The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A

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1 The ctin cytoskeleton of kidney podocytes is direct trget of the ntiproteinuric effect of cyclosporine A Christin Ful 1,2, Mry Donnelly 1,2, Sndr Merscher-Gomez 1,2, Yoon Hee Chng 2,5, Stefn Frnz 2,5, Jcqueline Delfguw 2,5, Jer-Ming Chng 3, Hoon Young Choi 2, Kirk N Cmpbell 1,2, Kwnghee Kim 2, Jochen Reiser 1,4 & Peter Mundel 1,2 The immunosuppressive ction of the clcineurin inhibitor cyclosporine A (CsA) stems from the inhibition of nucler fctor of ctivted T cells (NFAT) signling in T cells. CsA is lso used for the tretment of proteinuric kidney diseses. As it stnds, the ntiproteinuric effect of CsA is ttributed to its immunosuppressive ction. Here we show tht the beneficil effect of CsA on proteinuri is not dependent on NFAT inhibition in T cells, but rther results from the stbiliztion of the ctin cytoskeleton in kidney podocytes. CsA blocks the clcineurin-medited dephosphoryltion of synptopodin, regultor of Rho GTPses in podocytes, thereby preserving the phosphoryltion-dependent synptopodin b interction. Preservtion of this interction, in turn, protects synptopodin from cthepsin L medited degrdtion. These results represent new view of clcineurin signling nd shed further light on the tretment of proteinuric kidney diseses. Novel clcineurin substrtes such s synptopodin my provide promising strting points for ntiproteinuric drugs tht void the serious side effects of long-term CsA tretment. The kidney glomerulus is highly specilized structure tht ensures the selective ultrfiltrtion of plsm so tht essentil proteins re retined in the blood. The common denomintor in vriety of kidney diseses, including miniml chnge disese (MCD) nd focl segmentl glomerulosclerosis (FSGS), is podocyte dysfunction involving mssive loss of protein in the urine (proteinuri) 1,2.Dynmic regultion of the podocyte ctin cytoskeleton is vitl to norml kidney filter function, nd muttions ffecting severl podocyte proteins led to the rerrngement of the ctin cytoskeleton nd subsequent proteinuri 3. Proteins regulting podocyte ctin dynmics re therefore crucil for sustined glomerulr filter function 4. The ctin-binding protein synptopodin, which is highly expressed in podocytes 5, exists in three isoforms, neuronl Synpo-short, renl Synpo-long nd Synpo-T 6. Synptopodin-mutnt mice lcking Synpo-short nd Synpo-long upregulte Synpo-T protein expression in podocytes, thereby rescuing kidney filter function during development 6. They do, however, suffer from prolonged proteinuri when chllenged with lipopolyscchride () 6. Synptopodin is key regultor of podocyte function becuse bigenic heterozygosity for synptopodin nd CD2-ssocited protein results in proteinuri nd FSGS-like glomerulr dmge 7. Mechnisticlly, synptopodin induces stress fibers by stbilizing the GTPse RhoA 8 nd suppresses filopodi by disrupting cell division cycle-42 insulin receptor substrte p53 Men signling complexes 9. The serine/threonine phosphtse clcineurin is ubiquitously expressed in ll mmmlin tissues 10. The best chrcterized function of clcineurin is the regultion of NFAT signling. The immunosuppressive ction of the clcineurin inhibitor CsA stems from the inhibition of NFAT signling in T cells 11. In other cell types, clcineurin- NFAT signling cn lso cuse crdic hypertrophy, skeletl muscle differentition or chnges in synptic plsticity 11. In ddition, clcineurin-nfat signling is crucilly involved in ngiogenesis 10, pncretic bet cell function 12 nd hir growth 13. Likewise, inhibition of clcineurin-nfat signling in bone explins the bone loss often experienced during immunosuppressnt therpy tht trgets clcineurin 14. Notbly, CsA cn induce remission of proteinuri cused by such diseses s MCD nd FSGS 15. T cell dysfunction is ssocited with some forms of proteinuri, including subset of MCD in children. Therefore, it hs been ssumed tht T cells ct on podocytes to cuse proteinuri nd tht the ntiproteinuric effect of CsA results from the inhibition of NFAT signling in T cells 15. As it stnds, experimentl support of this hypothesis is missing. CsA cn lso reduce proteinuri in humn 16 nd experimentl 17 Alport s syndrome, nonimmunologicl disese, rising doubts bout the bove hypothesis. Moreover, -induced proteinuri cn develop independently of T cells 18,nd mice lcking synptopodin show impired recovery from -induced proteinuri 6. Collectively, these dt rgue for direct effect of 1 Deprtment of Medicine, University of Mimi Miller School of Medicine, 1600 Northwest Tenth Avenue, Mimi, Florid 33136, USA. 2 Deprtment of Medicine, Mount Sini School of Medicine, One Gustve L. Levy Plce, New York, New York 10029, USA. 3 Deprtment of Internl Medicine, Hsio-Kng Municipl Hospitl, Kohsiung, Medicl University, 482 Shn-ming Rod, Kohsiung, Tiwn. 4 Nephrology Division nd Progrm in Glomerulr Disese, Deprtment of Medicine, Msschusetts Generl Hospitl, Hrvrd Medicl School, 149 Thirteenth Street, Boston, Msschusetts 02129, USA. 5 Present ddresses: Deprtment of Medicine, Yle New Hven Hospitl, 20 York Street, New Hven, Connecticut 06510, USA (Y.H.C.); Roche Phrm Switzerlnd, Schönmttstrsse 2, CH-4153 Reinch, Switzerlnd (S.F.); Development Science, Grünenthl, Ziegelstrsse, D Achen, Germny (J.D.). Correspondence should be ddressed to P.M. (pmundel@med.mimi.edu). Received 14 April; ccepted 10 July; published online 24 August 2008; doi: /nm.1857 NATURE MEDICINE VOLUME 14 [ NUMBER 9 [ SEPTEMBER

