of podocyte ablation in the rat, data from this study vation of this receptor in kidneys from DT-treated mice demonstrate that ablation of ,20% of podocytes results in

434 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 429–437, 2012 www.jasn.org BASIC RESEARCH transient albuminuria followed by resolution. In most epithelial protein that can exist in the free state or complexed with sol- cell populations, this reparative response involves activation of uble Axl (sAxl, the extracellular domain of the Axl receptor). proliferation to increase cell numbers, as is seen in the proximal Binding of Gas6 to full-length Axl on the surface of cells results tubule after ischemia/reperfusion injury.24,25 However, consis- in receptor phosphorylation and activation of anti-apoptotic tent with prior studies in adult animals, we found no evidence and pro-proliferative signaling pathways such as the PI3K cas- for adaptive proliferation of surviving podocytes or a recovery cade.27,28 Consistent with the possibility that urinary Gas6 is of podocyte numbers along the GBM during the 4 weeks after playing a significant role in the tubular cell response to albu- ablation with 25 ng DT. Even though podocyte numbers never minuria, we observed that phosphorylated Axl (pAxl) is pres- recovered after partial ablation, the morphology of the podo- ent at the apical membrane of proximal tubular cells from cytes returned to normal over several weeks and albuminuria proteinuric kidneys. Interestingly, pAxl is also present at the resolved. This suggests that the early adaptive response to par- basolateral membrane in these kidneys. It is unclear whether tial podocyte loss may involve hypertrophy of surviving podo- this is due to increased local production of Gas6 in the renal cytes to cover a greater area of GBM. interstitium in response to the proteinuria or due to megalin- The second conclusion is that loss of a larger number of dependent transcytosis of the intact ligand.29,30 This second podocytes (.40% in our study) seems to exceed the capacity possibility is of particular interest in light of the observation by for glomerular adaptation and results in progressive glomer- Theilig et al. that proteinuria causes tubular cell proliferation ular damage. In this setting, some surviving podocytes lose in a megalin-dependent fashion.31 Wt1 expression and contribute to the formation of cellular In conclusion, the iDTR+;Pod-Cre+ mouse provides an ex- crescents. We found occasional examples of cells within cres- cellent model to study the effects of selective podocyte loss in cents that were positive for BrdU or Ki-67 and continued to the pathogenesis of glomerular injury. Our data indicate that express DTR even though they had lost Wt1 expression. Al- selective loss of a small number of podocytes leads to transient though we found no evidence that these DTR+ cells ever re- proteinuria with subsequent resolution despite the absence of express Wt1 or contribute to the podocyte population along podocyte proliferation. The proteinuria itself seems to stimu- the GBM, these data suggest that podocytes are not truly post- late proliferation of tubular cells without evidence of tubule mitotic, rather they are prevented from proliferating due to injury, scarring, or loss of kidney function. However, more some aspect of their microenvironment. substantial loss of podocytes results in scarring and crescent The third finding is that albuminuria due to podocyte loss formation, coinciding with severe tubular injury and kidney without persistent structural glomerular damage did not lead failure. More extensive studies are required to determine the to significant tubular injury or progressive renal dysfunction exact mechanism of tubule proliferation and to ascertain within the timeframe of our study. This is consistent with data whether the increase in cell numbers provides an adaptive from patients with minimal change disease, in whom massive advantage by increasing the number of cells available for re- albuminuria occurs after foot process fusion yet tubular cells absorption of abnormally filtered proteins and other small remain structurally intact unless ensuing hypotension leads to molecules. acute tubular necrosis or the glomeruli develop the structural changes of focal glomerulosclerosis. Because the longest duration of tubule exposure to albu- CONCISE METHODS minuria was approximately 5 days in our 25-ng DT ablation model, we cannot rule out the possibility that sustained Mouse Care and Genotyping albuminuria is eventually problematic for tubular cell sur- Mouse protocols were approved by the Yale University Institutional vival.However,wecanclearlysaythatevenabriefperiod Animal Care and Use Committee. The iDTR mouse was kindly pro- of glomerulosclerosis/crescent formation (group treated with vided by Dr. Waisman (Mainz, Germany)18 and the Podocin-Cre from 200 ng DT) leads to severe tubular damage that is not seen with Dr. Lawrence Holzman (University of Pennsylvania).19 The iDTR mice albuminuria alone. This observation is consistent with the were genotyped by PCR using primers 59-gaccatgaagctgctgccgtc-39 findings of Kriz and LeHir, who showed that tubular injury in (DTR sense) and 59-tcagtgggaattagtcatgc-39 (DTR antisense). The experimental proteinuric diseases correlates with the anatomic Podocin-Cre mice were genotyped by PCR using primers 59-acagctc- finding of occlusion of urine passage from Bowman’s space into caccaagacacag-39 (human podocin promoter, sense) and 59-tccggttatt- the proximal tubule.26 caacttgcacc-39 (Cre, antisense). On the basis of our in vitro and in vivo findings, it is our belief that serum factors other than albumin that are present in DT Treatment the proteinuric glomerular ultrafiltrate induce the observed DTwas dissolved in sterile 13PBS at 50 mg/ml. The stock solution tubule cell proliferation. There are multiple candidate factors was then aliquoted and frozen at 280°C until use. DT solution was present in serum that are known to have a proliferative effect thawed and diluted to 100 ng/ml, 250 ng/ml, 500 ng/ml, or 1000 ng/ml on tubular cells, including Hgf, Egf, Igf1, and Vegf. On the for intraperitoneal injections. To test the dose-response curve of DT basis of our non-biased approach, an additional mitogen, treatment, both male and female double transgenic mice aged 4–5 Gas6, is now implicated in this response. Gas6 is a secreted weeks were used (18–20 g body wt).

