SPECIAL ARTICLE www.jasn.org

Balancing Signals through TRPC5 and TRPC6 in Podocytes

Anna Greka and Peter Mundel

Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

ABSTRACT ϩ Calcium (Ca2 ) ions are important mediators of cellular homeostasis owing to their on an important exception: the crayfish ability to elicit a dynamic, transient, and tightly regulated range of biochemical muscle.1,5,6 In pioneering two microelec- ϩ responses. More than a decade ago, a nonselective, Ca2 -permeable, cationic trode intracellular recordings, the inves- ϩ conductance was identified in podocytes downstream of angiotensin II (Ang II) tigators were surprised when Na chan- signaling, but its molecular structure remained elusive. Six years ago, transient nel blockers did not abrogate action receptor potential canonical 6 (TRPC6) mutations were found in families with potentials, but instead engendered larger ϩ hereditary FSGS, and TRPC5 and TRPC6 channels are now known as the Ca2 and more prolonged spikes.5,6 Further influx pathways for this previously described, nonselective, cationic current in experiments revealed that the mysterious ϩ podocytes. Ang II activation engages this Ca2 influx to modulate the actin action potentials were generated by the ϩ cytoskeleton in podocytes. These discoveries dovetail with previously described entry of divalent Ca2 ions through volt- ϩ ϩ regulation of actin dynamics by the Ca2 -activated phosphatase, calcineurin, and age-gated Ca2 channels.1 the emergence of Rho GTPases as critical regulators of podocyte function in health Because of these initial experiments, ϩ and disease. Understanding the interconnected signaling regulated by Ca2 cur- important insights have been gained into ϩ rents offers potential new therapeutic targets and highlights the notion that the electrogenic role of Ca2 in excitable synergistic therapies targeting multiple levels of biochemistry may be useful in cells: Electricity is used to open (or gate) treating proteinuric kidney disease. the channels, and in turn the channels are used to make electricity (action po- ●●● ●●● J Am Soc Nephrol 22: – , 2011. doi: 10.1681/ASN.2011040370 tentials). Importantly, most excitable cells ultimately translate their electric ex- citation into another form of activity or A single can negotiate the ure 2). In particular, we explore recently cellular function. Although the list of passage of over 10 million ions per sec- uncovered insights into the activation of processes controlled by Ca2ϩ is lengthy, ond across the plasma membrane,1 and transient receptor potential canonical classical studies in excitable cells focus on calcium (Ca2ϩ) ions, in particular, are (TRPC) channels by Ang II and the re- three: contraction, secretion, and gat- ϩ important mediators of cellular homeo- sulting effects on podocyte signaling un- ing.1 The role of Ca2 in myocyte con- ϩ stasis.1,2 Ca2 permeates the membrane der physiologic and pathologic condi- traction is particularly important in the of virtually every cell to regulate diverse tions (Figure 3). Finally, we highlight the context of this review, as it provides in- vital processes such as muscle contrac- implications of balancing Ca2ϩ-con- sight into the Ca2ϩ-dependent mecha- tion, cytoskeletal structure, vesicle secre- trolled signaling pathways in podocytes

tion, transcription, and pro- for the development of novel antipro- Published online ahead of print. Publication date grammed cell death, to name a few.1,2 teinuric therapies (Figure 4). available at www.jasn.org. Thousands of Ca2ϩ channels on the Similar to sodium (Naϩ) and potas- ϩ Correspondence: Dr. Anna Greka, Division of Ne- plasma membrane precisely control the sium (K ) channels initially recorded in phrology, Massachusetts General Hospital and timing and entry of Ca2ϩ ions and cellu- the classical Hodgkin-Huxley studies of Harvard Medical School, 149 13th Street, Charles- ϩ 1,3,4 2 town, MA 02129, Phone: 617-726-9363; Fax: 617- lar homeostatic mechanisms modulate the squid giant axon, Ca channels 726-5669; E-mail: [email protected]; or the tight control and compartmentaliza- were identified in studies of excitable Dr. Peter Mundel, Division of Nephrology, Massa- tion of these intracellular Ca2ϩ transients cells from crustaceans. In the early 1950s, chusetts General Hospital and Harvard Medical ϩ th 2 School, 149 13 Street, Charlestown, MA 02129; (Figure 1). when Na was firmly established as the Phone: 617-643-8710; Fax: 617-726-5669; E-mail: Here, we review the emerging role of ion mediating action potentials, known [email protected] 2ϩ ϩ Ca signaling in the regulation of podo- as the Na theory of the action poten- Copyright © 2011 by the American Society of cyte function in health and disease (Fig- tial,1 Fatt and Katz accidentally stumbled Nephrology