2 b Synptopodin β Input GST β GST β Synpo-short GST Synpo-T Synpo-lt Synpo-long Merge Synptopodin Ltrunculin A d e f 116 kd 96 kd 66 kd Blot: GST 66 kd 45 kd 45 kd 31 kd β GFP β clcineurin nd its inhibitor CsA on podocytes. Here we show tht the ntiproteinuric effect of CsA is independent of its immunosuppressive function in T cells nd results directly from the stbiliztion of the podocyte ctin cytoskeleton. In prticulr, we show tht clcineurin regultes podocyte ctin dynmics by dephosphorylting synptopodin, thereby brogting the medited protection of synptopodin from cthepsin L (CtL)-medited proteolysis. RESULTS Synptopodin is nd clcineurin-binding protein proteins re chperone-like phospho serine/threonine binding proteins tht cn lter the structure or function of trget proteins 19. Humn Synpo-short nd Synpo-long contin two consensus binding motifs (mino cids : RAAATTP, nd mino cids : RPSRSSP; Fig. 1). Using yest two-hybrid screen 6, we found tht b nd Z bound synptopodin. In the kidney, coloclized with synptopodin in podocytes (Fig. 1b). In differentited cultured podocytes, coloclized with synptopodin long stress fibers, nd both proteins remined ssocited fter disruption of stress fibers by ltrunculin A (Fig. 1c). Biochemiclly, GST b specificlly bound synptopodin from isolted glomeruli (Fig. 1d), nd glomerulr b precipitted with endogenous synptopodin (Fig. 1e). To mp interction sites in synptopodin, we focused on the two binding sites (Fig. 1) tht fit the mode 2 consensus binding motif (RXXXpSXP or RXXXpTXP) 20. We hypothesized tht Thr216 nd Ser619 re phosphocceptor sites within the first (RAAApTTP) nd the second (RPSRpSSP) motif, respectively, whose phosphoryltion is required for binding. To test this hypothesis, we expressed Flgsynptopodin proteins with GFP b in HEK293 cells nd conducted coimmunoprecipittion studies. Wild-type synptopodin nd phosphomimetic Synpo-ED (contining T216E nd S619D muttions) intercted with b (Fig. 1f). In contrst, the destruction of the phosphocceptor sites by lnine substitution (T216A nd S619A; Synpo-AA) brogted b binding (Fig. 1f). Input Synpo c IP Blot: β Input Elute Elute Flg-synptopodin GFP β Merge WT AA ED IP: Flg Flgcontrol Blot: GFP Blot: GFP Figure 1 Synptopodin specificlly intercts with () Schemtic of synptopodin isoforms. The white box shows the first 676 mino cids tht re shred between Synpo-long nd Synposhort. This frgment contins two binding motifs (blck ovls) nd two CtL clevge sites (rrows). (b) In the dult mouse kidney, b coloclizes with synptopodin in podocytes. Scle br, 30 mm. (c) In differentited cultured podocytes, b coloclizes with synptopodin long stress fibers (top), nd both proteins remin ssocited fter disruption of ctin filments with ltrunculin A (bottom). Scle, 25 mm. (d) Synptopodin from isolted mouse glomerulr extrcts (input) specificlly binds GST b but not GST lone. (e) Coimmunoprecipittion experiments show tht endogenous synptopodin intercts with endogenous b in isolted mouse glomeruli. An ntibody to GFP serves s negtive control. IP, immunoprecipittion. (f) GFP b precipittes with wild-type (WT) Flgsynptopodin nd phosphomimetic Flg Synpo- ED but not with phosphoresistnt Flg Synpo-AA or Flg-rver (control). Myopodin, nother member of the synptopodin gene fmily, binds the ctlytic clcineurin subunits CnA nd CnAb 21. Similrly, GFP Synpo-lt, the C-terminl frgment of Synpo-long not present in Synpo-short (Fig. 1), precipitted with CnA from co-trnsfected HEK293 cells, wheres both GFP Synpo-short nd GFP Synpo-lt intercted with constitutively ctive clcineurin (CnA) 22 (Fig. 2). Furthermore, synptopodin could be immunoprecipitted with endogenous CnA from isolted glomeruli (Fig. 2b), nd clcineurin coloclized with synptopodin in podocytes in the kidney nd in culture (Fig. 2c). Phosphoryltion of synptopodin nd binding to Similr to the vst mjority of intercting proteins 23,myopodin is phosphoprotein tht binds to b in serine/threonine phosphoryltion dependent fshion 24. To test whether synptopodin is lso phosphoprotein, we incubted purified Flg Synpo-short from trnsfected HEK293 cells with lmbd protein phosphtse (l-ppse). As shown by SDS-PAGE nd western blot nlysis, l-ppse reduced the moleculr size of synptopodin (Fig. 3), thereby showing tht synptopodin is phosphorylted in vivo. Consensus binding motifs serve s substrtes for serine/threonine protein kinses 25 including protein kinse A (PKA) nd clcium-dependent protein kinse II (CMKII) 26, two kinses tht cn phosphorylte the binding motifs of myopodin 21. To test whether PKA nd CMKII cn phosphorylte synptopodin, we dephosphorylted purified wild-type Flg Synpo-short nd Flg Synpo-AA with l-ppse nd incubted it with PKA or CMKII in the presence of [g 32 P]ATP. 32 P lbeling of wild-type synptopodin by PKA ws first detected fter 2 min nd incresed over time (Fig. 3b). In contrst, 32 Plbelingof Synpo-AA ws strongly reduced (Fig. 3b). 32 P lbeling of synptopodin by CMKII ws first detected fter 15 min nd incresed therefter (Fig. 3b). In contrst, CMKII cused virtully no 32 P lbeling of Synpo-AA (Fig. 3b). To further explore the phosphoryltion of synptopodin by PKA nd CMKII, we trnsfected HEK293 cells with Flg Synpo-short nd incubted them in 32 P-contining 932 VOLUME 14 [ NUMBER 9 [ SEPTEMBER 2008 NATURE MEDICINE