J Am Soc Nephrol 23: 429–437, 2012 Tubular Proliferation after Podocyte Damage 435 BASIC RESEARCH www.jasn.org

BrdU Administration anti-E-cadherin (Cell Signaling Technology, Inc), anti-phospho-Axl BrdU was first dissolved in DMSO at 50 mg/ml. Aliquots were frozen at (US Biological), and anti-Gas6 (R&D Systems). Quantification of 220°C till use. BrdU was administered in drinking water at 200 mg/ml Western blot results was done using Image J software. unless otherwise stated. BrdU water was changed every 3 days. In Vitro Activation of Axl Urine Analysis and BUN Quantification MPT cells were serum starved overnight, washed, and then incubated in fi Urine albumin levels were quanti ed by ELISA using a kit purchased 15% urine (dilutedin serum-free culture medium) fromeitheruntreated ’ 2 2 from Bethyl Laboratories following the manufacturer s protocol. Serum control iDTR+/ ;Pod-Cre+/ mice or proteinuric urine on day 7 after BUN values were performed by the Mouse Metabolic Phenotyping treatment with 25 ng DT. Cells were lysed after 2 hours, proteins sepa- Center at Yale University using the COBAS Integra system (Roche, rated by SDS-PAGE, and immunoblotted following standard protocols. Indianapolis, IN). MTT Assay Podocyte and Proximal Tubular Cell Quantification Cell proliferation was quantified following the modified 3-(4,5- Podocyte numbers were estimated by counting the number of Wt1- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) positive cells per glomerular cross-section in 20 randomly selected method based on the protocol described by the Wallert and Provost.35 glomeruli/kidney as described by Shibata et al.32 The mean of these Briefly, MTT was dissolved in 13PBS at 4 mg/ml and filter sterilized counts thus represents the average number of podocytes per glomerular before use. Proximal tubular cells of approximately 75% confluence cross-section, rather than the total number of podocytes per glomerulus. cultured in six-well plates were serum starved for 24 hours and then Tocount proximal tubular cells, slides were stained by periodic acid– supplied with serum-free medium versus serum-free medium supple- Schiff (PAS) and counterstained with hematoxylin. At least 10 random mented with 15% normal urine or proteinuric urine. Eight hours later, fields from the renal cortex were imaged with one glomerulus in the MTTsolution was added to each well and incubated for 3.5 hours before center of the picture. Image J software was used to count nuclei of pink being terminated for spectrophotometer reading at 590 nm. Experiments (proximal tubular brush border stained by PAS) cells per image. An were carried out in triplicate for each condition and repeated twice. average of 10 images was used foreach mouse (n=5 mice for each group).