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nisms underlying all contractile cells, in- ing molecule because of its ability to me- diaphragm, and proteinuria, and is often cluding kidney podocytes.7 diate a dynamic, dramatic, transient, and a starting point for progressive kidney How are cells capable of sensing tightly regulated range of responses. disease.7,12 Thus, regulating the Ca2ϩ? In a resting cell, the levels of free Simply put, the entry or release of any plasticity of the podocyte actin cytoskel- cytoplasmic Ca2ϩ are tightly controlled amount of Ca2ϩ in the cytoplasm, large eton are critical for sustained function of and held extremely low. The resting in- or small, is a call to action.1,2,9 the glomerulus.7,12 Mutations in ϩ ϩ tracellular Ca2 concentration [Ca2 ] encoding nephrin,14 podocin,15 or phos- lies in the 20 to 300 nM range in virtually pholipase C epsilon16 give rise to congen- all cells.1 Calcium homeostasis mecha- PODOCYTES AND CALCIUM ital proteinuria.14,15 Additionally, adult- nisms (Figure 1) such as the Naϩ-Ca2ϩ onset focal segmental glomerulosclerosis exchanger (NCX) and the ATP-depen- The glomerular filtration unit of the kid- (FSGS) is associated with podocyte mu- ϩ dent plasma membrane Ca2 pump ney is a highly specialized corpuscle of tations in genes encoding ␣-actinin 4,17 ϩ (PMCA), along with Ca2 buffers (such capillaries capable of modulating hydro- CD2AP,18 INF2,19 TRPC6,20 and synap- as calbindins or parvalbumin) and inter- static ultrafiltration of blood plasma, al- topodin.21 Most recently, common vari- nal Ca2ϩ stores (endoplasmic reticulum lowing the passage of solutes but retain- ations in glypican 5 associate with ac- [ER], mitochondria uniporter [MiCa]), ing vital proteins.10–12 Among the three quired nephrotic syndrome.22 Taken labor to maintain the cytoplasmic Ca2ϩ layers of the glomerular filtration barrier, together, mutations in these moieties ϩ levels low.8 WhenaCa2 -permeable the podocyte layer comprises unique highlight the importance of the cytoskel- channel opens, whether in the plasma cells with a complex organizing pheno- eton in podocyte health and disease. membrane or on a Ca2ϩ-loaded organ- type, including characteristic interdigi- From the cell biologist’s perspective, elle (such as the inositol triphosphate re- tating foot processes with an interposed the podocyte’s refined repertoire of cyto- ceptor [IP3R] in the ER; Figure 1), Ca2ϩ slit diaphragm that cover the outer as- skeletal adaptations to environmental ions flow transiently into the cytoplasm, pect of the glomerular basement mem- cues12,23–28 renders it an ideal model sys- ϩ until the homeostatic mechanisms pre- brane.10,12,13 Podocyte dysfunction and tem to study Ca2 -dependent actin dy- vail once again to tie up or extrude the cytoskeletal disorganization leads to foot namics in a physiologically relevant con- excess Ca2ϩ ions. Ca2ϩ is a potent signal- process effacement, disruption of the slit text. A central working hypothesis has been that podocyte foot process efface- ment is mediated by rearrangement of PM the actin cytoskeleton.7,23,24 As early as TRPC5/TRPC6 GPCR RTK NCX, PMCA Other channels? 1978, elegant work by Kerjaschki pro- posed that an increase in [Ca2ϩ]i may 29 Ca2+ be an early event in podocyte injury (Figure 2). Complement C5b-9 com- Ca2+ Ca2+ CaC 2+ Ca2+ activated plex-mediated podocyte damage also as- 2+ signalingsig pathways: Ca bubuffersffers sociates with an increase in [Ca2ϩ]i and (parvalbumin,lbumin, Motility/contractionM calbindins)ndins) IP3R/RyR SERCA MiCa Secretion activation of phospholipase C (PLC).30 Channel gating GGene transcription Protamine sulfate, which can cause foot Cell death ER MitochondriaMitochondria process effacement in vivo,31 also in- ϩ 2 32 ϩ ϩ creases [Ca ]i in vitro (Figure 2). Dif- Figure 1. Schematic representation of Ca2 homeostasis in podocytes Ca2 is a potent ferentiated mouse podocytes in culture signaling molecule because of its ability to mediate a dynamic, dramatic, transient, and 2ϩ tightly regulated range of intracellular responses1,2,9 (PM: plasma membrane). Some increase [Ca ]i in response to brady- 33 proteins shown here have not yet been identified or studied in podocytes (calbindins, the kinin, an effect also observed in cul- mitochondrial uniporter or MiCa etc.), but they are likely to be present based on our tured rat podocytes in response to Ang ϩ understanding of calcium homeostasis in other cell types. The influx of Ca2 is likely to II34 (Figure 2). Importantly, in pioneer- be mediated by TRPC5 and TRPC6 channels, which were recorded at the single channel ing whole rat glomerular recordings, level in podocytes,37 but other influx pathways cannot be excluded. TRPC5 and TRPC6 Pavenstadt and colleagues described how are activated by upstream receptors such as G- coupled receptors (GPCR), in- ϩ Ang II evokes a nonselective cationic 62 2 cluding the AT1R, and receptor tyrosine kinases (RTK), similar to other cell types. Ca current in podocytes35 (Figure 2). The is tightly regulated upon entry into the cytoplasm. Calcium homeostasis relies on the ϩ ϩ molecular identity of this current re- Na -Ca2 exchanger (NCX), which has been described in podocytes,110 the ATP-depen- ϩ ϩ mained elusive until 2005, when Winn dent plasma membrane Ca2 pump (PMCA), plasma Ca2 buffers (calbindins, parvalbu- ϩ min, etc.) and internal Ca2 stores (endoplasmic reticulum (ER), mitochondria) to maintain and colleagues provided the first clues by ϩ ϩ low cytoplasmic Ca2 levels.8 When a Ca2 -permeable channel opens, whether in the describing a TRPC6 channel mutation in ϩ ϩ 20 plasma membrane or on a Ca2 -loaded organelle (e.g. the IP3R in the ER), Ca2 ions flow familial FSGS, followed by the identifi- transiently into the cytoplasm, until the homeostatic mechanisms take over once again to cation of five additional mutations by ϩ buffer or extrude the excess Ca2 ions. Pollak and colleagues36 (Figure 2). De-

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TIMELINE | Evolution of calcium signaling in podocytes

Bradykinin increases Antagonistic regulation intracellular Ca2+ in of podocyte actin differentiated mouse dynamics and cell motility podocytes in culture by TRPC5 and TRPC6

Ang II increases Activation of NFAT cytosolic Ca2+ signaling in podocytes activity in rat causes glomerulosclerosis podocytes in culture TRPC6 is a SD associated channel Synaptopodin is a Podocyte-specific PS induced FP Ang II depolarizes Characterization of a direct target of the overexpression of TRPC6 effacement is podocytes in the Na+-Ca2+ exchanger A mutation in TRPC6 anti-proteinuric causes glomerular Ca2+ dependent intact rat glomerulus in podocytes causes familial FSGS effect of CsA disease in mice

1978 1997 1999 2002 2004 2005 2007 2008 2009 2010 2011

PS induced Ca2+ AT1R overexpression Induction of TRPC6 Identification of a TRPC6 loss reduces signaling in podocytes in podocytes induces expression in TRPC6 mutation Ang II-induced glomerulosclerosis acquired proteinuric that causes albuminuria in mice in transgenic rats kidney disease childhood FSGS

