The roles of TRPC6 in renal tubular disorders: a narrative review.

The transient receptor potential canonical 6 (TRPC6) channel, a nonselective cation channel that allows the passage of Ca2+, plays an important role in renal diseases. TRPC6 is activated by Ca2+ influx, oxidative stress, and mechanical stress. Studies have shown that in addition to glomerular diseases, TRPC6 can contribute to renal tubular disorders, such as acute kidney injury, renal interstitial fibrosis, and renal cell carcinoma (RCC). However, the tubule-specific physiological functions of TRPC6 have not yet been elucidated. Its pathophysiological role in ischemia/reperfusion (I/R) injury is debatable. Thus, TRPC6 may have dual roles in I/R injury. TRPC6 induces renal fibrosis and immune cell infiltration in a unilateral ureteral obstruction (UUO) mouse model. Additionally, TRPC6 overexpression may modify G2 phase transition, thus altering the DNA damage checkpoint, which can cause genomic instability and RCC tumorigenesis and can control the proliferation of RCC cells. This review highlights the importance of TRPC6 in various conditions of the renal tubular system. To better understand certain renal disorders and ultimately identify new therapeutic targets to improve patient care, the pathophysiology of TRPC6 must be clarified.

Role of TRPC6 in apoptosis of skeletal muscle ischemia/reperfusion injury.

BACKGROUND: Skeletal muscle ischaemia-reperfusion injury (IRI) is a prevalent condition encountered in clinical practice, characterised by muscular dystrophy. Owing to limited treatment options and poor prognosis, it can lead to movement impairments, tissue damage, and disability. This study aimed to determine and verify the influence of transient receptor potential canonical 6 (TRPC6) on skeletal muscle IRI, and to explore the role of TRPC6 in the occurrence of skeletal muscle IRI and the signal transduction pathways activated by TRPC6 to provide novel insights for the treatment and intervention of skeletal muscle IRI. METHODS: In vivo ischaemia/reperfusion (I/R) and in vitro hypoxia/reoxygenation (H/R) models were established, and data were comprehensively analysed at histopathological, cellular, and molecular levels, along with the evaluation of the exercise capacity in mice. RESULTS: By comparing TRPC6 knockout mice with wild-type mice, we found that TRPC6 knockout of TRPC6 could reduced skeletal muscle injury after I/R or H/R, of skeletal muscle, so as therebyto restoringe some exercise capacity inof mice. TRPC6 knockdown can reduced Ca2+ overload in cells, therebyo reducinge apoptosis. In additionAdditionally, we also found that TRPC6 functionsis not only a key ion channel involved in skeletal muscle I/R injury, but also can affects Ca2+ levels and then phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway. by knocking downTherefore, knockdown of TRPC6, so as to alleviated the injury inducedcaused by skeletal muscle I/R or and H/R. CONCLUSIONS: These findingsdata indicate that the presence of TRPC6 exacerbatescan aggravate the injury of skeletal muscle injury after I/Rischemia/reperfusion, leading towhich not only causes Ca2+ overload and apoptosis., Additionally, it impairsbut also reduces the self- repair ability of cells by inhibiting the expression of the PI3K/Akt/mTOR signalling pathway. ETo exploringe the function and role of TRPC6 in skeletal muscle maycan presentprovide a novelew approachidea for the treatment of skeletal muscle ischemia/reperfusion injury.

TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway.

OBJECTIVE: Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage. Notably, the knockout of transient receptor potential channel 6 (TRPC6) has shown promise in alleviating renal fibrosis. However, the regulatory impact of TRPC6 on renal fibrosis remains unclear. METHODS: In vivo, TRPC6 knockout (TRPC6-/-) mice and age-matched 129 SvEv (WT) mice underwent unilateral renal ischemia-reperfusion (uIR) injury surgery on the left renal pedicle or sham operation. Kidneys and serum were collected on days 7, 14, 21, and 28 after euthanasia. In vitro, primary tubular epithelial cells (PTECs) were isolated from TRPC6-/- and WT mice, followed by treatment with transforming growth factor ß1 (TGFß1) for 72 h. The anti-fibrotic effect of TRPC6-/- and the underlying mechanisms were assessed through hematoxylin-eosin staining, Masson staining, immunostaining, qRT-PCR, and Western blotting. RESULTS: Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFß1. TRPC6-/- alleviated renal fibrosis by reducing the expression of fibrotic markers (Col-1, α-SMA, and vimentin), as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro. Additionally, we found that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK3ß) signaling pathway, a pivotal player in renal fibrosis, was down-regulated following TRPC6 deletion. CONCLUSION: These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3ß pathway. Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

A novel gain-of-function mutation in transient receptor potential C6 that causes podocytes injury.