Histology and Fluorescent Immunostaining Statistical Analyses After sacrifice, kidneys were gently sliced into two pieces, fixed in Data were presented at mean 6 SD throughout the text unless other- 13PBS buffered formalin for 12 hours, embedded in paraffin, and wise specified. Data were analyzed using either the t test or ANOVA sectioned at 3 mm. To review histology, slides were stained by PAS. where appropriate, with P,0.05 regarded as statistically significant. Immunostaining on paraffin embedded tissue was performed as de- scribed33 using rabbit polyclonal antibody against Wt1 (C-19; Santa Cruz Biotechnology, CA), rabbit anti-Ki-67 (Abcam, Cambridge, ACKNOWLEDGMENTS MA), rabbit anti-nephrin (kindly provided by Dr. Lawrence Holzman, University of Pennsylvania), phosphor-Axl (Y779; US Bio- We thank Dr. Ali Waisman (Mainz, Germany) for providing the iDTR logics), and goat anti-DTR (R&D Systems). mouse and Dr. Lawrence Holzman (University of Pennsylvania) for Microsome Preparation providing the podocin-Cre mouse and anti-nephrin antibody. The Total kidneys were minced into small pieces before being homog- Yale Mouse Metabolic Phenotyping Center performed the BUN and enized in homogenization buffer (13 PBS supplemented with creatinine measurements. phosphatase inhibitors [50 mM sodium fluoride, 15 mM sodium py- This study was supported by the following grants from the rophosphate]) and protease inhibitors (Roche, Indianapolis, IN) National Institute of Diabetes and Digestive and Kidney Diseases using a Potter-Elvehjem Teflon tissue grinder with a loose-fitting Teflon of the National Institutes of Health: U24 DK76169 (Yale Mouse pestle as described.34 The membrane fraction was collected from the Metabolic Phenotyping Center); DK075464 (J.K.G.); HL073742, supernatant after 2000 3 g centrifugation for 10 minutes. Microsomes DK061846, and DK072442 (D.S.K.); and DK065109 (L.G.C.). A.S., an were concentrated by centrifugation at 100,000 3 g for 1 hour. The undergraduate student from Brown University, was supported by a microsome pellet was then resuspended in 500 ml homogenization summer student program from the George M. O’Brien Kidney Center buffer and stored at 280°C until use. at Yale University (NIH/NIDDK P30DK079310).

Protein Array of Phosphorylated RTKs The mouse RTK array kit (catalog #ARY014; R&D Systems) was used DISCLOSURES to screen for kinases being activated in DT-treated kidneys following None. the manufacturer’s instructions. A total of four arrays with four different samples (two control and two podocyte damaged) were used. REFERENCES Western Blotting and Quantiﬁcation Western blotting was performed following standard protocols. The 1. Tryggvason K, Wartiovaara J: How does the kidney ﬁlter plasma? following antibodies were used: anti-phospho-tyrosine kinase and Physiology (Bethesda) 20: 96–101, 2005

436 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 429–437, 2012 www.jasn.org BASIC RESEARCH