TRPC6 mutations associcated with FSGS cause constitutive NFAT activation

Figure 2. Evolution of calcium signaling in podocytes from 1978 to today. tailed electrophysiology recordings dem- transactivation of the EGF receptor members, TRPC1 to TRPC7.9,49 On the onstrating TRPC channel activity in (EGFR) in tubular epithelia42 and basis of functional similarities and se- podocytes were not available until re- podocytes.43 AT1R-EGFR interactions quence alignment, two major subfam- cently, when Tian and colleagues37 un- also activate downstream serine/threo- ilies emerge, TRPC1/4/5 and TRPC3/ veiled TRPC5 and TRPC6 as the chan- nine kinases such as the MAPK path- 6/7 (TRPC2 is a pseudogene in nels downstream of Ang II–evoked way in a process that is known to re- ).49 Four TRP subunits are re- ϩ nonselective cationic conductance ini- quire an increase in cytosolic [Ca2 ].44 quired to form a functional channel, tially identified more than a decade ago35 Ang II induces membrane ruffling and rendering a cation-conducting pore (Figure 2). loss of stress fibers,45 thereby pheno- that is either a homomeric or hetero- copying the depletion of synaptopo- meric tetramer.49 TRPC channel acti- din46,47 or TRPC6.37 vation occurs primarily through acti- ANG II–MEDIATED Ca2؉ vation of phospholipase C (PLC), and SIGNALING IN PODOCYTES the cleavage of phosphatidylinositol bis- 2؉ TRPC CHANNELS AND Ca phosphate (PIP2) which gates the chan- In the context of proteinuric kidney SIGNALING nels directly, as in the case of TRPC4 and disease, angiotensin 1 receptor–medi- TRPC5, or indirectly through its hydro- ated (AT1R-mediated) signaling is of Transient receptor potential (TRP) channels lysis into diacylglycerol, as in the case of particular importance. In classical are receptor-operated, nonselective, cationic TRPC3,6,7.9,50 studies of diabetic nephropathy, an- channels first identified in Drosophila48 (see TRPC6 is closely related to TRPC7, giotensin-converting enzyme inhibi- below for some technical considerations with overlapping expression patterns in tors (ACEi) and angiotensin receptor regarding canonical TRP [TRPC] chan- the kidney.50 TRPC6 channels form ho- blockers (ARB) delay renal progres- nels). Although nonselective for a partic- momers with a characteristic doubly rec- sion.38,39 In 2004, Hoffmann and col- ular cation, from a cellular signaling per- tifying current-voltage relationship and leagues provided direct evidence that spective, the central focus in the study are six times more permeable for Ca2ϩ podocyte-specific overexpression of of TRPC channels has been their ability than Naϩ with a single-channel conduc- AT1R in transgenic rats is sufficient to to negotiate the influx of small but po- tance of 35 pS.49,51 TRPC6 channels, cause proteinuria and FSGS-type le- tent amounts of Ca2ϩ through the cell which inhibit endothelial cell migra- sions (Figure 2).40 At a cellular level, membrane.9,49 The TRP superfamily tion,52 are implicated in pulmonary arte- AT1R signaling is upstream of a num- consists of 28 members segregated into rial hypertension53 and cardiac hypertro- ber of pathways that may be important 7 families with diverse mechanisms of phy and fibrosis.41,54–56 TRPC6 also to TRPC signaling. One is the AT1R- activation, tissue-specific distribution, increases smooth muscle cell contrac- dependent activation of calcineurin.41 and functional properties.9,49 The ca- tion.57–59 TRPC6 null mice were there- Furthermore, AT1R signaling causes nonical TRPC channels include seven fore expected to have decreased vascular