Podocyte injury plays a vital role in focal segmental glomerulosclerosis (FSGS), and apoptosis is one of its mechanisms. The transient receptor potential channel 6 (TRPC6) is highly expressed in podocytes and mutations mediate podocyte injury. We found TRPC6 gene mutation (N110S) was a new mutation and pathogenic in the preliminary clinical work. The purpose of this study was to investigate the potential mechanism of mutation in TRPC6 (TRPC6-N110S) in the knock-in gene mouse model and in immortalized mouse podocytes (MPC5). Transmission electron microscopy was used to evaluate renal injury morphology. We measured 24-hour urinary albumin-to-creatinine ratios and major biochemical parameters such as serum albumin, urea nitrogen, and total cholesterol. The results of CCK-8 assay and apoptosis experiments showed that the TRPC6-N110S overexpression group had slower proliferative activity and increased apoptosis than the control group. FluO-3 assay revealed increased calcium influx in the TRPC6-N110S overexpression group. Podocin level was decreased in TRPC6-N110S group, while TRPC6 and desmin levels were increased in TRPC6-N110S group. The 24 h uACR at 6 weeks was significantly higher in the pure-zygotes group than in the WT and heterozygotes groups, and this difference was found at 8 and 10 weeks.TRPC6 levels showed no significant difference between homozygote and WT mice. Compared to homozygote group, expression of podocin and nephrin were increased in WT, but levels of desmin was decreased in WT. Our results suggest that this new mutation causes podocyte injury probably by enhancing calcium influx and podocyte apoptosis, accompanied by increased proteinuria and decreased expression of nephrin and podocin.

A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides.

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.

TRPC3 Is Downregulated in Primary Hyperparathyroidism.

Transient receptor potential canonical sub-family channel 3 (TRPC3) is considered to play a critical role in calcium homeostasis. However, there are no established findings in this respect with regard to TRPC6. Although the parathyroid gland is a crucial organ in calcium household regulation, little is known about the protein distribution of TRPC channels-especially TRPC3 and TRPC6-in this organ. Our aim was therefore to investigate the protein expression profile of TRPC3 and TRPC6 in healthy and diseased human parathyroid glands. Surgery samples from patients with healthy parathyroid glands and from patients suffering from primary hyperparathyroidism (pHPT) were investigated by immunohistochemistry using knockout-validated antibodies against TRPC3 and TRPC6. A software-based analysis similar to an H-score was performed. For the first time, to our knowledge, TRPC3 and TRPC6 protein expression is described here in the parathyroid glands. It is found in both chief and oxyphilic cells. Furthermore, the TRPC3 staining score in diseased tissue (pHPT) was statistically significantly lower than that in healthy tissue. In conclusion, TRPC3 and TRPC6 proteins are expressed in the human parathyroid gland. Furthermore, there is strong evidence indicating that TRPC3 plays a role in pHPT and subsequently in parathyroid hormone secretion regulation. These findings ultimately require further research in order to not only confirm our results but also to further investigate the relevance of these channels and, in particular, that of TRPC3 in the aforementioned physiological functions and pathophysiological conditions.

[Expansion of the genotypic and phenotypic spectrum and treatment of four children with Steroid-resistant nephrotic syndrome due to variants of TRPC6 gene].

OBJECTIVE: To summarize the clinical and genetic characteristics, treatment and prognosis of four children with Steroid-resistant nephrotic syndrome (SRNS) due to variants of TRPC6 gene. METHODS: Clinical data of four children with SRNS admitted to Children's Hospital Affiliated to Zhengzhou University between May 2020 and August 2022 were collected. Peripheral blood samples were collected from the children and their parents, and whole exome sequencing was carried out. Sanger sequencing was used to verify the pathogenicity of the candidate variants among the children and their parents. RESULTS: All of the four children were found to harbor heterozygous variants of the TRPC6 gene, including c.523C>T (p.R175W), c.1327T>A (p.F443I), c.430G>C (p.E144Q) (unreported previously), and c.523C>T (p.R175W), which were all missense variants. Two of the children have shown a simple type, whilst two have shown a nephritis type, none had extrarenal phenotype. Comprehensive renal pathology of three children revealed focal segmental glomerulosclerosis (FSGS). Two children were treated with steroids combined with calcineurin inhibitors (CNIs), among whom one showed significant improvement in symptoms. CONCLUSION: Discoveries of the novel c.430G>C variant and the new SRNS phenotype of the c.1327T>A variant have expanded the mutational and phenotypic spectrum of the TRPC6 gene, which has provided a reference for clinical diagnosis and genetic counseling for the families.

