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Inhibition of MTOR Disrupts Autophagic Flux in Podocytes

Davide P. Cinà,* Tuncer Onay,* Aarti Paltoo,* Chengjin Li,* Yoshiro Maezawa,* † ‡ Javier De Arteaga, Andrea Jurisicova,* and Susan E. Quaggin* §

*Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada; †Catholic University, Cordoba, Argentina; ‡Department of Medicine University Health Network, University of Toronto, Toronto, Ontario, Canada; and §Division of Nephrology, St. Michael’s Hospital, University of Toronto, Toronto, Canada

ABSTRACT Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs In one prospective trial, patients trea- with potent immunosuppressive, antiangiogenic, and antiproliferative properties. ted with or switched to siroli- De novo or worsening proteinuria can occur during treatment with these agents, mus from CNIs had comparable rates of but the mechanism by which this occurs is unknown. We generated and character- biopsy-confirmed acute allograft rejec- ized mice carrying a podocyte-selective knockout of the Mtor . Although Mtor tion and 2-year graft survival to those was dispensable in developing podocytes, these mice developed proteinuria at 3 treated with CNIs.7 In addition, siroli- weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice mus-treated patients had fewer malig- exhibited an accumulation of the autophagosome marker LC3 (rat microtubule- nancies and a better estimated GFR associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, (eGFR) at 24 months if their baseline and damaged mitochondria. Similarly, human podocytes treated with the MTOR eGFR was .40 ml/min. Because of their inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. antiproliferative and antiangiogenic ef- Taken together, these results suggest that disruption of the autophagic pathway fects, indications for MTOR inhibitors may play a role in the pathogenesis of proteinuria in patients treated with MTOR have expanded to include treatment of inhibitors. various cancers such as renal cell carci- noma, nonmalignant conditions such J Am Soc Nephrol 23: 412–420, 2012. doi: 10.1681/ASN.2011070690 as autosomal dominant polycystic kid- ney disease (AD-PKD), and primary glomerulopathies.8–12 The mammalian target of rapamycin and other MTOR inhibitors associate Despite its potential advantages in (MTOR) is an evolutionarily conserved with the FKBP12 protein, and together the transplant setting, evidence that serine-threonine kinase that interacts they directly bind MTOR to prevent the sirolimus causes de novo or worsening with regulatory associated protein of RPTOR-MTOR interaction and thus in- proteinuria is unequivocal. In one ran- MTOR (Rptor) or Rptor independent hibit mTORC1 function.3 In certain cell domized clinical trial in which patients companion of MTOR (Rictor) to form types, including the podocyte, chronic with AD-PKD received sirolimus or pla- mTORC1 and mTORC2 complexes, inhibition of MTOR by rapamycin also cebo, the group receiving an MTOR in- respectively. In turn, mTORC1 and results in downregulation of mTORC2 hibitor had a significantly higher median mTORC2 regulate different aspects of functions.4–6 Although this mechanism of MTOR function. mTORC1 is a key reg- action has not been completely elucidated, ulator of cellular metabolism, including data in podocytes suggest that prolonged Received July 15, 2011. Accepted October 8, 2011. protein , ribosomal biogene- rapamycin treatment directly downregu- D.P.C. and T.O. contributed equally to this work. sis, cell growth and proliferation, and lates MTOR and Rictor, both of which 6 Published online ahead of print. Publication date suppression of in response to are required for mTORC2 function. available at www.jasn.org. amino acids, growth factors, and ele- Sirolimus (rapamycin) was originally vated cellular ATP levels.1 mTORC2 is proposed as an immunosuppressant to Correspondence: Dr. Susan E. Quaggin, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, regulated primarily by growth factors prevent rejection of solid organ trans- 60 Murray Street, Box #41, Toronto, Ontario, M5T to promote actin cytoskeletal rearrange- plants. There were expectations that 3L9 Canada. Email: [email protected] ment, cell survival, and cell cycle progres- MTOR inhibitors would replace neph- Copyright © 2012 by the American Society of sion.2 In mammalian cells, rapamycin rotoxic calcineurin inhibitors (CNIs). Nephrology

