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TRPC5 Does Not Cause Or Aggravate Glomerular Disease

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TRPC5 Does Not Cause Or Aggravate Glomerular Disease BRIEF COMMUNICATION www.jasn.org 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 calcium 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 protein- 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 ion channel 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 humans 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).
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