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TRPC5 Does Not Cause or Aggravate Glomerular Disease

Xuexiang Wang , Ranadheer R. Dande, Hao Yu, Beata Samelko, Rachel E. Miller, Mehmet M. Altintas, and Jochen Reiser

Department of Medicine, Rush University Medical Center, Chicago, Illinois

ABSTRACT Transient receptor potential channel 5 (TRPC5) is highly expressed in brain and or pharmacologic inhibition of TRPC5 pro- kidney and mediates influx and promotes cell migration. In the kidney, loss tected mice from albuminuria. Yet, the di- of TRPC5 function has been reported to benefit kidney filter dynamics by balancing rect effect of TRPC5 overexpression in mice podocyte cytoskeletal remodeling. However, in vivo gain-in-function studies of was not shown and a direct pathogenic TRPC5 with respect to kidney function have not been reported. To address this role of TRPC5 for proteinuria remained gap, we developed two transgenic mouse models on the C57BL/6 background by obscure. overexpressing either wild-type TRPC5 or a TRPC5 ion-pore mutant. Compared with We generated two novel transgenic nontransgenic controls, neither transgenic model exhibited an increase in - mouse models by overexpression of ei- uria at 8 months of age or a difference in LPS-induced albuminuria. Moreover, acti- ther wild-type TRPC5 (TG) or pore mu- vation of TRPC5 by Englerin A did not stimulate proteinuria, and inhibition of TRPC5 tant dominant negative TRPC5 (DN) in by ML204 did not significantly lower the level of LPS-induced proteinuria in any mice on a C57BL/6 background (B/6). group. Collectively, these data suggest that the overexpression or activation of With these unique models, we have the the TRPC5 does not cause kidney barrier injury or aggravate such injury opportunity to investigate the functional under pathologic conditions. role of TRPC5 on kidney injury with ap- propriate controls. J Am Soc Nephrol 29: 409–415, 2018. doi: https://doi.org/10.1681/ASN.2017060682 The level of TRPC5 in these transgenic mouse models was detected at both the nucleic acid and protein levels using quantitative PCR (qPCR), Western TRPC5,a memberof the TRP channel fam- barrier. The transient receptor potential blot, andimmunofluorescence.Inthekid- ily, is a calcium permeant cation channel (TRP) superfamily is comprised of non- neys, TRPC5 mRNA expression was 8–10- thought to regulate the actin cytoskeleton selective calcium (Ca2+)-permeable cation fold higher in both TG and DN compared and cell shape in neurons. TRPC5 has been channels that are widely expressed in cells with B/6 (Figure 1A). The qPCR products proposed to influence permeability prop- and critical to cell behavior, physiology, on agarose gel showed a single band at erties of the glomerular filtration barrier and pathology. In kidney, the subfamily C approximately 100 bp (Supplemental Fig- through effects on the podocyte cytoskele- members 5 (TRPC5) and 6 (TRPC6) have ure 1A), indicating the integrity of the ton.Theauthorsmadetwonoveltransgenic been identified for having vital roles in product as well as the specificity of the mouse models overexpressing either wild- regulation of Ca2+ homeostasis in fibro- primers. In addition, mRNA levels of type (TG) or dominant negative (DN) blasts and podocytes. These channels TRPC5. Urinary protein excretion was were suggested to be the antagonistic reg- similar among TG, DN, and B/6 mice; ulators of actin dynamics and cell motil- 1 Received June 23, 2017. Accepted September 26, podocyte morphology was unaffected; ity in podocytes. Evidence suggests that 2017. and the proteinuric response to lipopoly- dysregulation of TRPC6 leads to kidney Published online ahead of print. Publication date saccharide (LPS) injection did not differ injury and proteinuria in and available at www.jasn.org. among the mouse lines. Injection of mice animals.2–4 However, there are a limited with the TRPC5 agonist Englerin A and number of studies on the role of TRPC5 in Correspondence: Dr. Jochen Reiser, Department of Medicine, Rush University Medical Center, 1717 antagonist ML204 did not modify pro- proteinuric kidney disease, and, in partic- W. Congress Pkwy., Kellogg Building, Suite:1035, teinuria. The findings do not add support ular, gain-in-function studies for TRPC5 Chicago, IL 60612. Email: [email protected] fi to a speci c role of TRPC5 in regulating are missing. In a recent study, Schaldecker Copyright © 2018 by the American Society of the properties of the glomerular filtration et al.5 demonstrated that genetic knockout Nephrology