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smooth muscle tone resulting in hypo- TRPC5 in the amygdala results in im- not clearly related to cytoskeletal regula- tension. Surprisingly, however, Birn- paired fear responses in young mice,68 tion, this study supports an important baumer and colleagues observed elevated suggesting a role for these channels in role for TRPC channel-mediated Ca2ϩ BP and increased vascular smooth mus- fear conditioning and during influx in activating calcineurin and its cle contractility in aortic rings from postnatal brain development. In podo- downstream effectors in podocytes. One TRPC6 null mice.60 These unexpected cytes, TRPC5 channels have been studied hypothesis involves a positive feedback effects were the result of compensatory in vitro,37 but no in vivo studies have been loop, whereby calcineurin-NFAT–medi- upregulation of constitutively active performed to date. ated increases in transcription lead to TRPC3 channels.60 In the kidney, TRPC more TRPC channels on the membrane, channels came to the forefront when thus more Ca2ϩ influx and perpetuation gain-of-function TRPC6 mutations were CALCINEURIN SIGNALING IN of the cycle, leading to podocyte injury. linked to familial FSGS20,36 and TRPC6 PODOCYTES Such a feed forward cycle has been dem- was shown to be associated with the slit onstrated in a mouse model of car- ϩ diaphragm.36 An important link between Ca2 and diac hypertrophy, where calcineurin- Recent work has also implicated podocyte injury is the finding that acti- NFAT–mediated increases in TRPC6 TRPC5, in addition to TRPC6, as an im- vation of the Ca2ϩ-dependent phosphatase, transcription promote pathologic car- portant mediator of cytoskeletal changes calcineurin, leads to cathepsin L-mediated diac remodeling.41 More recently, in podocytes.37 Although TRPC5 is ubiq- cleavage of synaptopodin and proteinuria47 however, other TRPC channels, in- uitously expressed, its highest levels of (Figure 2). The calcineurin inhibitor, cyclo- cluding TRPC4 (which is highly ho- expression are in brain and kidney.9,50 sporine A (CsA), prevents synaptopodin mologous and functionally similar to ϩ TRPC5 channels generate Ca2 tran- degradation in vitro, and mice resistant to ca- TRPC564,65), were also shown to medi- sients implicated in neuronal growth thepsin-mediated synaptopodin degrada- ate calcineurin-NFAT activation in car- cone motility61,62 and vascular smooth tion are protected from proteinuria in vivo.47 diac myocytes downstream of Ang II and muscle cell migration.63 Vesicular inser- Conversely, the activation of calcineurin AT1R signaling,41,55,56 strongly suggest- tion of TRPC5 from a reserve pool is in podocytes is sufficient to cause degra- ing this pathway is not specific to downstream of EGF-RTK signaling in a dation of synaptopodin and protein- TRPC6. Recent work by Chen and col- pathway that involves Rac1 and phos- uria.47 Taken together, these findings re- leagues also shows that activation of phatidylinositol 4-phosphate 5-kinase.62 veal a T cell and NFAT-independent NFAT signaling in podocytes is sufficient TRPC5 subunits form homomeric chan- mechanism for the long known antipro- to cause glomerulosclerosis in mice.72 nels with a unique current-voltage (I–V) teinuric effect of CsA: Preservation of This finding begs the question of signature curve in response to stimula- synaptopodin and the podocyte actin cy- whether, by analogy to cardiac myocytes, tion through G protein coupled recep- toskeleton.47 podocytes have also evolved a feed for- tors (GPCR).64,65 TRPC5 channels con- In light of the proposed central role of ward loop linking TRPC channels to duct ten Ca2ϩ ions for one Naϩ ion into synaptopodin in podocytes, we must NFAT activation and subsequent TRPC cells with a single-channel conductance take a moment to revisit the question of transcriptional upregulation. As it stands of 38 pS.66 The current-voltage relation is synaptopodin in null mice. Synaptopo- now, however, there is no direct evidence doubly rectifying, with substantial in- din mutant mice lacking Synpo-short showing that TRPC6 or another member ward current, little outward current up and Synpo-long upregulate Synpo-T of the TRPC family causes FSGS through to ϩ40 mV, and steeply outwardly recti- protein expression in podocytes, thereby the activation of NFAT in podocytes. fying current above ϩ40 mV. rescuing kidney filter function during TRPC5-mediated currents are in- development.69 Moreover, bigenic here- ϩ creased in the presence of 100 ␮MLa3 , tozygosity for synaptopodin and CD2AP Ca2؉-DEPENDENT RHO GTPase in contrast to virtually all other TRPC results in proteinuria and FSGS-like glo- SIGNALING IN PODOCYTES channels (except TRPC4), which are merular damage, underscoring the im- inhibited by La3ϩ at even lower con- portance of synaptopodin and CD2AP The Rho family of GTPases (RhoA, Rac1, centrations.67 TRPC5 is also strongly for sustained kidney filter function.70 In and Cdc42) controls pathways that mod- ϩ potentiated by intracellular Ca2 .66 In- humans, heterozygous mutations in the ulate cytoskeletal dynamics.73,74 Rac1 terestingly, coexpression of TRPC1 promoter of the synpo gene in patients and Cdc42 promote cell motility through and TRPC5 in HEK293 cells results in with idiopathic FSGS reduce gene tran- the formation of lamellipodia and filop- heteromeric TRPC1/5 channels with scription in vitro and protein abundance odia at the leading edge, respectively.74 In voltage dependence similar to N-methyl- in vivo.21 contrast, RhoA promotes the formation D-aspartate receptor channels: At polar- FSGS-causing TRPC6 mutations but of stress fibers and focal contacts, gener- ized potentials the inward current is not wild-type TRPC6 induce constitu- ating a contractile phenotype.74 In podo- gently inwardly rectifying, but steeply tive activation of calcineurin-NFAT–de- cytes, predominance of RhoA activity outwardly rectifying above 0 mV. Loss of pendent gene transcription.71 Although produces a stationary phenotype, sug-

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gesting stable foot processes, whereas exchange factors (GEFs), catalyzing the gests that unopposed TRPC5 activity is predominance of Cdc42/Rac1 activity exchange of bound GDP with free responsible for the Ca2ϩ-calcineurin– mediates a disease-associated motile GTP.74 In vascular smooth muscle dependent degradation of synaptopodin phenotype, suggesting unstable or re- cells, the Rho GEF, Arhgef1, mediates and loss of stress fibers in TRPC6-de- tracted foot processes.7 the effects of Ang II on vascular tone pleted podocytes. These results signifi- ϩ Synaptopodin promotes RhoA sig- and BP in a Ca2 -dependent manner.80 cantly extended our mechanistic under- naling through competitive inhibition Although these studies establish an inti- standing of TRPC in of Smurf1-mediated ubiquitination of mate association between Ca2ϩ influx podocytes by revealing that rather than fo- RhoA.46 Synaptopodin thus protects and Rho GTPase-mediated cytoskeletal cusing on a single channel, we have to con- RhoA from proteosomal degradation, reorganization in other cell types, the sider the balance between TRPC5 and and preserves stress fibers in vitro, mechanisms by which the podocyte TRPC6 in understanding the effects of while safeguarding against proteinuria senses and transduces extracellular cues Ca2ϩ signaling in podocytes (Figure 3). in vivo.46,47 This notion is supported by that modulate synaptopodin and Rho How can this new insight be placed the observation that synaptopodin- GTPase activity, cell shape, and motility in the context of what we have collec- depleted podocytes display loss of remained unclear. In particular, the tively learned thus far? Biochemical stress fiber formation and aberrant Ca2ϩ entry mechanism responsible for and detailed electrophysiology experi- filopodia.69 Synaptopodin also sup- mediating these signaling pathways had ments identify TRPC6 as the predomi- presses Cdc42 signaling through the been elusive. nant TRPC channel in the plasma inhibition of Cdc42:IRSp53:Mena membrane of podocytes, which sup- complexes.75 Overall, synaptopodin ports the notion that at baseline podo- stabilizes the kidney filter by preventing ANTAGONISTIC REGULATION OF cytes maintain an adaptive TRPC6 and the reorganization of the podocyte foot PODOCYTE ACTIN DYNAMICS BY RhoA-predominant, contractile actin process cytoskeleton into a migratory TRPC CHANNELS: A BALANCING cytoskeleton37 (Figure 3). This baseline phenotype.47 ACT? condition, characterized by relative Elegant work by Fujita and col- TRPC6 predominance, is likely to be- leagues76 recently showed that Rho GDP Recently, TRPC5 and TRPC6 were un- come severely biased toward TRPC6 in dissociation inhibitor ␣ null mice77 also veiled as conserved antagonistic regula- the presence of gain of function TRPC6 develop heavy albuminuria, which is at- tors of actin dynamics and cell motility in mutations, where the podocyte’s ho- tributed to increased, constitutively ac- podocytes and fibroblasts through the meostatic mechanisms may be over- ϩ tive Rac1 signaling in podocytes.76 The regulation of RhoA and Rac1, respec- whelmed, leading to Ca2 overload– proposed mechanism involves the Rac1- tively.37 The latter study identified con- mediated cellular injury or death, and dependent accumulation of mineralo- served, antagonistic, mutually inhibitory ultimately, to FSGS (Figure 3). Alter- corticoid receptor into the podocyte signaling pathways triggered by AT1R- natively, an unopposed or overactive nucleus through p21 activated kinase mediated TRPC5 and TRPC6 channel TRPC6-RhoA pathway may tip the bal- phosphorylation.76 Pharmacologic in- activity to control the balance between ance too far in favor of a contractile tervention with a Rac1-specific small motile and contractile phenotypes. Gene phenotype: Podocytes that are too stiff molecule inhibitor (NSC23766) dimin- silencing of TRPC6 results in loss of may have trouble adapting to fluctua- ishes mineralocorticoid receptor hyper- stress fibers, activation of Rac1, and in- tions in glomerular filtration pressure, activity and ameliorates proteinuria and creased motility, which is rescued by and may suffer from a disrupted cyto- renal damage in this mouse model of constitutively active RhoA.37 Conversely, skeleton, disassembly of the actin fila- proteinuria.76 gene silencing of TRPC5 results in en- ment network, and cell death. Interest- Although Ca2ϩ and synaptopodin- hanced stress fiber formation, activation ingly, TRPC6 activity above or below mediated Rho GTPase signaling are im- of RhoA, and decreased motility, which physiologic levels may lead to disease, a portant in the modulation of the podo- is reversed by constitutively active notion supported by the observation cyte cytoskeleton, the question remains Rac1.37 Remarkably, TRPC5 specific- that TRPC6 overexpression in podo- as to how these pathways intersect. Pre- ally interacts with and activates Rac1, cytes results in loss of stress fibers,81 vious studies in other cell types reveal whereas TRPC6 specifically interacts similarly to the silenced TRPC6 mor- that spatially and temporally restricted with and activates RhoA in another, dis- phology.37 This concept is in keeping changes in the concentration of free cal- tinct molecular complex.37 Consistent with neuronal plasticity models, where ϩ ϩ cium (Ca2 flickers) are enriched near with previous studies,47 CsA rescues the either increased or decreased Ca2 the leading edge of migrating cells.78,79 loss of synaptopodin in TRPC6-depleted channel activity ultimately has the ϩ Rho GTPases can be regulated by Ca2 cells.37 In contrast, synaptopodin expres- same effect on the cytoskeleton, result- and by GTPase-activating proteins sion is preserved in TRPC5-depleted ing in the weakening or pruning of a (GAPs), catalyzing the hydrolysis of podocytes.37 The antagonistic relation- synaptic contact.82 Whatever the pre- GTP to GDP, and guanine nucleotide ship between TRPC5 and TRPC6 sug- cise mechanism for podocyte loss may