3 Elute Elute Input Flg-clcineurin WT WT CnA CnA Flg-control Short Alt Short Alt Short GFP-synptopodin Alt IP: Flg Blot: GFP Blot: GFP medium for 2 h. After immunoprecipittion, Flg Synpo-short showed strong 32 P lbeling, which ws mrkedly reduced in the presence of the PKA inhibitor H89 or the CMKII inhibitor KN62 (Fig. 3c). Combined inhibition of both kinses further decresed the 32 P lbeling of synptopodin (Fig. 3c). Consensus binding motifs cn be dephosphorylted by serine/threonine protein phosphtses, including clcineurin 26.Clcineurin dephosphoryltes myopodin, thereby brogting the myopodin interction 21. To test whether synptopodin is lso substrte of clcineurin, we used PKA to lbel purified Flg Synposhort with 32 P before incubtion for 30 or 60 min in the presence or bsence of clcineurin. 32 P-synptopodin bundnce decresed over time only in the presence of clcineurin (Fig. 3d). To further test whether clcineurin cn dephosphorylte synptopodin, we expressed Flg Synpo-short in HEK293 cells with dominnt ctive GFP-CnA 20 in the presence of 32 P followed by immunoprecipittion with n ntibody to Flg. 32 P lbeling of Flg Synpo-short immunoprecipitted from cells expressing GFP-CnA ws lmost completely bolished when compred to cells expressing GFP (Fig. 3e). Next, we exmined the bility of purified Flg Synpo-short to bind immobilized GST b before nd fter dephosphoryltion. Flg Synpo-lt, which cnnot bind b (dt not shown), served s negtive control. In the bsence of phosphtse tretment, synptopodin strongly nd specificlly bound to immobilized GST b (Fig. 3f). In contrst, the dephosphoryltion of Flg Synpo-short with l-ppse or clcineurin brogted the binding of synptopodin to b (Fig. 3f). Synptopodin binding mintins stress fibers Synptopodin, which is required for stress fiber formtion in podocytes 6,8, cn be phosphorylted by PKA nd CMKII (Fig. 3b,c) nd dephosphorylted by clcineurin (Fig. 3d,e). Therefore, we tested IP Synpo Clcineurin GFP Blot: clcineurin Synptopodin Clcineurin Merge Figure 2 Identifiction of synptopodin s clcineurin binding protein. () GFP Synpo-short nd GFP Synpo-lt precipitte with Flg-tgged CnA from co-trnsfected HEK293 cells. GFP Synpo-lt cn lso interct with WT clcineurin. No binding is found with Flg-rver (control). (b) Endogenous coimmunoprecipittion experiments show tht synptopodin intercts with clcineurin in isolted mouse glomeruli. An ntibody specific for GFP serves s negtive control. (c) In the dult mouse kidney (top) nd differentited cultured podocytes (bottom), clcineurin prtilly coloclizes with synptopodin. Scle brs, 30 mm (top pnels) nd 25 mm (bottom pnels). b c whether the phrmcologicl inhibition of clcineurin, PKA or CMKII modultes podocyte ctin dynmics. Inhibition of clcineurin with CsA incresed stress fiber content nd synptopodin expression (Supplementry Fig. 1 online) without inducing podocyte poptosis (Supplementry Tble 1 online). The effect on stress fiber content ws medited by synptopodin, becuse it did not occur in synptopodin-knockdown podocytes 6. The PKA inhibitor H89 nd, more strongly, the combined ppliction of H89 nd the CMKII inhibitor KN62 cused the loss of stress fibers nd synptopodin expression, which could be rescued by CsA (Supplementry Fig. 1). Similrly, the trnsfection of differentited podocytes with yellow fluorescence protein tgged difopein, specific inhibitor of lignd interctions 27, cused the loss of stress fibers nd synptopodinexpressionintrnsfectedpodocytesbut not in neighboring nontrnsfected cells (Supplementry Fig. 1b). Synptopodin proteolysis by CtL nd protection by Upregultion of CtL ctivity in podocytes is ssocited with the development of proteinuri 28,29. Glomerulr synptopodin is highly unstble, leding to the genertion of 44-kD frgment 5. Synptopodin contins two high-scoring CtL clevge sites 30 (mino cids : ALGAE nd mino cids : ALPRPS), the first of which predicts the genertion of n N-terminl 44-kD frgment (Fig. 1). Therefore, we tested whether CtL cn cleve purified Flg-synptopodin. At ph 4.5, which reflects lysosoml milieu, we found concentrtion-dependent complete degrdtion of synptopodin (Fig. 4). At ph 6.5, we observed dosedependent ccumultion of the 44-kD frgment (Fig. 4). At ph 7.0, which llows for cytosolic-like CtL ctivity 29, we found prtil degrdtion of synptopodin (Fig. 4). To test whether decresed synptopodin expression fter protein kinse inhibition (Supplementry Fig. 1) resulted from CtLmedited clevge, we repeted the kinse inhibition experiment in the presence of the cthepsin inhibitor E64, which prevented the H89- nd KN62-induced loss of stress fibers nd synptopodin expression (Fig. 4b). Next, we monitored