412 ISSN : 1046-6673/2303-412 J Am Soc Nephrol 23: 412–420, 2012 www.jasn.org BRIEF COMMUNICATION urine protein/creatinine ratio.9 Simi- Given the well recognized proteinuric of age, suggesting that Mtor is dispens- larly, in a recent open-label randomized effect of MTOR inhibitors, we were in- able in developing podocytes. By 4 clinical trial in which 503 renal trans- terested in understanding its mecha- weeks of age, however, whole animal plant patients were randomized to an nism. To explore the role of MTOR in growth restriction was evident in Mtor everolimus-based CNI-free regime or vivo, we developed a mouse model with pod-KO mice (11.54 g [Mtor pod-KO] standard CNI therapy, those taking a podocyte-selective deletion of the versus 18.13 g [control]; P=0.03) (Figure everolimus had a significantly higher Mtor gene (Mtor pod-KO). 2, A and B). The protein/creatinine ratio 24-hour urine protein excretion.13 Al- A conditional Mtor allele was gener- was significantly increased in 3-week- though subnephrotic increases in pro- ated using BAC recombineering to old mutants (22.32 mg/mg for Mtor teinuria may result from glomerular or place loxP sites around the first three pod-KO mice) compared with controls tubular injury, the small incidence of exons of the Mtor gene (Figure 1A). (7.117 mg/mg) (P=0.025) (Figure 2C). reportedcasesofpatientsdeveloping The successful generation of a floxed Glomeruli from 2- to 3-week-old mu- full-blown nephrotic syndrome after Mtor allele was confirmed by Southern tant mice were histologically similar to treatment with rapamycin14 suggests blot (Figure 1B). In this mouse, Cre- controls but proteinaceous casts were that the glomerular filtration barrier is mediated excision results in a null Mtor observed in the tubules of mutant mice affected, at least in this subset of pa- allele, abolishing function of both (Figure 2D). However, by 4 weeks of age, tients. mTORC1 and mTORC2 complexes. Be- glomeruliinmutantmiceshoweddra- Several in vitro and patient biopsy cause chronic use of mTOR inhibitors matic pathologic changes with areas of studies have addressed a role for MTOR downregulates both mTORC1 and sclerosis and numerous vacuolated po- in the glomerulus. One group described mTORC2 functions, we chose to delete docytes (Figure 2D). By 5 weeks of age, thrombotic microangiopathic glomeru- the Mtor gene—as opposed to its part- complete destruction of the glomerular lar lesions in renal biopsies from five ners Rptor or Rictor—to obtain the most tuft was seen, with distended Bowman’s patients who developed proteinuria when complete knockdown and simulate the space and widespread vacuolization of treated with sirolimus.15 These lesions clinical effects of MTOR inhibitors. podocytes (Figure 2E). These histologic were associated with downregulation To confirm functional deletion of the findings are similar to those reported in fl fl of vascular endothelial growth factor Mtor gene, we bred the Mtor ox/ ox mice lacking both mTorc1 and mTorc2 A (VEGFA) expression in podocytes, a mouse to a pCaggsCre-driver strain in their podocytes (Rptor/Rictor pod- molecular mechanism that has been as- that deletes the gene at the one cell em- KO).19 However, unlike Rptor/Rictor sociated with thrombotic microangiop- bryo stage, giving a germline deletion of pod-KO, the phenotype in Mtor pod- athy in patients with pre-eclampsia16 Mtor (Mtor del) (Figure 1C). Heterozygous KO mice is fully penetrant and strain and in those treated with anti-VEGFA Mtorwt/del mice appear healthy but have independent, which is likely