2. Faul C, Asanuma K, Yanagida-Asanuma E, Kim K, Mundel P: Actin up: depletion in rats expressing human diphtheria toxin receptor transgene. Regulation of podocyte structure and function by components of the J Am Soc Nephrol 16: 2941–2952, 2005 actin cytoskeleton. Trends Cell Biol 17: 428–437, 2007 21. Yamaizumi M, Mekada E, Uchida T, Okada Y: One molecule of diph- 3. Wiggins RC: The spectrum of podocytopathies: A unifying view of theria toxin fragment A introduced into a cell can kill the cell. Cell 15: glomerular diseases. Kidney Int 71: 1205–1214, 2007 245–250, 1978 4. Gubler MC: Podocyte differentiation and hereditary proteinuria/ 22. Yamaizumi M, Uchida T, Takamatsu K, Okada Y: Intracellular stability nephrotic syndromes. J Am Soc Nephrol 14[Suppl 1]: S22–S26, 2003 of diphtheria toxin fragment A in the presence and absence of anti- 5. Johnstone DB, Holzman LB: Clinical impact of research on the podo- fragment A antibody. Proc Natl Acad Sci USA 79: 461–465, 1982 cyte slit diaphragm. Nat Clin Pract Nephrol 2: 271–282, 2006 23. Erkan E, Devarajan P, Schwartz GJ: Mitochondria are the major targets 6. LeHir M, Kriz W: New insights into structural patterns encountered in albumin-induced apoptosis in proximal tubule cells. JAmSoc in glomerulosclerosis. Curr Opin Nephrol Hypertens 16: 184–191, Nephrol 18: 1199–1208, 2007 2007 24. Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, 7. Nosadini R: Hypertension and renal complications in type 2 diabetes. Bonventre JV: Restoration of tubular epithelial cells during repair of the Semin Vasc Med 2: 109–119, 2002 postischemic kidney occurs independently of bone marrow-derived 8. Shah SN, He CJ, Klotman P: Update on HIV-associated nephropathy. stem cells. JClinInvest115: 1743–1755, 2005 Curr Opin Nephrol Hypertens 15: 450–455, 2006 25. Lin F, Moran A, Igarashi P: Intrarenal cells, not bone marrow-derived 9. Fogo AB: Animal models of FSGS: lessons for pathogenesis and cells, are the major source for regeneration in postischemic kidney, treatment. Semin Nephrol 23: 161–171, 2003 JClinInvest115: 1756–1764, 2005 10. Lu TC, He JC, Klotman P: Animal models of HIV-associated nephrop- 26. Kriz W, LeHir M: Pathways to nephron loss starting from glomerular athy. Curr Opin Nephrol Hypertens 15: 233–237, 2006 diseases-insights from animal models. Kidney Int 67: 404–419, 2005 11. Erkan E, Devarajan P, Schwartz GJ: Apoptotic response to albumin 27. Goruppi S, Ruaro E, Varnum B, Schneider C: Requirement of phos- overload: Proximal vs. distal/collecting tubule cells. Am J Nephrol 25: phatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl 121–131, 2005 mitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol 17: 12. Donadelli R, Zanchi C, Morigi M, Buelli S, Batani C, Tomasoni S, Corna 4442–4453, 1997 D, Rottoli D, Benigni A, Abbate M, Remuzzi G, Zoja C: Protein overload 28. Goruppi S, Ruaro E, Varnum B, Schneider C: Gas6-mediated survival in induces fractalkine upregulation in proximal tubular cells through NIH3T3 cells activates stress signalling cascade and is independent of nuclear factor kappaB- and p38 mitogen-activated protein kinase- Ras. Oncogene 18: 4224–4236, 1999 dependent pathways. JAmSocNephrol14: 2436–2446, 2003 29. Pohl M, Kaminski H, Castrop H, Bader M, Himmerkus N, Bleich M, 13. Shalamanova L, McArdle F, Amara AB, Jackson MJ, Rustom R: Albumin Bachmann S, Theilig F: Intrarenal renin angiotensin system revisited: overload induces adaptive responses in human proximal tubular cells role of megalin-dependent endocytosis along the proximal nephron. through oxidative stress but not via angiotensin II type 1 receptor. Am J Biol Chem 285: 41935–41946, 2010 J Physiol Renal Physiol 292: F1846–F1857, 2007 30. Marinó M, Andrews D, Brown D, McCluskey RT: Transcytosis of retinol- 14. Ohse T, Inagi R, Tanaka T, Ota T, Miyata T, Kojima I, Ingelfinger JR, binding protein across renal proximal tubule cells after megalin Ogawa S, Fujita T, Nangaku M: Albumin induces endoplasmic re- (gp 330)-mediated endocytosis. J Am Soc Nephrol 12: 637–648, 2001 ticulum stress and apoptosis in renal proximal tubular cells. Kidney Int 31. Theilig F, Kriz W, Jerichow T, Schrade P, Hähnel B, Willnow T, Le Hir M, 70: 1447–1455, 2006 Bachmann S: Abrogation of protein uptake through megalin-deficient 15. Caruso-Neves C, Pinheiro AA, Cai H, Souza-Menezes J, Guggino WB: proximal tubules does not safeguard against tubulointerstitial injury. PKB and megalin determine the survival or death of renal proximal JAmSocNephrol18: 1824–1834, 2007 tubule cells. Proc Natl Acad Sci USA 103: 18810–18815, 2006 32. Shibata S, Nagase M, Yoshida S, Kawarazaki W, Kurihara H, Tanaka H, 16. Iglesias J, Abernethy VE, Wang Z, Lieberthal W, Koh JS, Levine JS: Miyoshi J, Takai Y, Fujita T: Modification of mineralocorticoid receptor Albumin is a major serum survival factor for renal tubular cells and function by Rac1 GTPase: implication in proteinuric kidney disease. Nat macrophages through scavenging of ROS. Am J Physiol 277: F711– Med 14: 1370–1376, 2008 F722, 1999 33. Charles AK, Mall S, Watson J, Berry PJ: Expression of the Wilms’ tumour 17. Iglesias J, Levine JS: Albuminuria and renal injury—Beware of proteins gene WT1 in the developing human and in paediatric renal tumours: an bearing gifts. Nephrol Dial Transplant 16: 215–218, 2001 immunohistochemical study. Mol Pathol 50: 138–144, 1997 18. Buch T, Heppner FL, Tertilt C, Heinen TJ, Kremer M, Wunderlich FT, 34. Biemesderfer D, DeGray B, Aronson PS: Active (9.6 s) and inactive (21 s) Jung S, Waisman A: A Cre-inducible diphtheria toxin receptor mediates oligomers of NHE3 in microdomains of the renal brush border. JBiol cell lineage ablation after toxin administration. Nat Methods 2: 419– Chem 276: 10161–10167, 2001 426, 2005 35. Provost J, Wallert M: MTT Proliferation Protocol, Moorehead, MN, 19. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB: Podocyte- Minnesota State University, 2007 specific expression of cre recombinase in transgenic mice. Genesis 35: 39–42, 2003 20. Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, Saunders TL, Dysko RC, Kohno K, Holzman LB, Wiggins RC: Podocyte This article contains supplemental material online at http://jasn.asnjournals. depletion causes glomerulosclerosis: Diphtheria toxin-induced podocyte org/lookup/suppl/doi:10.1681/ASN.2011040396/-/DCSupplemental.