synptopodin stedy-stte protein levels in the presence or bsence of H89 nd KN62 with or without CsA, E64 or E64D ( specific inhibitor of CtL). Full-length synptopodin bundnce ws decresed by combined ppliction of H89 nd KN62 nd could be restored by CsA, E64 or E64D (Fig. 4c). In plnts, cn increse the stedy-stte protein levels of its intercting prtners by blocking their clevge 30. To test whether binding protects synptopodin from proteolytic degrdtion by CtL in similr mnner, we digested purified Flg Synpo-short t ph 7.0 with CtL in the presence or bsence of purified Flg b or Flg -ctinin-4, nother synptopodin-binding protein 6. The clevge of synptopodin by CtL resulted in decrese in the mount of full-length synptopodin nd the ppernce of the predicted 44-kD frgment (Fig. 4d). We detected protection of full-length synptopodin in the presence of b but not -ctinin-4 (Fig. 4d). To test whether b conferred true protection insted of simply NATURE MEDICINE VOLUME 14 [ NUMBER 9 [ SEPTEMBER

4 b c Flg-synpo + λ-ppse Flg-synptopodin (WT) Flg-synptopodin (AA) Min PKA Con Flg-synptopodin H89 KN62 H89+ KN62 f Flg Synpo-short λ-ppse Clcineurin + + Flg Synpo-lt Coomssie Flg-synptopodin (WT) Flg-synptopodin (AA) Figure 3 The synptopodin interction is ntgonisticlly regulted by PKA, CMKII nd clcineurin. () SDS-PAGE (top) nd western blot (bottom) nlyses show reduced moleculr weight of purified Flgsynptopodin (Flg-Synpo) fter dephosphoryltion with l-ppse. (b) Timedependent phosphoryltion of purified Flg Synpo-short by PKA (top pnels) nd CMKII (bottom pnels). Alnine substitution of Thr216 nd Ser619 (AA) strongly reduces 32 P lbeling when compred to WT Min CMKII competing with synptopodin s substrte of CtL, we seprtely incubted b nd -ctinin-4 with CtL. Of note, neither b nor -ctinin-4 ws cleved by CtL (Fig. 4d). The first CtL clevge site of synptopodin (mino cids : ALGAE) is conserved phylogeneticlly from fish to humns, the second (mino cids : ALPRPS) is conserved mong mmmls (Fig. 4e). To explore the function of these sites, we mutted them individully (CM1: AQDSE; CM2: ARTEPS) or in combintion (CM1+2) nd determined the effect on the proteolytic processing by CtL (Fig. 4f). We detected no degrdtion in the bsence of CtL (Fig. 4f). The muttion of ech clevge site individully conferred prtil resistnce ginst proteolysis, nd the strongest protection ws found for Synpo-CM1+2 (Fig. 4f). Therefore, we concluded tht the muttion of the CtL clevge sites stbilizes synptopodin stedystte protein levels nd hypothesized tht the expression of such protected synptopodin mutnt in podocytes would preserve stress fibers. In keeping with this hypothesis, Synpo-CM1+2 protected podocytes ginst H89- nd KN62-induced or -induced 18 loss of stress fibers (Supplementry Fig. 2 online). The degrdtion of synptopodin by CtL is fcilitted by the clcineurin-medited dephosphoryltion of synptopodin, rising the possibility tht synptopodin is direct trget of the ntiproteinuric effect of CsA 15 nd E As shown before 18, (n ¼ 6; ± microgrms lbumin per milligrm cretinine) but not PBS (n ¼ 6; ± microgrms lbumin per milligrm cretinine) cused proteinuri in SCID mice tht re devoid of T nd B cells 33 (P o 0.001; Fig. 5). The -induced proteinuri ws significntly reduced by tretment with CsA (n ¼ 10, ± microgrms lbumin per milligrm cretinine; P o 0.001) or E64 (n ¼ 8; ± microgrms lbumin per milligrm cretinine; P o 0.001) fter injection (Fig. 5). Western blot nlysis of isolted glomeruli showed tht cused nerly complete loss of full-length synptopodin nd its downstrem effector RhoA 8, which could be blocked by CsA or E64 (Fig. 5b). Genetic overexpression of synptopodin prevents proteinuri To test whether the restortion of synptopodin protein bundnce in mice protects ginst -induced proteinuri, we injected wild-type Flg- Clcineurin Flg-synpo GFP GFP-CnA Min GST β synptopodin. Flg blots show equl protein loding. (c) Phosphoryltion of synptopodin by PKA nd CMKII in HEK293 cells cultured in the bsence of inhibitors (Con), KN62 nd H89. (d) Time-dependent dephosphoryltion of purified 32 P-lbeled Flg Synpo-short by clcineurin (left) or in its bsence (right). (e) Dephosphoryltion of 32 P Flg Synpo-short in HEK293 cells by clcineurin. (f) Dephosphoryltion of purified Flg Synpo-short by l-ppse or clcineurin brogtes the binding of synptopodin to immobilized GST b. Flg Synpo-lt does not bind nd serves s negtive control. d e Blot: GST mice with fter in vivo gene delivery 9,29 of Flg-synptopodin. Delivery solution lone served s negtive control. First, we monitored Flg-synptopodin protein bundnce in the bsence of by immunoblotting (Fig. 5c). At fter gene delivery, we detected the recombinnt Flg-synptopodin proteins in extrcts from isolted glomeruli nd livers. Wheres the protein bundnce of wildtype synptopodin nd its mutnt forms were comprble in the liver, they were mrkedly different in glomeruli (Fig. 5c). Synpo-CM1+2 nd Synpo-ED showed the highest expression, wheres wild-type synptopodin nd Synpo-AA expression ws much lower nd comprble to endogenous synptopodin in control mice (Fig. 5c). Notbly, the delivered Flg-synptopodin proteins