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A B C podocytes in vivo, it also raises some in- C5 C6 teresting questions: Is the significance of C5 the protective effect lost because of the C6 inherent variability in proteinuria that C6 C5 some animal models display or could this be the sign of a biologic phenomenon? Is Physiologic FSGS Proteinuria? (TRPC6 gain of (Ang II-driven perhaps TRPC3 (over)compensating for function mutations) or acquired) the loss of TRPC6, thus conferring a pro- tective effect from exposure to AngII? In Figure 3. Antagonistic activities of TRPC5 versus TRPC6 signaling in podocytes in health light of the antagonistic effects of TRPC5 and disease: Is it a balancing act? This working model attempts to synthesize published and TRPC6 on podocyte actin dynam- data and underscore the areas in which future experiments are likely to enhance our 37 understanding of TRPC signaling in podocytes. (A) Under physiologic conditions, active ics, it will be interesting to study the TRPC6 channels are more abundant on the podocyte cell membrane, as demonstrated role of TRPC5 in podocytes derived from on the single channel level,37 which underscores their importance for maintaining podo- TRPC6 knockout mice. Future studies cyte integrity, through their selective activation of RhoA.37,46 (B) TRPC6 gain of function will be necessary to elucidate the role of mutations20,36,111 result in overactive TRPC6 channels, the cell is overwhelmed by TRPC6 channels in podocytes in vivo, ϩ TRPC6-mediated Ca2 influx, which ultimately leads to FSGS.83 The observed podo- perhaps by developing podocyte-spe- ϩ cyte injury may result either broadly from Ca2 cytotoxicity and cell death or specif- cific, inducible TRPC6 knockout mice. ically from excessive RhoA-mediated contraction, for example, increased “stiffness” leading to a “broken” actin cytoskeleton, and ultimately, cell death. (C) Given the 76 experimental evidence that (a) constitutive Rac1 activity leads to proteinuria, TRPC CHANNEL TRAFFICKING IN (b) TRPC5 activates Rac1 in podocytes,37 and (c) Rac1 is required for TRPC5 insertion PODOCYTES into the plasma membrane in podocytes,37 it is reasonable to hypothesize that, in states of excess AngII, TRPC5/Rac1–mediated overactivity drives proteinuria. This notion generates interest in TRPC5 channels as mediators of acquired, Ang II–driven Another important aspect of TRPC proteinuria. channel biology is the regulation by upstream receptor pathways. GPCR signaling promotes TRPC6 plasma be, an in vivo study showed that mice studies are needed to delineate the role of membrane insertion in endothelial overexpressing wild-type TRPC6 or TRPC5 in podocytes in health and dis- cells.86 TRPC6 can traffic to the cell TRPC6 gain-of-function mutants de- ease. membrane as part of a large molecular velop albuminuria and FSGS-type le- A recent study reported that TRPC6 complex that includes the large con- ϩ ϩ sions.83 Although proteinuria in these null mice were significantly protected ductance Ca2 -activated K (BK) mice was modest with low and variable from the proteinuric effects of Ang II.84 A channel.87,88 In neurons, BK conduc- penetrance, and structural abnormali- well-designed protocol included the tance results in hyperpolarization, ties were not observed before 6 to 8 analysis of 18 null animals, showing sig- which terminates voltage gated Ca2ϩ months of age,83 these results are con- nificantly mitigated proteinuria to Ang II channel activity.89 The same inhibitory sistent with a dominant TRPC6 hypoth- infusion for the first 4 weeks of infusion. role for BK has been shown for esis (Figure 3). Clearly, more mechanis- The observed protective role of TRPC6 TRPC1-BK interactions in vascular tic work is required to delineate the deletion is surprising given that TRPC6 smooth muscle cells,90 and TRPM4/ precise mechanisms leading to FSGS in null mice were initially reported to be hy- 5-BK interactions in mast cells.91,92 In TRPC6 transgenic mice. pertensive at baseline,60 and thus one contrast, the speculation in podocytes Constitutively active Rac1 signaling would expect increased albuminuria, if is that BK promotes TRPC6 channel in podocytes results in enhanced translo- not at baseline, certainly after Ang II in- activity through membrane hyperpo- cation of the mineralocorticoid receptor fusion. In the original TRPC6 deletion larization, which allows for increased to the nucleus, an effect efficiently study, TRPC3 (over)compensated for Ca2ϩ permeability through TRPC6 blocked by a Rac1 inhibitor.76 Intrigu- the loss of TRPC6.60 Significant upregu- pores.87,93 This would effectively pro- ingly, TRPC5 channels depend on Rac1 lation of mRNA encoding TRPC3 was long TRPC6 channel activity,87,93 for their insertion into the plasma mem- also found in the latter study.84 Indeed, thereby tipping the balance in favor of brane.62 If TRPC5 is indeed an important the well-known compensation by the a RhoA-dominant, contractile pheno- mediator of Ca2ϩ signaling in podocytes, various TRPC channels has plagued the type. This hypothesis has not yet been could the enhanced insertion and over- TRPC deletion field for more than a de- tested experimentally in podocytes, activity of TRPC5 channels in the podo- cade, making it impossible to assign spe- and thus we cannot exclude the alter- ϩ cyte plasma membrane contribute to the cific functions to specific channels.85 nate possibility that Ca2 -mediated in- proteinuria observed in RhoGDI␣ null Although this study has extended our activation of TRPC6 channels precedes mice (Figure 3)? Clearly, future in vivo understanding of TRPC6 channels in or prevails over the effects of BK activ-