J Am Soc Nephrol 23: 429–437, 2012 Tubular Proliferation after Podocyte Damage 437 100 * 90

30 ** ***

20 *

Albumin (mg/dl) noralized to creatinine (mg/dl) creatinine to (mg/dl) noralized Albumin 1 3 5 7 9 1 3 5 7 11 13 days after DT control 25 ng DT 200 ng DT

Figure S1. A con 50ng DT, 1 wk 50ng DT, 4 wk a b c

d e f

B control 100 ng DT *

SMA + DTR + DAPI

Figure S2 control

6 min exposure pPDGFRa DT treated

control

pAxl

30 min exposure DT treated

Figure S3. Supplemental Figures

Figure S1. Normalized albumin loss in the urine. Y-axis, urinary albumin concentration (mg/dl) normalized to creatinine (mg/dl). Mice on day 7 after 200ng DT displayed oliguric renal failure with a marked decrease in urine creatinine (14+/-4 mg/dl as compared to 56+/-12 mg/dl on day 5) and resultant increase in the urine albumin/creatinine ratio. *p<0.05; **p<0.01; ***p<0.001 (vs. control)

Figure S2. Glomerulosclerosis, crescent formation and tubular injury in mice with

>40% Podocyte Loss.

A. 50ng DT is sufficient to trigger progressive glomerular damage. a, untreated control; b,

1 week after a single dose of 50ng injection; c, 4 weeks after a single dose of 50ng DT administration. d, e, and f are higher magnifications of the boxed glomeruli in a, b, and c, respectively, showing glomerulosclerosis (arrow).

B. Anti-αSMA immunostaining is only detected at the vascular pole (asterisk) in the control glomerulus and DTR staining is restricted to normal podocytes (arrow, left panel), while wide spread αSMA signal is present in the fibrocellular crescent 4 weeks after 100 ng DT treatment. Note αSMA+/DTR+ cells are found in the crescent outside of the glomerular tuft (arrow), indicating that podocytes contributed to crescent formation.

Figure S3. RTK array identified two receptor kinases that were differentially phosphorylated after DT treatment (pPDGFRα at short exposure and pAxl at longer

1 exposure). 200ug microsomal protein was loaded to each filter, two controls and two treated samples were processed side by side. Only one pair of the array is shown.