were functionl, becuse they incresed glomerulr mounts of RhoA (Fig. 5c). Hence, synptopodin undergoes constnt physiologicl turnover vi CtL-medited degrdtion, even in the bsence of. In keeping with the biochemicl results, we detected Flg Synpo-ED in kidney cells including podocytes, s reveled by double-lbeling deconvolution microscopy with the podocyte mrker podocin 34 (Fig. 5d). Next, we conducted studies fter in vivo gene delivery of Flg-synptopodin constructs or delivery solution serving s negtive control (Fig. 5e). The expression nd ctivity of recombinnt Flg-synptopodin ws monitored by immunoblotting (Fig. 5f). As expected 9,18, no significnt proteinuri ws detected in PBSinjected control mice receiving delivery solution (n ¼ 6;, ± 8.55 microgrms lbumin per milligrm cretinine versus, ± microgrms lbumin per milligrm cretinine; not significnt). In contrst, significnt proteinuri developed in injected mice receiving delivery solution (n ¼ 5, ± microgrms lbumin per milligrm cretinine; P o ). induced proteinuri ws significntly reduced by gene trnsfer of Synpo-CM1+2 (n ¼ 8, ± microgrms lbumin per milligrm cretinine; P o 0.05) or Synpo-ED (n ¼ 6; ± microgrms lbumin per milligrm cretinine; P o 0.05) but not by wild-type synptopodin (n ¼ 7, ± microgrms lbumin per milligrm cretinine; not significnt) or Synpo-AA (n ¼ 8, ± microgrms lbumin per milligrm cretinine; P o 0.05; not significnt) (Fig. 5e). Biochemiclly, we detected comprble mounts of ll Flg-synptopodin proteins nd endogenous RhoA in liver Input Elute Elute 934 VOLUME 14 [ NUMBER 9 [ SEPTEMBER 2008 NATURE MEDICINE

5 ph: 4.5 ph: 6.5 ph: 7.0 Ct L (µl) b Phlloidin E64 Synptopodin Phlloidin + E64 Synptopodin c Con H89 + KN62 Con CsA E64 E64D 44 kd H kd d Synpo Actinin β CtL 110 kd 44 kd Blot: Synpo β Actinin-4 extrcts (Fig. 5f). In contrst, in isolted glomeruli, expression of Synpo-CM1+2 nd Synpo-ED ws preserved fter tretment, wheres wild-type synptopodin nd Synpo-AA showed nerly complete loss (Fig. 5f). The preservtion of Synpo-CM1+2 or Synpo-ED expression ws ssocited with the preservtion of glomerulr RhoA expression (Fig. 5f), thereby confirming tht the delivered recombinnt proteins were functionl. The lrge GTPse dynmin is nother trget of CtL, nd gene trnsfer of CtL-resistnt dynmin lso protects ginst -induced proteinuri 29, suggesting functionl link between synptopodin nd dynmin. Therefore, we nlyzed the bove-mentioned protein extrcts (Fig. 5b,f) for dynmin. CsA nd E64 preserved not only synptopodin nd RhoA (Fig. 5b) but lso dynmin stedy-stte protein levels (Supplementry Fig. 3 online). Similr to RhoA (Fig. 5f), Synpo-CM1+2 or Synpo-ED gene trnsfer ws lso sufficient to stbilize dynmin (Supplementry Fig. 3b). In light of nother report suggesting tht CsA interferes with the expression of the tight junction protein ZO-1 in podocytes 35, we nlyzed the protein extrcts for ZO-1 (Fig. 5f). CsA nd E64 (Supplementry Fig. 3) or gene trnsfer of Synpo-CM1+2 nd Synpo-ED stbilized ZO-1 (Supplementry Fig. 3b), thereby suggesting tht the effect of CsA on ZO-1 expression is indirect vi the stbiliztion of synptopodin. In keeping with the biochemicl results (Fig. 4d), the expression of -ctinin-4 ws not ffected by, CsA, E64 or gene trnsfer (Supplementry Fig. 3). Tken together, expression of clcineurinresistnt Synpo-ED or CtL-resistnt Synpo-CM1+2 stbilizes glomerulr RhoA, dynmin nd ZO-1 stedy-stte levels, thereby protecting ginst -induced proteinuri. These experiments lso show tht synptopodin protein levels in the liver re stble nd not ffected by. e H89 + KN62 H. spiens P. pygmeus P. troglodytes R. norvegicus M. musculus D. reo H. spiens P. pygmeus P. troglodytes R. norvegicus M. musculus kd CtL WT CM1 CM2 CM1+2 Figure b, E64 nd CsA block the CtL-medited degrdtion of synptopodin. () Dose-dependent degrdtion of purified Flg Synpo-short by CtL t ph 4.5, 6.5 or 7.0 leds to the genertion of 44-kD frgment. (b) H89-medited reduction of stress fibers nd synptopodin bundnce re prevented by the cthepsin inhibitor E64 (right, top). Similrly, E64 prevents the loss of synptopodin expression nd stress fibers cused by simultneous inhibition of PKA nd CMKII (right, bottom). Scle br, 25 mm. (c) Western blot nlysis of synptopodin stedy-stte bundnce in differentited WT podocytes. In control cells (Con) the 110-kD full-length protein is visible. Inhibition of PKA nd CMKII (H89 + KN62) cuses prtil degrdtion of full-length synptopodin. Tretment with CsA, E64 or E64D blocks the H89- nd KN62-medited degrdtion of synptopodin. Immunoblotting for glycerldehyde-3- phosphte dehydrogense (GAPDH) shows equl protein loding. (d) Binding of b but not of -ctinin-4 prtilly protects synptopodin ginst proteolytic processing by CtL (left) b (middle) or -ctinin-4 (right) re not cleved by CtL. (e) Synptopodin contins two evolutionrily conserved CtL clevge sites. The first motif (ALGAE) is conserved from fish to humns (top). The second motif (ALPRPS) is preserved from mice to humns (bottom). (f) Site-directed mutgenesis of CtL clevge sites seprtely (CM1 or CM2) or together (CM1+2) increses resistnce of synptopodin ginst CtLmedited proteolytic