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ity. Additionally, BK-TRPC5 interac- mediated store-operated Ca2ϩ entry in should be avoided, as experience in tions have not yet been tested in podo- podocytes.93,94 In the past decade, there other cell types has shown that it is cytes. Future studies will be required to were indeed reports suggesting that prone to erroneous conclusions. As it address these unresolved issues. various TRPC channels could serve as stands, there is compelling evidence Receptor-mediated insertion of store-operated channels (SOCs). How- that TRPC channels are receptor-oper- TRPC5 into the plasma membrane de- ever, the identification of Orai-STIM ated and there is no need to evoke them pends on Rac1.62 The reciprocal de- as the best-characterized SOC channel as components of SOCs. pendence between TRPC5 and Rac1 complex and the channel responsible Another important consideration ϩ activities suggests there may be a feed for Ca2 release activated current involves the idea that TRPC6 channels 95–97 forward loop: Rac1 promotes channel (ICRAC) has cast serious doubt on may act as mechanosensors in podo- localization to the membrane,37,62 the idea that TRPC channels are com- cytes.98 The term mechanosensor de- which in turn triggers enhanced ponents of SOCs.9,50 It is worth noting scribes a channel directly gated by me- ϩ TRPC5-mediated Ca2 influx, thereby that most data postulating TRPCs as chanical force, thus acting as its own ϩ increasing Rac1 activity.37 The cycle SOCs are solely based on Ca2 imag- force sensor, to be distinguished from a ϩ may be terminated when the Ca2 sig- ing, whereas most of the electrophysi- mechanically sensitive channel that is nal wanes, perhaps because of deple- ology studies refute the idea that activated by second messengers down- ϩ tion of the pool of TRPC5-containing TRPCs are SOCs.9 Ca2 imaging alone stream of true mechanical force sen- vesicles, or because of channel inacti- is prone to false positives, and electro- sors.99 By this definition, a review of vation. What could be the physiologic physiology alone is prone to false neg- the literature reveals little evidence to ϩ significance of such a feed forward atives.9 Ca2 imaging is an indirect support the notion that TRPC6 chan- model? When the hydrostatic pressure readout of channel function because it nels are mechanosensors. One study ϩ across the glomerular filter changes, reflects the total cytosolic [Ca2 ]re- reported that the interaction of podo- ϩ dictated by the cardiac cycle or under gardless of the cause. Ca2 entry into cin with TRPC6 results in enhanced conditions of increased systemic BP, the unbuffered cytoplasm can affect TRPC6 channel activity in oocytes in a ϩ foot process remodeling is required to the activity of numerous Ca2 -acti- cholesterol-dependent manner.100 An- maintain an intact filter. According to vated channels, transporters, or other other report suggested that receptor this model, BP-driven increases in Ang molecules, which in turn influence the and mechanically mediated TRPC6 ac- ϩ II may signal to TRPC5 channels, trig- imaging results.9 In Ca2 imaging ex- tivity share a common underlying gering their insertion into the plasma periments, voltage levels, which fuel mechanism, which involves lipid sens- ϩ membrane, thereby increasing Rac1 the entry of Ca2 ions into the cell, are ing by the channel.101 Similarly, in an activity. This event would counter the uncontrolled. In contrast, patch clamp elegant study, Mederos y Schnitzler effects of the more abundant TRPC6/ electrophysiology offers a direct mea- and colleagues102 showed that TRPC6 RhoA pathway and therefore promote surement of channel activity if and is not a mechanosensitive channel, but transient, adaptive foot process actin when these channels are present in the rather, that the AT1R imparts mecha- remodeling. Under maladaptive plasma membrane.9 Perfusion of the nosensitivity to vascular smooth mus- pathophysiology of excessive AT1R40 membrane with defined solutions, cle cells through its effects on TRPC6 or Rac176 signaling, excess TRPC5 tight control (clamping) of the voltage, channels, to mediate the Bayliss ef- channel insertion may promote exces- and the opportunity to apply com- fect,103 the intrinsic property of arterial sive foot process remodeling, which pounds on either side of the mem- blood vessels to constrict in response to causes podocyte injury (Figure 3). Fur- brane, coupled with excellent time and rises in intraluminal pressure. Thus, ther work is needed to elucidate the pre- current resolution, are clear advan- with regard to TRPC6, the weight of cise mechanisms for TRPC5 channel tages of this technique.9 However, in the evidence suggests that, rather than trafficking in podocyte health and dis- standard patch clamp, the intracellular direct mechanical gating, there is likely ease. contents are dialyzed out, removing indirect sensitivity to mechanical important diffusible signaling mole- forces through signaling mechanisms cules.9 Perforated patch recordings can involving plasma membrane lipids. In TRPC CHANNELS IN PODOCYTES: be performed to overcome this diffi- light of the discovery of TRPC5 chan- A FEW TECHNICAL culty, but even in their absence, elec- nels as important mediators of Ca2ϩ CONSIDERATIONS trophysiology is the technique of influx in podocytes,37 it is also worth choice to record TRP channel activity.9 considering this question more ϩ Although TRPC channels are firmly es- Thus, a combination of Ca2 imaging broadly. There is one report of osmo- tablished as receptor-operated chan- and electrophysiology may ultimately mechanical stimulation of TRPC5, nels (ROCs)50 in a wide array of cell be the most illuminating approach for which was however dependent on al- 37 104 types including podocytes, some in- the characterization of TRP channels terations in PIP2 levels, and thereby vestigators have implicated TRPC- in podocytes, but Ca2ϩ imaging alone