J Am Soc Nephrol 29: 409–415, 2018 ISSN : 1046-6673/2902-409 409 BRIEF COMMUNICATION www.jasn.org other TRPC channels were examined and similarly to LPS challenge as the DN or Significance Statement no significant differential expression was the B/6 group did. In the second experi- found in TRPCs 1–4, 6, and 7 (Supplemen- ment, two lower doses of LPS (235mg/kg) TRPC5, a member of the TRP channel family, tal Figure 2). Further analysis using brain were injected 24 hours apart and pro- isa calcium permeant cation channel thought lysates from 6-week-old mice showed that teinuria was measured 24 hours after to regulate the actin cytoskeleton and cell shape in neurons. TRPC5 has been proposed TRPC5 protein was overexpressed in TG each injection. Similarly, all three groups to influence permeability properties of the and DN mice as detected by a customized of animals exhibited increased albumin- glomerular filtration barrier through effects antibody (Figure 1, B and C). The Western uria after LPS challenge. But we did not on the podocyte cytoskeleton. The authors blots were validated by a monoclonal anti- observe any differences in proteinuria made two novel transgenic mouse models body from NeuroMAB. Only the custom- among all groups (Figure 2C). Kidney overexpressing either wild-type (TG) or dominant negative (DN) TRPC5. 2Protein ized antibody exhibited weak TRPC5 bands samples from the second experiment excretion was similar among TG, DN and B/6 on primary podocyte lysates (Supplemental were used for transmission electron mi- mice,podocyte morphologywas unaffected, Figure 1, B–D). Immunofluorescence in croscopy (TEM) to analyze podocyte and the proteinuric response to lipopoly- kidney sections revealed colocalization of foot process (FP) effacement. By count- saccharide injection did not differ among the TRPC5 with podocyte marker synaptopodin ing the number of FPs and slits over a mouse lines. Injectionofmice withtheTRPC5 agonist Englerin A and antagonist ML204 did and significantly stronger TRPC5 staining in measured length of glomerular basement notmodifyproteinexcretion.Thefindingsdo TG and DN groups (Figure 1, D and E). membrane (GBM), we detected a similar not add support to a specific role of TRPC5 in Using Ca2+ imaging in primary podocytes degree of effacement and FP width regulating the properties of the glomerular from transgenic mice, we identified that 100 among all groups (Figure 2, F–H). This filtration barrier. mM carbachol (Cch) evoked a higher rise of result was in accordance with the pro- 2+ fi intracellular Ca in TG mice than that in teinuria ndings and indicated that over- that antagonizing TRPC5 by ML204 did DN mice (Figure 1, F and G). These data expression of TRPC5 would not result in not rescue glomerular injury from LPS demonstrated that the novel transgenic higher susceptibility or aggravated kid- challenge. mouse models had increased expression of ney injury after LPS administration. Ca2+ homeostasis is indispensable for TRPC5 and TG mice exhibited increased Injection of drugs either activating or orchestrating multiple cellular functions functional TRPC5 channels in podocytes. inhibiting TRPC5 was implemented to because of its role as a spatial and tem- To characterize the effects of overex- further assess the relationship between poral second messenger in various cell fi pressed TRPC5 on kidney ltration, we TRPC5 and proteinuric disease. Englerin types.12 The TRP superfamily par- evaluated albuminuria in TG, DN, and A, a reported potent and selective TRPC4/ ticipates in Ca2+ homeostasis and is 8 B/6 mice (Figure 2A). At 2 months old, TRPC5 activator, was utilized to stimu- involved in critical physiologic and all mice had little albuminuria without a late the TRPC5 channels in TG, DN, and pathologic cellular events.13 There is significant difference among the groups. B/6 animals. Five micromolar of Englerin convincing evidence that dysregulation By month 