processing. Flg-synptopodin nd its mutnt forms were purified from HEK293 cells (left) nd incubted with CtL t ph 7.0 (right). f + CtL WT CM1 CM2 Blot: GAPDH CM1+2 To further confirm the results of the gene delivery studies in nother in vivo setting (Fig. 5), we creted podocyte-specific nd tetrcycline-inducible trnsgenic mouse line expressing GFP Synpo-CM1+2 by crossing podocin-rtta mice 36 with teto- GFP-Synpo-CM1+2 strin (Fig. 6). We gve the resulting doubletrnsgenic offspring doxycycline or vehicle solution for 7 d followed by injection 36. Biochemiclly, synptopodin remined detectble fter tretment (Fig. 6b). The preservtion of synptopodin expression ws ssocited with the preservtion of endogenous RhoA, dynmin nd ZO-1 protein levels fter tretment (Fig. 6b) nd protected ginst -induced proteinuri (Fig. 6c). Significnt proteinuri developed in vehicle-treted control mice (n ¼ 4;, ± microgrms lbumin per milligrm cretinine versus, ± microgrms lbumin per milligrm cretinine; P o 0.005) but not in doxycycline-treted, Synpo-CM1+2 expressing mice (n ¼ 5;, ± microgrms lbumin per milligrm cretinine versus, ± microgrms lbumin per milligrm cretinine; not significnt). At, we lso detected significnt difference (P o 0.005) between both groups (Fig. 6c). Podocyte-specific expression of CnA cuses proteinuri The inhibition of clcineurin by CsA protected ginst -induced proteinuri (Fig. 5) nd prevented the degrdtion of synptopodin (Fig. 5b). Additionlly, clcineurin cused the dephosphoryltion of synptopodin nd the loss of binding (Fig. 3). Therefore, we hypothesized tht the ctivtion of clcineurin in podocytes should hve n -like effect leding to the reduction of synptopodin stedy-stte protein levels nd to the disruption of glomerulr brrier function. To test this hypothesis, we creted tetrcycline-inducible 36 NATURE MEDICINE VOLUME 14 [ NUMBER 9 [ SEPTEMBER

6 Proteinuri (microgrms lbumin per milligrm cretinine) CsA E64 BSA (µg) ** ** + PBS ** + CsA + E64 Proteinuri (microgrms lbumin per milligrm cretinine) 1, trnsgenic mouse line expressing CnA 22, constitutively ctive form of clcineurin, s GFP fusion protein specificlly in podocytes in mnner similr to the experiments described in the previous prgrph (Fig. 6d). The resulting double-trnsgenic offspring were dministered doxycycline or vehicle 36. Similrly to injection (Fig. 5 nd Supplementry Fig. 3), the induction of GFP-CnA expression in podocytes cused the degrdtion of endogenous synptopodin nd the downregultion of RhoA, dynmin nd ZO-1 (Fig. 6e). Most notbly, the mice expressing the trnsgene (doxycycline-treted) but not the control mice (vehicle-treted) developed significnt proteinuri (vehicle-treted: n ¼ 10; ± microgrms lbumin per milligrm cretinine versus doxycycline-treted: n ¼ 15; ± microgrms lbumin per milligrm cretinine; P o 0.005) (Fig. 6f). DISCUSSION Our dt unveil new clcineurin signling pthwy tht opertes in podocytes nd is essentil for the mintennce of kidney filter function (Fig. 6g). In contrst to most other clcineurin-controlled signling events 10 13, the ntiproteinuric effect of CsA described here does not result from the inhibition of NFAT signling. Insted, CsA blocks the clcineurin-medited dephosphoryltion of the ctinorgnizing protein synptopodin 6,8,9. Our dt lso show tht ctivtion of clcineurin in the podocyte is sufficient to cuse proteinuri vi the degrdtion of synptopodin. Our findings lso identify b e PBS CsA E kd Blot: RhoA Blot: GAPDH c *** Isolted glomeruli Liver CM CM Con WT 1+2 AA ED Con WT 1+2 AA ED Blot: NS * WT * NS CM1+2 AA ED Synpo RhoA GAPDH f d Flg synpo-ed Flg Podocin Merge CM Con Con WT 1+2 AA ED Con Con WT 1+2 CM AA ED Blot: Isolted glomeruli Figure 5 CsA nd E64 meliorte -induced proteinuri by blocking the CtL-medited degrdtion of synptopodin. () SDS-PAGE nlysis of urines from control nd -injected mice (top). Quntittive nlysis of lbuminuri (bottom). (b) Western blot nlysis of isolted glomeruli showing tht cuses the degrdtion of endogenous synptopodin nd RhoA. CsA or E64 block the -induced degrdtion of synptopodin nd RhoA. GAPDH shows equl protein loding. (c) Immunoblot nlysis of Flg-synptopodin proteins from isolted glomeruli (left) nd liver extrcts (right) fter in vivo gene delivery. Glomerulr RhoA levels re positively correlted with Flg-synptopodin bundnce. (d) Expression of Flg Synpo-ED in podocytes fter in vivo gene delivery s detected by double-lbeling deconvolution microscopy with ntibodies to Flg nd the podocyte mrker podocin. No Flg signl is seen in podocytes of delivery solution injected mice (control). Scle br, 50 mm. (e) Gene trnsfer of Synpo-CM1+2 or Synpo-ED but not WT synptopodin, Synpo- AA or delivery solution (control) protects ginst -induced proteinuri. NS, not significnt. (f) Immunoblot nlysis of synptopodin protein expression fter injection nd gene delivery. cuses degrdtion nd decrese of synptopodin ( + Con) when compred to PBS-injected mice (Con). High levels of synptopodin cn be found in -injected mice fter gene trnsfer of Synpo-CM1+2 or Synpo-ED but not of WT synptopodin or Synpo-AA (left). Synptopodin protein in the liver is not ffected by (right). Mice tht did not receive