due to a agents.17 Another small case series de- reduced Mtor protein levels (Supple- more complete excision and knockdown scribes three instances of de novo FSGS mental Figure 1). Homozygous Mtor del/del of gene function. in patients treated with sirolimus, char- mice die in utero, similar to the conven- Electron micrographic (EM) studies acterized by focal loss of PAX2, synapto- tional knockout (Figure 1D).20,21 In all showed focal effacement of foot pro- podin, and VEGFA.14 Although not all subsequent breeding, one Mtordel allele cesses in mutant mice beginning at 3 patients with proteinuria who take was utilized to enhance the degree of weeks of age although the endothelium sirolimus have a distinct glomerular le- Cre-mediated excision in the podocyte. and glomerular basement membranes sion, Stallone et al. performed a biopsy To generate an Mtor pod-KO, Podocin- were preserved (Figure 2F). Nonetheless, study showing that sirolimus treatment Cremicewerebredtomicecarrying by 5 weeks of age, profound structural fl was associated with decreased expression one conditional allele (Mtor ox) and one changes were observed in podocytes, en- of synaptopodin, podocin, CD2AP, and deleted (Mtordel) allele (Figure 1C). Glo- dothelial and mesangial compartments, nephrininpodocytes.18 Cell culture merular deletion of Mtor was verified by consistent with light microscopic find- studies support the results of these analysis of genomic DNA isolated from ings (not shown). biopsy studies and further suggest a the renal cortex and identification of Despite the presence of foot process role for MTOR in regulating actin and the deleted band (Figure 1E). Further effacement, previous studies indicate slit-diaphragm–associated proteins in confirmation was obtained by Western that this is unlikely to be the primary the podocyte.6 Finally, genetic deletion blot analysis of glomerular protein show- mechanism of glomerular injury because of Rptor alone or both Rictor and Rptor ing downregulation of Mtor (Figure 1F). other transgenic mouse models with from podocytes results in glomerular Mice of all genotypes were born in expec- even more marked foot process defects injury in mice by an unknown mech- ted Mendelian frequency (Supplemental do not exhibit such a dramatic disease anism.19 These data suggest that inhi- Table 1) and mutants appeared as course or the vacuolated podocyte phe- bition of MTOR signaling within the healthy as wild-type or mixed genotype notype. For example, mice lacking the podocyte may play a complex role to controls at birth. Interestingly, no overt adaptor proteins, Nck1/2, in their podo- promote proteinuria in patients. phenotype was observed until 3 weeks cytes do not form any foot processes.

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Figure 1. Generation of podocyte-selective knockout of the Mtor gene. (A) BAC recombineering construct. LoxP sites (triangles) are inserted around the first three exons of the Mtor gene. The neomycin selection cassette is used to select for positive embryonic stem cell clones, then removed by FLPe recombinase-mediated excision. Cre-mediated excision results in a null Mtor allele. (B) Southern blot analysis confirms correctly targeted alleles. (C) A null Mtor allele (Mtordel) is generated by breeding floxed mice to a pCaggs-Cre driver strain that deletes at the one cell stage (germline deletion). A podocyte-selective knockout is generated by breeding Mtorflox/del mice to 2 2 a Podocin-Cre driver strain. (D) Table showing correlation between genotype and survival. As predicted, Mtordel/del (Mtor / )micedie in utero. (E) Southern blot analysis of renal cortical genomic DNA confirms the presence of a deleted Mtor allele in Mtor pod-KO mice. (F) Western blot analysis of isolated glomerular protein confirms downregulation of Mtor protein in the glomeruli from Mtor pod-KO mice compared with Mtorwt/flox mice. Each lane contains glomeruli pooled from four mice. Wt, wild-type allele; Tg, transgenic Mtorflox allele.