8, there was no change in al- A evoked a modest rise in intracellular Ca2+ of one particular TRPC member, buminuria, which remained low in all in both TG and DN podocytes in vitro TRPC6, leads to FSGS by either gain- groups. Histologic analysis by hematox- (Figure 3A). On the basis of the previous or loss-of-function mutation.3,4,14,15 ylin and eosin (HE) and periodic acid– in vivo studies,9,10 we chose to inject two Other TRPC channels are relatively less Schiff (PAS) stain did not show any evident doses of Englerin A (233 mg/kg; i.p.) 24 studied in the context of kidney diseases. morphologic changes at the level of glo- hours apart and measure proteinuria 24 merulus in all mice (Figure 2, D and E). hours after each injection. As displayed in It was reported that TRPC5 and TRPC6 These data suggested that overexpression Figure 3B, activating TRPC5 by Englerin had antagonistic effects on angiotensin of TRPC5 does not lead to natural progres- A did not cause any noticeable increase II (AngII) receptor overexpressing po- 1 sion in kidney filtration barrier injury. in proteinuria in all groups. Next, we tested docytes upon AngII treatment. How- The LPS model was used to examine the TRPC4/TRPC5 inhibitor ML20411 in ever, AngII-evoked TRPC5 activation the kidney filtration barrier defects and the “two-doses” LPS model. A 20-minute cannot be seen in primary podocytes albuminuria in our transgenic animals.6 preincubation of primary podocytes with from rats or mice.16,17 Additionally, In the first experiment, a high dose of 20 mM ML204 blocked Cch-induced Ca2+ one study showed that plasma and sera LPS (10 mg/kg) was injected intraperi- transients significantly in both TG and from patients with recurrent FSGS pro- toneally (i.p.) and proteinuria was eval- DN groups (Figure 3C). Physiologic doses duced robust effects on podocyte TRPC6 uated at 24 and 48 hours. As reported of ML204 (332 mg/kg) were injected (i.p.) channels but had minimal effects on po- before,6,7 there was a significant increase 6 hours before the first and second LPS docyte TRPC5 channels.18 The first di- in albuminuria at 24 hours, which sub- treatments and 6 hours after the second rect evidence linking TRPC5 to kidney stantially decreased at 48 hours in all LPS treatment. There was no statistical injury came recently, when a study re- three groups (Figure 2B). LPS induced a distinction of albuminuria in ML204- ported that inhibition or loss of TRPC5 reversible change in podocyte cytoskele- treated groups compared with vehicle- protected podocytes from cytoskeletal ton. However, the TG group responded treated groups (Figure 3D), indicating remodeling upon injury and decreased

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Figure 1. TRPC5 was overexpressed in the novel transgenic mice. (A) TRPC5 mRNA in kidney was significantly elevated in transgenic (TG)anddominantnegative(DN)micecomparedwithcontrol(B/6)animals,wherethe(average)TRPC5mRNAwassetto1.0.RNAfrom HEK 293 cells that were transfected with either wild-type or dominant negative TRPC5 was used as positive control. (B) Representative Western blot images showed the expression levels of TRPC5 and GAPDH housekeeping in brain lysates of TG, DN, and B/6 mice. The whole cell lysate of HEK 293 transfected with an expression plasmid for mouse TRPC5 was presented as control. (C) Quantitative analysis of TRPC5 expression (normalized with GAPDH) exhibited that TRPC5 was overexpressed in TG and DN mice compared with that in B/6 mice. (D) Representative immunofluorescence images showing the expression of synaptopodin (Synpo) and TRPC5 in the glomeruli of TG, DN, and B/6 mice. (E) Quantitative analysis of TRPC5 expression (normalized with synaptopodin) exhibited that TRPC5 levels were significantly higher in TG and DN mice. (F) Time course Ca2+ transients in primary podocytes after Cch treatment showed a higher response in TG mice than DN mice. (G) Quantification of average peak amplitude exhibited higher Ca2+ transients in primary podocytes of TG mice than that of DN mice. n$5 per group, *P,0.05 compared with B/6; n$150 cells per group, #P,0.05 compared with DN; error bars are 6SEM. ab, antibody; CTL, control; DF/F0, change in florescence normalized by the baseline fluorescence.