synptopodin cdna (Con, Con + ) do not express synptopodin in the liver. *P o 0.05; **P o 0.001; ***P o synptopodin s substrte of PKA nd CMKII. Together with clcineurin, these kinses control the phosphoryltion stte of synptopodin, thereby regulting binding nd CtL-medited proteolysis (Fig. 6g). Although it ws previously shown tht binding cn increse protein stedy-stte levels 31,37, to our knowledge, the present study is the first to provide mechnistic insight into how binding cn stbilize trget proteins. Furthermore, our results unveil new role of cytoplsmic CtL under not only pthologicl 29 but lso physiologicl conditions. Collectively, our dt show tht the expression of CtL-resistnt synptopodin by in vivo gene trnsfer or by n inducible trnsgene in podocytes is sufficient to protect ginst -induced proteinuri. Finlly, our study hs uncovered mechnism underlying the ntiproteinuric effect of CsA, n estblished drug in the tretment of proteinuric humn kidney diseses, in prticulr in cses of MCD or FSGS tht re resistnt to tretment with corticosteroids or tht relpse fter tretment 15. Our findings suggest tht the ntiproteinuric effect of CsA is independent of its immunosuppressive effect nd insted stems from direct effect on synptopodin in podocytes (Fig. 6g). It is possible tht, similr to the ctivtion of clcineurin by trnsient receptor potentil chnnel-6 (TRPC6) in the hert 38, incresed TRPC6-medited clcium influx my cuse FSGS 39,40 by enhncing clcineurin ctivtion in podocytes, which, in turn, would led to the loss of synptopodin vi the bove-described mechnism. This ide is supported by the observtion tht overexpression of Liver Synpo RhoA GAPDH 936 VOLUME 14 [ NUMBER 9 [ SEPTEMBER 2008 NATURE MEDICINE

7 Podocin promoter d pcmv Podocin promoter g teto teto pcmv rtetr rtetr CMKII P PKA Cthepsin Synptopodin Synptopodin Syn pto podin CN Intct glomerulr filter VP16 PolyA GFP Synpo-CM1+2 PolyA VP16 GFP-CnA CsA PolyA PolyA b c Figure 6 Expression of Synpo-CM1+2 in podocytes protects ginst e Veh Dox 110 kd Veh Dox Blot: RhoA Blot: dynmin Blot: ZO-1 Blot: α-ctinin Blot: GAPDH 110 kd Blot: RhoA Blot: dynmin Blot: ZO-1 Blot: α-ctinin Blot: GAPDH Proteinuri TRPC6 in podocytes cuses the loss of stress fibers 41,therebyphenocopying the loss of synptopodin 8. A role for reduced synptopodin expression s contributing fctor in the pthogenesis of FSGS is further supported by the observtion tht heterozygosity for synptopodin is sufficient to cuse proteinuri nd FSGS-like disese 7. Altogether, the ntiproteinuric effect of CsA results, t lest in prt, from the mintennce of synptopodin protein bundnce in podocytes, which, in turn, is sufficient to mintin the integrity of the glomerulr filtrtion brrier nd to sfegurd ginst proteinuri. These findings should open new venues for the development of ntiproteinuric drugs tht directly nd selectively trget the podocyte, thereby voiding the serious side effects of prolonged NFAT inhibition cused by long-term CsA tretment 16,42. METHODS Cthepsin L digestion. For the in vitro digestion studies, we dded 28, 70, or 140 ng humn CtL (Sigm-Aldrich) to 1 mg purified Flg-synptopodin in rection buffer (100 mm sodium cette ph 4.5 or 100 mm sodium phosphte monobsic ph 6.5 or 7.0, 1 mm EDTA, nd 2 mm dithiothreitol) in totl volume of 50 ml 29,43. In some experiments, we dded 1 mg purified Flg b or Flg -ctinin-4 before the digest. After incubtion on ice for 30 min, we dded 84 ng CtL nd 10 ml rection buffer nd further incubted the proteins t 37 1C for 30 min. We stopped the rection by boiling in smple buffer. After resolving the smples by 8 12% grdient SDS-PAGE, we nlyzed them by immunoblotting. Lipopolyscchride-induced proteinuri. All niml studies were pproved by the Mount Sini School of Medicine Animl Institute Committee. We induced proteinuri in femle SCID mice (n ¼ 6 10 per group) by injection s E64 Proteinuri (microgrms lbumin per milligrm protein) f Proteinuri (microgrms lbumin per milligrm protein) * * Veh * NS Dox Veh Dox proteinuri, wheres ctivtion of clcineurin in podocytes cuses proteinuri. () Schemtic of constructs used for the genertion of Synpo- CM1+2 trnsgenic mice. rtetr, reverse Tet repressor; VP16, herpes simplex VP16 protein; teto, tet opertor sequences; pcmv, cytomeglovirus promoter. (b) Immunoblot nlysis of isolted glomeruli from doxycycline (Dox)- or vehicle (Veh)-treted double-trnsgenic mice. After injection, synptopodin protein bundnce remins stble in Dox-treted but not in Veh-treted mice. Protein bundnce of RhoA, dynmin nd ZO-1 re positively correlted with synptopodin bundnce. GAPDH shows equl protein loding. (c) GFP Synpo-CM1+2 (Dox +) protects ginst induced proteinuri. (d) Schemtic of constructs used for the genertion of CnA-trnsgenic mice. (e) Immunoblot nlysis of isolted glomeruli showing reduced stedy-stte protein levels of synptopodin, RhoA, dynmin nd ZO-1 but not of -ctinin-4 in GFP-CnA expressing (Dox) mice. (f) Detection of proteinuri in GFP-CnA expressing (Dox) but not in control (Veh) mice. (g) Model for the regultion of podocyte function by clcineurin. Phosphoryltion of synptopodin by PKA or CMKII promotes binding, which