Pharmacological Activation of TRPC6 Channel Prevents Colitis Progression.

We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.

TRPV4 Channels Promote Pathological, but Not Physiological, Cardiac Remodeling through the Activation of Calcineurin/NFAT and TRPC6.

TRPV4 channels, which respond to mechanical activation by permeating Ca2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca2+-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca2+-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

Renal ischaemia-reperfusion injury is promoted by transcription factor NF-kB p65, which inhibits TRPC6 expression by activating miR-150.

AIM: To investigate the mechanism by which NF-κB p65 activates miR-150 to suppress TRPC6 expression and promote renal ischemia-reperfusion injury. METHODS: To assess the transcription of miR-150, NF-B p65, and TRPC6 in HK-2 cells treated with hypoxia reperfusion and rat kidney tissue damaged by ischemia-reperfusion (I/R), qPCR was implemented. The protein production of NF-κB p65 and TRPC6 was assessed by Western blot (WB) analysis. The histological score of rat kidney tissue was assessed using H&E (hematoxylin and eosin) staining. To assess the rate of apoptosis of renal tissue cells following I/R injury, we used the TACS TdT In Situ Apoptosis Detection Kit. To find out the impairment of renal function, blood levels of creatinine (Cr) and blood urea nitrogen (BUN) were tested in rats. Concentrations of inflammatory cytokines, including IL-1ß, IL-10, and TNF-α, were detected in HK-2 cells and rat renal tissue cells utilizing ELISA kits. FITC and CCK-8 were employed to analyze the death rate and cellular proliferation of HK-2 cells. To analyse the mechanism of engagement between NF-κB p65 and the miR-150 promoter, coupled with the detrimental impact of miR-150 on TRPC6, we adopted the dual-luciferase reporter assay. To confirm the activating effect of NF-κB p65 on miR-150,we implemented the ChIP assay. RESULTS: NF-κB p65 expression was significantly upregulated in rat renal tissue following IRI. Applying the dual-luciferase reporter assay, we demonstrated that the specific attachment of NF-B p65 with the miR-150 promoter location is viable, resulting in the promotion of the activity of the promoter. When miR-150 was overexpressed, we observed a notable reduction in cell proliferation. And it notably increased the rate of cellular apoptosis rate and amounts of the proinflammatory cytokines IL-1ß, IL-10, and TNF-α. Employing the dual-luciferase reporter assay, we demonstrated that miR-150 transfection diminished the function of luciferase in the TRPC6-WT group, whereas luciferase activity in the TRPC6-MUT group remained unchanged, indicating that miR-150 is a targeted inhibitor of TRPC6. In the rat renal I/R model, when miR-150 was inhibited or TRPC6 was overexpressed in the rat kidney I/R model, the histological score of rat kidney tissue significantly decreased, so did the quantities of proinflammatory cytokines IL-1ß, IL-10, TNF-α, creatinine (Cr) and blood urea nitrogen (BUN) contents and the rate of cell apoptosis in kidney tissue. CONCLUSION: Activation of miR-150 by NF-κB p65 results in downregulation of TRPC6 expression and promotion of IRI in the kidney.

Trpc6 knockout protects against renal fibrosis by restraining the CN­NFAT2 signaling pathway in T2DM mice.

Diabetic kidney disease (DKD), one of the common complications of type­2 diabetes mellitus (T2DM), has become the principal cause of end­stage kidney disease. Transient receptor potential channel 6 (TRPC6), one of non­selective cation channels with significant calcium­permeability, is associated with renal fibrosis. However, the mechanism of TRPC6 in T2DM­induced renal fibrosis is still not entirely understood. The present study explored the potential mechanism of Trpc6 knockout in T2DM­induced renal fibrosis in Trpc6­/­ mice. The results showed that Trpc6 knockout inhibited the loss of body weight and the increase of fasting blood glucose (FBG) and significantly improved renal dysfunction and glomerular fibrosis in T2DM mice. The present study also indicated that Trpc6 knockout significantly lowered the expression of phosphorylated (p­)SMAD2/3, TGF­ß, calcineurin (CN), nuclear factor of activated T­cell (NFAT)2 and Nod­like receptor (NLR) 3 inflammasome­associated proteins. Calcium imaging results revealed that Trpc6 knockdown could decrease the levels of [Ca2+]i and inhibited calcium homeostasis imbalance. Moreover, it was found that knockout of Trpc6 had no significant influence on lipid disposition and reactive oxygen species generation in the kidney cortex. The present study suggested that knockout of Trpc6 may alleviate glomerular fibrosis and delay DKD progression by reducing [Ca2+]i overload and inhibiting the CN­NFAT2 pathway in T2DM mice.