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Figure 2. Natural progression and LPS challenge caused similar level of kidney filtration injury in TG, DN, and B/6 mice. (A) No noticeable change in albuminuria over time was found for TG, DN, and B/6 mice. (B) Single dose LPS challenge induced substantial increase in al- buminuria at 24 hours followed by a decrease at 48 hours. There is no difference between each of TG, DN, and B/6 mice at each time point. (C) Two doses of LPS challenge caused a similar increase in albuminuria for all three groups. (D) Representative images of hematoxylin and eosin and periodic acid–Schiff staining of kidneys from 8-month-old mice (original magnification, 320). (E) Glomerular injury scoring on the basis of percentage of sclerosis showed no noticeable damage. (F) Analysis of TEM images exhibited equal degree of FP effacement after two doses of LPS challenge. (G) Average FP widths calculated for TG, DN, and B/6 were all similar. (H) Representative TEM images of kidneys after two doses of LPS challenge (15,0003). Analysis of TEM images exhibited equal degree of FP effacement. n$6 per group; error bars are 6SEM. Alb/Cr, albumin/creatinine ratio; HE, hematoxylin and eosin; PAS, periodic acid–Schiff. proteinuria in mice.5 Of note, unlike investigate TRPC5 function using a TRPC5 was constructed with a mutation TRPC6, there is no evidence thus far in- gain-of-function model of TRPC5. in the pore region by replacing the con- dicating that the pathologic effect of Recently, we developed these novel served LFW motif, which is essential for TRPC5 is associated with patients who transgenic mouse models by overex- the channel function, with three ala- suffer from kidney disease. To better un- pressing either wild-type or pore mutant nines.19,20 This nonfunctional mutation derstand the potential role of TRPC5 TRPC5 (Supplemental Figure 3). The was confirmed in our in vivo Ca2+ imag- in kidney injury, it is necessary to nonfunctional, dominant negative ing data, where the pore mutant TRPC5

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Figure 3. Englerin A and ML204 treatments have no effect on proteinuria in TG, DN, and B/6 mice. (A) Englerin A evoked a significant increase in Ca2+ transients in primary podocytes from TG and DN. (B) TRPC5 agonist Englerin A injection resulted in no change in al- buminuria in TG, DN, and B/6 mice. (C) Average peak amplitude of Cch- and Cch+ML204-induced Ca2+ transients in TG and DN primary podocytes exhibited a markedly decreased Ca2+ transient in both groups. (D) TRPC5 antagonist ML204 injection had no significant effect on albuminuria levels between each of TG, DN, and B/6 mice. N mice $6 per group; N cells $100 per group; #P,0.05 compared with baseline or Cch; error bars are 6SEM. Alb/Cr, albumin/creatinine ratio; DF/F0, change in florescence normalized by the baseline fluorescence. overexpression DN mice had 40% lower albuminuria 24 hours after challenge. challenge model. It was reported that a Cch-induced Ca2+ transientthanthatin LPS causes podocyte cytoskeletal re- high dose (2320 mg/kg) of ML204 (i.p.) wild-type TRPC5 overexpression TG modeling via aCa2+-mediated path- ameliorated LPS-induced proteinuria.5 mice (Figure 1F). This characteristic way.22,23 Thus, we selected this model Our physiologic dose (332mg/kg)of made our TG and DN mice an ideal pair to examine the effect of TRPC5 in vivo ML20424 demonstrated a slight decrease of controls. Considering a comparable and found that overexpression of TRPC5 in albuminuria compared with vehicle- amount of TRPC6 overexpression was did not aggravate glomerular injury. treated animals. Of note, the original re- sufficient to produce kidney filtration Next, we chose to interfere with the port on ML204 indicated that it also injury in mice,21 this amount of TRPC5 activity of TRPC5 by either activation or modestly inhibited TRPC6 channels.11 overexpression in TG mice should inhibition of the channel. Englerin A is a In a recent publication, it was stated have been adequate to detect a kidney potent and selective stimulator for that ML204 was unlikely to be a specific phenotype. TRPC4/TRPC5 and is widely tested on TRPC5 channel inhibitor because it pro- LPS injection is a well established cancer therapies.8,10 Our results showed duced an acute and potent Ca2+ release model to induce kidney filtration barrier that a tolerated dose (233mg/kg)of from intracellular stores.17 TRPC family defects and proteinuria independent of B Englerin A (i.p.) did not exhibit a change members are known to be store-operated,25 or T cells.6,22 This reversible injury is as- in albuminuria. Then, we tested TRPC4/ which could all be affected by ML204. sociated with FP effacement and peak TRPC5 antagonist ML204 in the LPS Therefore, inhibition of TRPC5 by