protects synptopodin ginst CtL-medited clevge, thereby contributing to the intct glomerulr filtrtion brrier. Dephosphoryltion of synptopodin by clcineurin (CN) brogtes the interction with This renders the CtL clevge sites of synptopodin ccessible nd promotes the degrdtion of synptopodin. CsA nd E64 sfegurd ginst proteinuri by stbilizing synptopodin stedy-stte protein levels in podocytes. *P o described before 9,18. We injected the mice with ultrpure (300 mg intrperitonelly (i.p.); InvivoGen) nd strted tretment with CsA (25 mg per kilogrm body weight i.p.) or E64 (3 mg per kilogrm body weight i.p.) 1 h fter disese induction. We repeted the, CsA nd E64 injections t 24 h nd killed the mice fter. At 12 nd 36 h, we injected ll mice with 0.8 ml of isotonic sline (i.p.) to prevent hypovolemi. To quntify the level of proteinuri, we diluted 5 ml of urine with 5 ml of double-distilled wter, boiled the diluted urine in smple buffer nd nlyzed the smples by SDS-PAGE followed by Coomssie blue stining. We rn BSA stndrds (0.1, 0.5, 1.0 nd 5.0 mg) on the sme gel nd used them to identify nd quntify urinry lbumin bnds. We quntified the lbumin bnds with ImgeJ softwre (US Ntionl Institutes of Helth) nd determined the cretinine bundnce from the sme urine smples with commercil kit (Exocell) ccording to the mnufcturer s protocol. We clculted lbuminuri s microgrms lbumin per milligrm cretinine. In vivo gene delivery. We chieved in vivo hydrodynmic gene delivery by til vein injection s previously described 9, with some modifictions. We determined bseline urinry protein excretion of 8-week-old femle mice s described bove. We then injected the mice (n ¼ 5 8 per group) with 50 mg of Flg-synptopodin cdna (wild-type, Synpo-CM1+2, Synpo-AA or Synpo-ED) in 2 ml of isotonic sline. In control experiments, we injected the mice with 2 ml of isotonic sline only. We then inoculted the mice i.p. with 300 mg t 1 h nd 24 h fter gene delivery. Forty-eight hours fter gene delivery, we mesured proteinuri gin s described bove. We killed the mice nd monitored the efficcy of the gene trnsfer by western blot nlysis of synptopodin in protein extrcts from livers nd isolted glomeruli nd by double-lbeling immunofluorescence microscopy for Flg nd the podocyte mrker podocin 34. Genertion of podocyte-specific nd tetrcycline-inducible trnsgenic mice. We creted the cdna constructs for the genertion of the teto GFP Synpo- CM1+2 nd teto GFP-CnA trnsgenic mice using the BD Cretor Cloning System (BD Biosciences, Clontech). We confirmed the proper orienttion of the cdna by restriction enzyme digest mpping. We then trnsferred the teto GFP Synpo-CM1+2 nd teto GFP-CnA cdnas from the donor vector to plp-tre2, n inducible tetrcycline-responsive expression vector, using CreloxP site-specific recombintion. We relesed the inducible teto GFP Synpo- CM1+2 nd teto GFP-CnA cdna constructs from the vector bckbone by enzymtic digest nd purified them by gel extrction (QIAquick Gel Exctrction Kit, Qigen) before injecting them into pronuclei of fertilized oocytes of FvB/NJ mice. We identified trnsgenic founder mice by PCR nd mted them NATURE MEDICINE VOLUME 14 [ NUMBER 9 [ SEPTEMBER

8 to podocin-rtta mice on n FvB/NJ bckground 36 to generte doubletrnsgenic doxycycline-inducible podocin-rtta teto GFP Synpo-CM1+2 nd podocin-rtta teto GFP-CnA mice, respectively. We identified doubletrnsgenic mice by PCR 36. We mted podocin-rt-ta teto GFP Synpo-CM1+2 or podocin-rtta teto GFP-CnA F 1 littermtes to obtin F 2 double-trnsgenic mice for experimentl procedures. To induce trnsgene expression in podocytes, we dministered doxycycline (Sigm-Aldrich) t concentrtion of 2 mg ml 1 in 7% sucrose (ph B5) to double-trnsgenic mice in the drinking wter for 7 d 36. Simultneously, we fed the mice specil chow diet contining doxycycline (2,000 p.p.m.; Reserch Diets). We fed the control mice (vehicle) norml chow nd 7% sucrose in the drinking wter. We treted five podocin-rta teto GFP Synpo-CM1+2 mice with doxycycline nd four with vehicle. We lso treted 15 podocin-rtta teto GFP-CnA mice with doxycycline nd 10 with vehicle. Sttisticl nlyses. The results of ll niml studies were ssessed by Student s t-test nd re expressed s mens ± s.d. Additionl methods. Detiled methodology is described in Supplementry Methods online. Note: Supplementry informtion is vilble on the Nture Medicine website. ACKNOWLEDGMENTS We thnk C. Chiu nd S. Rtner for excellent technicl ssistnce nd T. Reinheckel for the nlysis of CtL clevge sites. We thnk the Mount Sini School of Medicine Mouse Genetics Reserch Fcility for performing pronucler injections. We lso thnk J.B. Kopp (US Ntionl Institutes of Helth) for providing the podocin-rtta mice, H. Fu (Emory University) for yellow fluorescence protein tgged difopein nd E.N. Olson (The University of Texs Southwestern Medicl Center t Dlls) for wild-type nd constitutively ctive clcineurin cdna constructs. Y.H.C. ws supported by reserch fellowship from the Albert Einstein College of Medicine, S.F. ws supported by Krger Stiftung nd J.D. ws supported by the Deutsche Forschungsgemeinschft. 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