Although they are born with severe pro- expression was decreased at 3 weeks in Given the observations of decreased teinuria, their renal injury progresses glomeruli of Mtor pod-KO mice (Sup- VEGFA levels in renal biopsies from more slowly than that seen in Mtor plemental Figure 2A). However, Wt1 patients treated with MTOR inhibitors pod-KO mice.22 In keeping with struc- and Nphs1 expression were unaffected at and regulation of VEGFA by mTORC1 tural changes observed by EM, podocin this time point (Supplemental Figure 2B). through upregulation of hypoxia-inducible

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Figure 2. Disease course and glomerular histology in Mtor pod-KO mice. (A) Mtor pod-KO mice appear smaller than their control lit- termates at 3 weeks of age. (B) Mtor pod-KO mice are significantly growth restricted by 4 weeks of age (*P,0.01; **P,0.05). (C) Protein/ creatinine ratios are significantly increased by 3 weeks of age in Mtor pod-KO mice compared with wild-type (*P,0.01; **P,0.05). (D) At 3 weeks of age, glomeruli look similar in mutants and controls although proteinaceous casts are visible in tubules (periodic acid–Schiff stain, magnification 340). By 4 weeks of age, glomerular scarring and vacuolization of podocytes are evident. (E) By 5 weeks of age, glomeruli are end stage with widespread vacuolization of podocytes. (F) Electron micrographs from control and mutant glomeruli at 3 weeks of age show focal foot process effacement in mutants compared with controls. The endothelium and glomerular basement membrane appear largely intact. Scale bars represent 1 mm. (G) Electron micrographs of glomeruli from 2-week-old and 3-week-old Mtor pod-KO mice show frequent double-membraned cytoplasmic vesicles characteristic of autophagosomes. Scale bar in the top left panel represents 500 nm. All other scale bars represent 1 mm. (H) Isolated glomeruli from Mtor pod-KO mice show increased levels of LC3-II compared with controls. AV, autophagosomal vesicles; ALV, autophagolysosomal vesicles.

J Am Soc Nephrol 23: 412–420, 2012 Mtor Inhibition Disrupts Autophagic Flux in Podocytes 415 BRIEF COMMUNICATION www.jasn.org factors,15,23 we next examined whether as autophagosomal vesicles (AV), which autophagy through inhibition of MTOR, reduced Vegfa in podocytes might be can be seen on electron micrographs.29 continued starvation, paradoxically, driving the disease in Mtor pod-KO Importantly, AVs were readily observed leads to upregulation of MTOR activity, mice. In our model, Veg fa levels were in podocytes from Mtor pod-KO mice establishing a negative feedback loop similar in mutants and controls at the (Figure 2G) beginning at 2 weeks of age that is important for autolysosomal ves- onset of disease (between 2 and 3 weeks but were not seen in controls. To quanti- icle recycling and cell survival (Figure of age). However, by 3 weeks of age, Veg fa tate the number of AVs, we isolated pro- 3A). In the setting of rapamycin or a levels were visibly reduced but not absent tein from the glomeruli of wild-type or knockout of the Mtor gene, this negative (Supplemental Figure 1C). Despite this Mtor pod-KO mice. LC3 (rat microtu- feedback loop is blocked and autophagic finding, a primary reduction of Veg fa bule-associated protein 1 light chain 3) vesicles will accumulate, will fail to re- alone in podocytes results in a very dif- is the mammalian homolog of the es- cycle, and the process will arrest, result- ferent phenotype and disease course in sential yeast autophagy protein Atg8 ing in toxicity to the cell. On the basis mice, including marked endothelial and is the most commonly used molec- of these data, we posit that disruption swelling with mesangiolysis,17,24 and, in ular marker of the autophagosome. of both steps of the autophagic cycle the case of developmental deletion, com- There are two LC3 isoforms generated contribute to the development of dra- plete loss of the glomerular endothelium. by post-translational modification: matic glomerular injury observed in Similarly, reduction of Veg fa by approxi- LC3-I and LC3-II. LC3-I localizes to the Mtor pod-KO mice. mately 75% of normal levels results in cytoplasmic compartment, whereas We tested this model in human po- dramatic mesangiolysis by 3 weeks of LC3-II localizes to the autophagosome docytes treated with rapamycin. Human age.25 None of these pathologic