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not directly gated by mechanical gated channel candidates, it is essential as a mechanosensor lacks experimental forces. to differentiate between channels that evidence and is thus in need of further In general, TRP ion channels are are directly gated by mechanical forces investigation. emerging as candidate transduction and those that are downstream of a channels in a wide variety of sensory force sensor. It is also essential to show systems, particularly those involved in these candidate proteins fulfill a num- sensing mechanical stimuli. As re- ber of specific criteria (reviewed in de- CONCLUSIONS AND CLINICAL viewed in great detail by Christensen tail99). To date, therefore, the question IMPLICATIONS and Corey,99 for these TRP channels, of whether TRPC5, TRPC6, or any and generally for all mechanically other ion channel in podocytes serves A fundamental question in cell biology is how a signal maintains specificity in Level 1 time and space. This is particularly im-

ACEI/ARB Ang II/AT1R portant in contractile cells such as podocytes, where the continuous re- Level 2 modeling of the actin cytoskeleton is

New target? TRPC5 vs. TRPC6 required to adapt to ever-changing en- vironmental cues. This review high- Level 3 lights the critical role played by the tiny 2ϩ CsA Calcineurin vs. PKA/CaMKII but potent Ca ion in this dynamic Input from other pathways process. Although more work is needed (nephrin, TLRs, RTKs, Level 4 to develop a mechanistic understand- Wnt/β-catenin etc.?) New target? Synaptopodin NFAT ing of TRPC channelopathies in podo- cytes, a novel emerging paradigm sug- Level 5 gests the balance between TRPC5 and

NSC23766 Rac1 vs. RhoA vs. Cdc42 TRPC6 may be particularly important in understanding the effects of Ca2ϩ Level 6 signaling in podocytes.

Eplerenone MR TRPC5 Actin TRPC6 Unresolved questions of current in- terest include the role of synaptopodin in Figure 4. A model for multiple signaling pathways in podocyte injury: Is a multidrug, the TRPC-dependent regulation of Rho synergistic therapy the answer to proteinuria? A synthesis of work by many groups GTPase activity in podocytes. Long-term suggests that multiple signaling levels are involved in the Ang II–mediated regulation of studies should reveal if and how, similar ϩ podocyte function in health and disease. Level 1 consists of the binding of Ang II, whose to myocytes, podocytes employ Ca2 as a availability is limited by ACE inhibitors, to AT1Rs, which are blocked by ARBs. Subse- selective activator of kinases or phospha- ϩ quent activation of TRPC5 and TRPC6 channels on level 2 results in Ca2 influx into tases to mediate contraction versus relax- podocytes. TRPC5-driven signaling may predominate under pathologic conditions of ation or increased foot process motility. excess Ang II and/or Rac1 activity. TRPC5-targeted agents may therefore be a novel 2ϩ Importantly, a systematic investigation therapeutic approach to proteinuria. On level 3, Ca -activated phosphatases (calcineu- of all Ca2ϩ influx, release, and efflux rin) or kinases (PKA and CamKII) battle for downstream effects on their mutual targets, mechanisms is needed to ultimately ob- synaptopodin and NFAT (level 4). Synaptopodin-preserving agents such as CsA exert 2ϩ their antiproteinuric effect by blocking the calcineurin-initiated degradation of synap- tain a comprehensive view of Ca sig- topodin. Stabilization of synaptopodin protein abundance or inhibition of NFAT signaling naling in podocytes (Figures 1 and 3). may be another therapeutic approach to proteinuria. On level 5, Rac1, RhoA, and Cdc42 Another open question is the rela- battle for downstream effects on the actin cytoskeleton. Inhibition of Rac1 has an tion of TRPC/Rho GTPase-mediated antiproteinuric effect in vivo, perhaps because of the decreased activity of mineralocor- actin remodeling to nephrin and Nck- ticoid receptors (MR), similar to treatment with Eplerenone (level 6). Synergistic mecha- mediated remodeling of podocyte actin nisms on level 6 include (a) the Rac1-driven insertion of TRPC5 channels in the plasma dynamics.7,23,105,106 A recent report membrane (level 6), which potentiates the activity of Rac1 in a positive feedback loop, suggests that nephrin may suppress and (b) the effects of Rac1 on the actin cytoskeleton, resulting in maladaptive podocyte TRPC6 channel activity under physio- foot process motility, which correlates with proteinuria (level 6). RhoA and Cdc42 also logic conditions, whereas TRPC6 mu- affect the actin cytoskeleton (level 6). NFAT promotes TRPC6 transcription (level 6). ϩ tations causing FSGS escape the inhib- Although AngII and AT1Rs are centrally important in podocyte Ca2 signaling, inputs 107 from many other pathways are likely to modulate the molecular events in each of the itory effect of nephrin. Future signaling levels, for example, signaling through the Nephrin pathway. This model also studies should test whether slit dia- offers an explanation why the combined inhibition of several levels is necessary for phragm-mediated actin dynamics can effective and sustained antiproteinuric treatment (TLRs, toll-like receptors; RTKs, receptor modulate TRPC signaling, and vice tyrosine kinases). versa, whether TRPC signaling can