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ML204 did not salvage the glomerular Immunofluorescence Englerin A Injection injury and the effect of ML204 was prob- Frozen kidney tissues were fixed with ice-cold Twodoses of 3 mg/kg Englerin Awere injected ably not due to antagonizing TRPC5 alone. acetone for 10 minutes. After 30 minutes of (i.p.) to 14–16-week-old female mice (20–25 g) Overall, this study reveals that over- blocking (2.5% donkey normal serum and 24 hours apart. Urine samples were collected 24 expression or activation of TRPC5 is 2.5% FBS in PBS), samples were incubated hours after each injection. not the culprit for de novo or aggravat- with synaptopodin (1:100; Santa Cruz, Dallas, ing proteinuric kidney disease. Future TX) and TRPC5 (1:100; GenScript) primary an- ML204 Injection research will be required to clarify a tibodiesfor2hourseach,followedbyAlexaFluor Two-doses of LPS were injected to female mice potential role for TRPC5 in renal secondaryantibodies (1:1500; Molecular Probes, weighing 20–25 g, as described above. Then, pathophysiology. Eugene,OR)for1hour.Imageswereobtained 2 mg/kg ML204 were injected (i.p.) 6 hours and analyzed using an LSM 700 confocal micro- before the first and second LPS treatments scope (Zeiss, Thornwood, NY). and6hoursafterthesecondLPStreatment CONCISE METHODS (three injections total). Urine samples were 2+ In Vitro Ca Imaging collected 48 hours after the first LPS injection. Glomeruli were isolated by cell strainer and cul- Mice tured in media at 37°C for 5 days. The primary All animal experiments and protocols were podocytes were transferred to collagen-coated TEM Analysis approved by the Rush University Institutional Kidneys were cut into 2–3mm3 pieces and coverslips to grow for 3 more days. Before im- Animal Care and Use Committee. B/6 mice were fixed in Trumps Fixative (Electron Microscopy aging, podocytes were loaded with Fluo-4AM obtained from Jackson Laboratory (Bar Harbor, Sciences, Hatfield, PA). They were embedded (1.8 mM; Thermo Fisher Scientific, Waltham, ME). TRPC5-overexpressing mice were gener- in LX112 epoxy resin and polymerized at 60°C MA) for 30 minutes in serum-free medium. A atedbytheTransgenicAnimalModelCoreatthe for 2–3 days. Ultrathin sections (70 nm) from subset of coverslips were preincubated with University of Michigan. cDNA of wild-type or kidney cortex were obtained using an EM UCT ML204 (20 mM; Sigma-Aldrich) for 20 minutes. pore-mutated TRPC5 were constructed19,20 and Ultramicrotome (Leica, Buffalo Grove, IL) and A balanced salt solution (140 mM NaCl, 10 mM cloned into the pCAGEN vector (Addgene, stained with 5% uranyl acetate and 0.1% lead Hepes, 2 mM CaCl ,1mMMgCl,10mMGlu- Cambridge, MA).26 The transgene was under 2 2 citrate. Specimens were examined using a JEM- cose, 5 mM KCl, pH 7.4) was used for cell rins- the control of the CAG promoter/enhancer.27 1220 transmission electron microscope ing and perfusing. The response of primary Purified DNA was microinjected into fertilized (JEOL, Peabody, MA). Digital images were podocytes to 2.5 minutes of perfusion of Cch eggs.28 The original founder mice were bred acquired using an Erlangshen ES1000W (100 mM; Abcam) or Englerin A (5 mM; PanReac with B/6 to develop offspring. Each generation model 785 CCD camera (Gatan, Pleasanton, AppliChem) was recorded through intracellu- was genotyped before 6 weeks of age. All groups CA) and Digital Micrograph. The length of lar Ca2+ imaging. Three independent exper- of experimental mice were age, weight, and sex the GBM was measured by ImageJ and FPs iments from three TG and DN mice were matched. and slits were counted. FP width was calcu- performed. ImageJ (National Institutes of lated as W =p∑GBM length/4∑slits.30 Health, Bethesda, MD) was used to determine FP qPCR the change in fluorescence intensity with time. Total RNA was extracted from kidney with Cells with spontaneous responses to perfu- Statistical Analyses RNeasy Midi kit (QIAGEN, Germantown, sion buffer were excluded. Statistical analysis was performed using Prism MD). cDNA was synthesized using iScript 5.0 software (GraphPad, La Jolla, CA). Signif- cDNA Synthesis kit (Bio-Rad, Hercules, CA). Urine Sampling and Analysis icance of the difference between two groups qPCR was performed using SsoAdvanced Mouse urine samples were collected at times de- was assessed using the unpaired two-tailed SYBR Green Supermix (Bio-Rad) in a CFXCon- scribed in the manuscript. Urinary albumin and t test. For multiple group comparisons, data nect real-time system (Bio-Rad). The relative creatinine were measured by mouse albumin were evaluated by one-way ANOVA using gene fold expression changes were determined ELISA (Bethyl Laboratories, Montgomery, TX) Tukey multiple comparison test or Bonferroni using the comparative CTmethod by calculating and creatinine assay kits (Cayman Chemical, test. Data are shown as mean6SEM. –DDCT 29 2 for each gene. Ann Arbor, MI).