findings membrane as well as the cytoplasm. podocytes were stably transfected with were observed in Mtor pod-KO mice. LC3-II is increased in glomeruli isolated an LC3-GFP construct. After exposure to If primary cytoskeletal defects and from mutant animals (Figure 2H) con- rapamycin, accumulation of fluorescent Vegfa deficiency are unlikely to be the sistent with an increase in AVs. ringed structures was observed (Supple- initiating events of the glomerular injury After engulfment of the intracellular mental Movie 1 and Figure 4A) consis- in Mtor pod-KO mice, what explains the targets, the AV fuses with lysosomes to tent with an increased number of AVs dramatic phenotype? MTOR is known generate the autophagolysosomal vesicle and enhanced level of autophagy. West- to regulate and inhibit many cellular pro- (ALV), permitting degradation of intra- ernblotanalysisconfirmed an upregula- cesses, including autophagy (reviewed by cellular organelles. The process is suc- tion of the AV-associated isoform LC3-II Zoncu et al.1). Autophagy is a lysosomal- cessfully terminated after reformation of in the rapamycin-treated human podo- dependent cellular survival response to the autophagosome and lysosomes from cytes. These findings were associated starvation or lack of growth factors in the ALV (Figure 3A). Failure to regener- with coincident inhibition of phosphory- which cells degrade cellular constituents ate these components will disrupt the lated S6K, a marker of mTORC1 activity from proteins to entire organelles, such autophagic cycle, with consequent accu- (Figure 4B). as mitochondria, to provide a supply of mulation of acidophilic ALVs and dam- Todetermine whether ALVsaccumulate nutrients under conditions of stress. A aged organelles such as mitochondria, after MTOR inhibition (i.e., arrest of the basal level of autophagy is, however, nec- ultimately leading to cell death. A recent autophagic cycle), we studied distribution essary to remove damaged organelles, cell culture study showed that MTOR of LC3-GFP puncta and Lysotracker, excessive lipids, and long-lived or mis- plays a role in both phases of the auto- a lysosomal marker. After 5 days of expo- folded proteins. Terminally differenti- phagic cycle and that inhibition of sure to rapamycin, there was almost ated, nondividing cells such as neurons MTOR therefore has two distinct effects complete co-localization of LC3-GFP have an elevated basal level of autoph- on the process. First, MTOR inhibition and acidophilic Lysotracker Red (Figure agy.26 Similarly to neurons, the basal level causes enhanced autophagosome for- 4A). In addition, there was evidence of of autophagy also seems to be increased mation, followed by failure of MTOR- enhanced Mitosox staining indicative in podocytes.27,28 In the podocyte, it has dependent lysosomal reformation, which of superoxide activity derived from been suggested that autophagy may be is accompanied by decreased clearance mitochondria in podocytes treated with required to protect the cell from injury. of damaged organelles due to accumula- rapamycin. This suggests an accumu- In this regard, deletion of Atg5, a key tion of autophagolysosomes.30 In this lation of dysfunctional mitochondria component of the autophagic pathway, sense, autophagy stimulated by naturally that would normally be cleared by au- results in late onset of glomerular disease occurring endogenous factors or events tophagy (Figure 4A). Consistent with in mice at 20–24 months of age, pre- such as starvation or growth factor with- the model in Figure 3A, ALV accumu- sumably due to the accumulation of drawal has a very different effect on the lation in podocytes is transient under damaged organelles and ubiquitinated cell than autophagy that is initiated starved conditions, coinciding with a protein complexes.27 Active autophagy is through pharmacologic or genetic in- transient downregulation of MTOR ac- characterized by the presence of double- hibition of MTOR. Although starvation tivity (Figure 4, C and D). At 4 hours of membraned cytoplasmic vesicles known or loss of growth factor signals initiate starvation, the autophagosome and