8 Journal of the American Society of Nephrology J Am Soc Nephrol 22: ●●●–●●●, 2011 www.jasn.org SPECIAL ARTICLE modulate slit diaphragm protein func- ated regulation of podocyte actin dy- 5. Fatt P, Katz B: The effect of inhibitory nerve tion. namics involves at least six signaling impulses on a crustacean muscle fibre. J Physiol 121: 374–389, 1953 Proteinuria is a cardinal sign and a levels, each of which can receive addi- 6. Fatt P, Katz B: The electrical properties of prognostic marker of kidney disease, tional input from multiple other signal- crustacean muscle fibres. J Physiol 120: and also an independent risk factor for ing pathways (Figure 4). This model may 171–204, 1953 cardiovascular morbidity and mortal- help explain why ACEi and ARBs have 7. Faul C, Asanuma K, Yanagida-Asanuma E, ity.108 For decades, proteinuric kidney only limited efficacy as antiproteinurics. Kim K, Mundel P: Actin up: Regulation of podocyte structure and function by com- disease, such as hypertensive and dia- It also highlights why the combined or ponents of the actin cytoskeleton. Trends betic nephropathy, have been treated synergistic effect of specific agents tar- Cell Biol 17: 428–437, 2007 with essentially the same agents, target- geted to specific levels within the signal- 8. Schwaller B: Cytosolic Ca2ϩ buffers. Cold ing the renin-angiotensin system.109 ing cascade, similar to oncologic treat- Spring Harb Perspect Biol 2, 2010, How can our enhanced knowledge of ments, may ultimately be our best a004051 2ϩ 9. Clapham DE: TRP channels as cellular sen- Ca signaling in podocytes translate answer for the effective treatment of pro- sors. Nature 426: 517–524, 2003 into new therapeutic interventions that teinuria. 10. Kriz W, LeHir M: Pathways to nephron loss may help our patients in the clinic? The starting from glomerular diseases-insights discovery that Ang II acts through from animal models. Kidney Int 67: 404– TRPC5 and TRPC6 to target Rho GT- NOTE ADDED IN PROOF 419, 2005 11. Mundel P, Shankland SJ: Podocyte biology 37 Pase activity offers a molecular, and response to injury. J Am Soc Nephrol mechanistic understanding of how The following studies were published after ac- 13: 3005–3015, 2002 ACEi and ARB treat proteinuria in a ceptance of this manuscript: Vassiliadis J, 12. Somlo S, Mundel P: Getting a foothold in podocyte-specific manner, above and Bracken C, Matthews D, O’Brien S, Schiavi S, nephrotic syndrome. Nat Genet 24: 333– beyond their systemic BP lowering ef- Wawersik S: Calcium mediates glomerular 335, 2000 13. Mundel P, Kriz W: Structure and function of 39 filtration through calcineurin and mTORC2/ fects (Figure 4). podocytes: An update. Anat Embryol (Berl) A new disease paradigm thus Akt signaling. J Am Soc Nephrol 22: 1453– 192: 385–397, 1995 emerges whereby proteinuria arises as a 1461, 2011; Zhu L, Jiang R, Aoudjit L, Jones 14. Kestila M, Lenkkeri U, Mannikko M, Lamer- result of dysfunctions in a multilevel sig- N, Takano T: Activation of RhoA in podo- din J, McCready P, Putaala H, Ruotsalainen naling cascade, which bring together up- cytes induces focal segmental glomeruloscle- V, Morita T, Nissinen M, Herva R, Kashtan rosis. J Am Soc Nephrol 22: 1621–1630, 2011. CE, Peltonen L, Holmberg C, Olsen A, stream receptor pathways, TRPCs, syn- Tryggvason K: Positionally cloned gene for aptopodin, NFAT, Rho GTPases, and a novel glomerular protein–nephrin–is mu- downstream targets such as the actin cy- tated in congenital nephrotic syndrome. toskeleton or the mineralocorticoid re- ACKNOWLEDGMENTS Mol Cell 1: 575–582, 1998 ceptor (Figure 4). The detailed mecha- 15. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, nistic understanding of podocyte- We apologize to our colleagues whose work Niaudet P, Antignac C: NPHS2, encoding specific signaling validates the podocyte we were not able to cite in this review because the glomerular protein podocin, is mutated as the target of choice for the treatment of space limitations. Reviews were often in autosomal recessive steroid-resistant ne- phrotic syndrome. Nat Genet 24: 349–354, of proteinuria. Although still speculative, quoted at the expense of original work. We this schematic understanding of key 2000. thank D. Corey, W. Kriz, M. Winn, and M.A. 16. Hinkes B, Wiggins RC, Gbadegesin R, Vlan- molecules provides a framework and Arnaout for helpful discussions. A.G. is gos CN, Seelow D, Nu¨rnberg G, Garg P, should pave the road for the identifica- funded by a Nephcure Young Investigator Verma R, Chaib H, Hoskins BE, Ashraf S, tion of novel drug targets in podocytes Grant and NIH Grant DK083511 and P.M. by Becker C, Hennies HC, Goyal M, Wharram that may act synergistically with estab- NIH Grants DK57683 and DK062472. BL, Schachter AD, Mudumana S, Drum- lished agents (Figure 4). For example, we mond I, Kerjaschki D, Waldherr R, Dietrich A, Ozaltin F, Bakkaloglu A, Cleper R, Ba- can imagine a new agent acting as an in- sel-Vanagaite L, Pohl M, Griebel M, Tsygin hibitor of TRPC5 channels: This would AN, Soylu A, Muller D, Sorli CS, Bunney abrogate Rac1 signaling, shut down REFERENCES TD, Katan M, Liu J, Attanasio M, O’toole Rac1-mediated TRPC5 membrane inser- JF, Hasselbacher K, Mucha B, Otto EA, tion, and thus act synergistically with 1. 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J Am Soc Nephrol 22: ●●●–●●●, 2011 Balancing Ca2ϩ Signals through TRPC5 and TRPC6 11 SPECIAL ARTICLE www.jasn.org

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12 Journal of the American Society of Nephrology J Am Soc Nephrol 22: ●●●–●●●, 2011