Western Blotting LPS Challenge The brain tissue was lysed in Laemmli buffer In the “single dose LPS” experiment, 10 mg/kg ACKNOWLEDGMENTS with homogenization and sonication (QSon- LPS (Sigma-Aldrich, St. Louis, MO) was injected ica, Newtown, CT). Fifty-microgram samples (i.p.) to 14–16-week-old females weighing 20–25 We acknowledge Galina Gavrilina, Wanda were loaded in gels (Thermo Scientific). Tar- g. Urine samples were collected 24 and 48 hours Filipiak, and Thom Saunders for preparation get were visualized and quantitated after LPS injection. In the second experiment, of transgenic mice and the Transgenic Animal with Odyssey CLx infrared imaging system two doses of 5 mg/kg LPS were injected (i.p.) Model Core of the University of Michigan’s (LI-COR Biosciences). TRPC5 primary anti- 24 hours apart. Urine samples were collected Biomedical Research Core Facilities. bodies were from GenScript (customized; 24 hours after each injection. Kidneys were har- Core support was provided by the National Piscataway, NJ) and NeuroMab (Davis, CA). vested for TEM at the end of the experiment. Cancer Institute of the National Institutes of

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Health (NIH) under Award Number 8. Akbulut Y, Gaunt HJ, Muraki K, Ludlow MJ, and sera from patients with recurrent FSGS and P30CA046592; the University of Michigan Gut Amer MS, Bruns A, Vasudev NS, Radtke L, by putative glomerular permeability factors. Willot M, Hahn S, Seitz T, Ziegler S, Christmann Biochim Biophys Acta 1863: 2342–2354, 2017 Peptide Research Center, NIH grant number M, Beech DJ, Waldmann H: (-)-Englerin A is a 19. Hofmann T, Schaefer M, Schultz G, Gudermann DK34933; and the University of Michigan potent and selective activator of TRPC4 and T: Subunit composition of mammalian transient George M. O’Brien Renal Core Center, NIH TRPC5 calcium channels. Angew Chem Int Ed receptor potential channels in living cells. Proc grant number P30DK08194. This work was Engl 54: 3787–3791, 2015 Natl Acad Sci U S A 99: 7461–7466, 2002 supported by funds from the Department of 9. Carson C, Raman P, Tullai J, Xu L, Henault M, 20. Strübing C, Krapivinsky G, Krapivinsky L, Thomas E, Yeola S, Lao J, McPate M, Verkuyl Clapham DE: Formation of novel TRPC Medicine, Rush University Medical Center. JM, Marsh G, Sarber J, Amaral A, Bailey S, channels by complex subunit interactions in R.E.M. was supported by the NIH/National Lubicka D, Pham H, Miranda N, Ding J, Tang embryonic brain. JBiolChem278: 39014– Institute of Arthritis and Musculoskeletal HM, Ju H, Tranter P, Ji N, Krastel P, Jain RK, 39019, 2003 and Skin Diseases (K01AR070328). Schumacher AM, Loureiro JJ, George E, 21. Krall P, Canales CP, Kairath P, Carmona- Berellini G, Ross NT, Bushell SM, Erdemli G, Mora P, Molina J, Carpio JD, Ruiz P, Mezzano Solomon JM: Englerin A agonizes the TRPC4/ SA, Li J, Wei C, Reiser J, Young JI, Walz K: C5 cation channels to inhibit tumor cell line Podocyte-specific overexpression of wild DISCLOSURES proliferation. 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