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large intravesicular structures character- istic of AVs and ALVs together with ac- cumulation of damaged mitochondria in podocytes of mutant mice (Figure 2G) (data not shown) consistent with a sub- stantive defect in lysosomal reformation. Our data support a model whereby MTOR inhibition in podocytes results in enhanced initiation of autophagy followed by failure to regenerate key components of this process (Figure 3B). Why might this be important? Although reduced autophagy can lead to cell dam- age in podocytes, activation and dys- regulation of autophagy are also likely to result in susceptibility to glomerular disease in patients. This is notably ap- parent in patients with lysosomal storage diseases, such as Fabry’s disease, aspartyl- glucosaminuria, or Scheie’sdisease,in which failure of lysosomes to acidify causes failure of lysosomal reforma- tion30 and these patients are also prone to developing proteinuria.31 In addition to the kidney, there are a number of other pathologic states, including cystic fibrosis,32 certain myopathies,33 and neurodegenerative diseases,26 character- ized by dysfunctional or incomplete autophagy causing tissue damage. Our results suggest that dysregulation of autophagy may be an important com- ponent in the pathogenesis of protein- uria and glomerular injury in patients treated with MTOR inhibitors and per- Figure 3. Model explaining the effect of MTOR inhibition in podocytes. (A) Physiologic haps other types of glomerular disease. level of MTOR activity inhibits autophagy (step 1), and maintains it at a basal level (step 2) Although actin cytoskeletal defects and in podocytes. MTOR reactivation allows autophagolysosomal reformation and the cycle reduction of Vegfa may contribute to of autophagy to complete itself. (B) MTOR inhibition (step 1) disrupts the autophagic this damage, the Mtor pod-KO pheno- pathway at two points. First, it relieves chronic suppression, resulting in activation and type does not resemble the phenotypes enhanced autophagy (step 2). However, MTOR inhibition will also lead to suppression of observed in mouse models with primary the reformation of lysosomes and autophagosomes, ultimately resulting in an accumulation defects in either of these pathways. In of ALVs (step 3), damaged intracellular organelles such as mitochondria, and cell death. turn, these data suggest that strategies Lyso, lysosome; AV, autophagosomal vesicle; ALV, autophagolysosomal vesicle. to protect the cell from enhanced and/or dysregulated autophagy may be useful in lysosomes often co-localized. After 10 of ALVs in podocytes as demonstrated protecting the kidney during treatment hours of starvation, there was almost by LC3 and Lysotracker markers (Figure with MTOR inhibitors. complete clearance of LC3-GFP–labeled 4C) and robust suppression of phos- Finally, it is important to consider vesicles and restoration of lysosomal phorylated S6K that does not recover these results in light of recent data size. Although phosphorylated S6K is even by 6 hours (Figure 4D). These find- demonstrating a critical role for Mtor downregulated after 4 hours of starvation ings were recapitulated in vivo in the activation in the development and pro- alone, it recovers by 6 hours, thereby dem- Mtor pod-KO mouse model (Figure 2). gression of diabetic nephropathy.19,34 onstrating reactivation of mTORC1. Specifically, LC3-II is increased in glo- Haploinsufficiency for Rptor slowed the By contrast, administration of rapa- meruli isolated from mutant animals development of glomerular changes in mycin results in massive accumulation (Figure 2H) and EM studies revealed diabetic mice, suggesting that rapalogs

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the Mtor gene using BAC recombineering36 to generate a floxed Mtor gene. The first loxP site was introduced into the unconserved 59 flanking region of the Mtor promoter and the second loxP site into an unconserved region in intron 3. Correctly targeted clones analyzed by Southern blot were used for ag- gregation to produce chimeras. After identi- fication of F1 offspring carrying the targeted allele, the neomycin selection cassette was fl removed by breeding Mtorwt/ ox neo-in mice to a mouse carrying the FLPe recombi- fl nase transgene.37 Heterozygous Mtorwt/ ox mice were bred to mice carrying a pCaggs-Cre transgene to generate Mtorwt/del mice and then bred to the Podocin-Cre driver mouse strain. Mtorwt/del Podocin-Cre mice were fl fl fl bred to Mtor ox/ ox mice to obtain Mtor ox/del Podocin-Cre double-transgenic mice.

Phenotypic Analysis Spot urine was collected from mice 1, 2, 3, and 4 weeks of age. We used a urine dipstick (Chemstrip7; Roche Diagnostics Corp) to detectthepresenceorabsenceofprotein in the urine. A standard colorimetric assay for urine protein and urine creatinine was performed according to the manufacturer’s instructions (Sigma) to estimate urine protein/ creatinine ratios.

Histologic Analysis and Immunofluorescence Kidneys from mice 1, 2, 3, and 4 weeks of age were dissected and fixed in 10% formalin in Figure 4. Chronic MTOR inhibition disrupts the autophagic pathway in podocytes in vitro. PBS and then embedded in paraffinfor (A) Human podocytes stably transfected with LC3-GFP and treated with rapamycin exhibit m fl histologic analysis. Sections 4 m in thick- increased uorescent-tagged ringed structures characteristic of autophagosomes. The – AVs colocalize with the lysosomal marker Lysotracker Red. Podocytes exposed to rapa- ness were cut and stained with periodic acid mycin also demonstrate increased superoxide activity as shown by Mitosox Red, sug- Schiff or hematoxylin and eosin and then gestive of mitochondrial damage. (B) Western blot analysis confirms downregulation of examined and photographed with a DC200 phospho-S6K (pS6K) and inhibition of mTOR activity in podocytes exposed to rapamycin Leica camera and Leica DMLB microscope along with upregulation of the autophagosome-associated LC3 isoform. (C) Podocytes ex- (Leica Microsystems Inc). posed to starvation show only a transient increase in ALVs. In contrast, rapamycin-treated Immunofluorescent tissue staining for cells exhibit prolonged and marked accumulation of ALVs. ALVs are marked by co- podocin was performed using a rabbit anti- localization of the lysosomal dye (Lysotracker Red) and GFP-LC3 (green). (D) Western blot podocin antibody (P0372; Sigma). analysis shows recovery of mTOR activity after 6 hours of starvation as evidenced by in- creasing levels of phospho-S6K. In contrast, rapamycin-treated cells show continued sup- In Situ pression of phospho-S6K. Hybridization Kidneys from mice at 2, 3, and 4 weeks of age were dissected and washed briefly in RNAse- may be useful agents to treat diabetic ne- CONCISE METHODS free PBS and fixed overnight in diethyl phropathy. Our findings highlight the pyrocarbonate–treated 4% paraformalde- importance of tight MTOR regulation Generation of Mtor Pod-KO hyde. These tissues were then transferred to in podocytes and suggest that the use of Podocin-Cre recombinase transgenic mice 15% sucrose for 24 hours followed by 30% rapalogs to treat renal disease must be were used as previously described.35 Ade- sucrose for 24 hours, after which they were considered with caution. tailed targeting strategy was designed for embedded in Tissue-Tek OCT and snap

418 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 412–420, 2012 www.jasn.org BRIEF COMMUNICATION frozen. Sections 10 mm in thickness were cut Western Blotting 5. Sarbassov DD, Ali SM, Sengupta S, Sheen on a Leica Jung cryostat (model CM3050; Protein electrophoresis was performed on JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM: Prolonged rapamycin treatment inhibits Leica Microsystems Inc) and transferred to protein isolated from mouse glomeruli or mTORC2 assembly and Akt/PKB. Mol Cell cultured human podocytes. Antibodies Superfrost microscope slides (Fischer Scien- 22: 159–168, 2006 tific Co.). Digoxigenin-labeled probes were against MTOR (#2972; Cell Signaling), LC3 6. Vollenbröker B, George B, Wolfgart M, prepared according to the Roche Molecular (PM046; MBL), phospho-P70S6K (#9205; Saleem MA, Pavenstädt H, Weide T: mTOR Biochemicals protocol (Roche Molecular Cell Signaling,), total P70S6K (#9202; Cell regulates expression of slit diaphragm pro- teins and cytoskeleton structure in podocytes. Biochemicals). Probes used for in situ analysis Signaling), and b-actin (ab8227; Abcam) Am J Physiol Renal Physiol 296: F418–F426, were used. A goat anti-rabbit IgG-horseradish were Nphs1, Veg fa, and Wt1. Further details 2009 regarding the in situ protocol can be obtained peroxidase secondary antibody was used for 7. Schena FP, Pascoe MD, Alberu J, del Carmen on request. all of the above primary antibodies (sc-2054; Rial M, Oberbauer R, Brennan DC, Campistol Santa Cruz). JM, Racusen L, Polinsky MS, Goldberg- Cell Culture Alberts R, Li H, Scarola J, Neylan JF Sirolimus The immortalized human podocyte cell line CONVERT Trial Study Group: Conversion from was a kind gift from Moin Saleem (University calcineurin inhibitors to sirolimus mainte- nance therapy in renal allograft recipients: of Bristol, Bristol, UK) and was previously ACKNOWLEDGMENTS 24-month efficacy and safety results from the 38 characterized. Cells were maintained in CONVERT trial. Transplantation 87: 233–242, RPMI 1640 medium supplemented with We thank Doug Holmyard and Ken Harpal 2009 10% FBS and insulin-transferrin-selenium for electron microscopy and histologic stains 8. Tumlin JA, Miller D, Near M, Selvaraj S, Hennigar R, Guasch A: A prospective, open- at 33°C. and Anne-Claude Gingras, John Brumell, and label trial of sirolimus in the treatment of focal Human podocytes were co-transfected the entire Quaggin laboratory for helpful dis- segmental glomerulosclerosis. Clin J Am Soc with a pCMV-GFP-LC3 expression vector cussions. Wealso thank Jeffrey Zaltzman whose Nephrol 1: 109–116, 2006 (Cell Biolabs Inc) and a marker plasmid ex- thoughtful question stimulated this study. 9. Serra AL, Poster D, Kistler AD, Krauer F, Raina pressing a puromycin-resistant gene. Two days This work was supported by the Kid- S, Young J, Rentsch KM, Spanaus KS, Senn O, Kristanto P, Scheffel H, Weishaupt D, Wüthrich after transfection, cells were selected in 2 mg/L neyFoundation of Canada (KFOC110014), RP: Sirolimus and kidney growth in autosomal of puromycin (Sigma) until resistant colonies Canadian Institutes of Health Research dominant polycystic kidney disease. N Engl of cells appeared. GFP positive clones were (CIHR MOP62931 and MOP77756), and CIHR JMed363: 820–829, 2010 selected. Cells were treated with 50–1000ng/ml Team Grant/Terry Fox TFF105268 to S.E.Q. 10. Walz G, Budde K, Mannaa M, Nürnberger J, of rapamycin (R878; Sigma) in RPMI. D.P.C. is supported by an American Society Wanner C, Sommerer C, Kunzendorf U, Banas B, Hörl WH, Obermüller N, Arns W, Lysotracker Red (Molecular Probes) or of Nephrology Student Scholar Grant. S.E.Q. Pavenstädt H, Gaedeke J, Büchert M, May C, Mitosox Red (Invitrogen) was incubated with holds the Gabor-Zellerman Chair in Renal Gschaidmeier H, Kramer S, Eckardt KU: Ev- GFP-LC3 stably transfected podocytes at con- Research in the Department of Medicine at the erolimus in patients with autosomal domi- centrations of 75 nM and 5 mM, respectively, University of Toronto. nant polycystic kidney disease. NEnglJMed for 20 minutes. After 20 minutes, cells were 363: 830–840, 2010 washed with PBS and visualized by confocal 11. Perico N, Antiga L, Caroli A, Ruggenenti P, Fasolini G, Cafaro M, Ondei P, Rubis N, microscopy. DISCLOSURES Diadei O, Gherardi G, Prandini S, Panozo A, Untransfected human podocytes were None. Bravo RF, Carminati S, De Leon FR, Gaspari used for Western blotting. 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420 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 412–420, 2012