TRPC6 Deletion Enhances eNOS Expression and Reduces LPS-Induced Acute Lung Injury.

Acute lung injury (ALI) is characterized by endothelial barrier disruption and associated inflammatory responses, and transient receptor potential cation channel 6 (TRPC6)-mediated Ca2+ influx is critical for endothelial hyperpermeability. In this study, we investigated the role of TRPC6 in LPS-induced ALI, analyzed gene expression in WT and TRPC6-/- lungs using RNA sequencing, and explored the effects of TRPC6 in the LPS-induced hyperpermeability in human umbilical vein endothelial cells (HUVECs) to elucidate the underlying mechanisms. Intratracheal instillation of LPS caused edema in the mouse lungs. Deletion of TRPC6 reduced LPS-induced lung edema and decreased cell infiltration. RNA sequencing analysis suggested that downregulated cell adhesion molecules in TRPC6-/- lungs may be responsible for their resistance to LPS-induced injury. In addition, downregulation of TRPC6 significantly alleviated the LPS-induced decrease in eNOS expression in lung tissue as well as in HUVECs. Moreover, inhibition of TRPC6 with the channel antagonist larixyl led to a decrease in LPS-induced hyperpermeability and ROS production in HUVECs, which could be reversed by blocking eNOS. Our findings suggest that inhibition of TRPC6 ameliorates LPS-induced ALI, which may be achieved by acting on the cell adhesion molecule signaling pathway and participating in the regulation of eNOS levels in endothelial cells.

A Putative Role for TRPC6 in Immune-Mediated Kidney Injury.

Excessive activation of the immune system is the cause of a wide variety of renal diseases. However, the pathogenic mechanisms underlying the aberrant activation of the immune system in the kidneys often remain unknown. TRPC6, a member of the Ca2+-permeant family of TRPC channels, is important in glomerular epithelial cells or podocytes for the process of glomerular filtration. In addition, TRPC6 plays a crucial role in the development of kidney injuries by inducing podocyte injury. However, an increasing number of studies suggest that TRPC6 is also responsible for tightly regulating the immune cell functions. It remains elusive whether the role of TRPC6 in the immune system and the pathogenesis of renal inflammation are intertwined. In this review, we present an overview of the current knowledge of how TRPC6 coordinates the immune cell functions and propose the hypothesis that TRPC6 might play a pivotal role in the development of kidney injury via its role in the immune system.

An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis.

Transient receptor potential cation channel-6 (TRPC6) gene mutations cause familial focal segmental glomerulosclerosis (FSGS), which is inherited as an autosomal dominant disease. In patients with TRPC6-related FSGS, all mutations map to the N- or C-terminal TRPC6 protein domains. Thus far, the majority of TRPC6 mutations are missense resulting in increased or decreased calcium influx; however, the fundamental molecular mechanisms causing cell injury and kidney pathology are unclear. We report a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite a largely truncated TRPC6 protein. We studied the molecular effects of V691Kfs* TRPC6 mutant using the tridimensional cryo-EM structure of the tetrameric TRPC6 protein. The results indicated that V691 is localized at the pore-forming transmembrane region affecting the ion conduction pathway, and predicted that V691Kfs* causes closure of the ion-conducting pathway leading to channel inactivation. We assessed the impact of V691Kfs* and two previously reported TRPC6 disease mutants (P112Q and G757D) on calcium influx in cells. Our data show that the V691Kfs* fully inactivated the TRCP6 channel-specific calcium influx consistent with a complete loss-of-function phenotype. Furthermore, the V691Kfs* truncation exerted a dominant negative effect on the full-length TRPC6 proteins. In conclusion, the V691Kfs* non-functional truncated TRPC6 is not sufficient to cause FSGS. Our data corroborate recently characterized TRPC6 loss-of-function and gain-of-function mutants suggesting that one defective TRPC6 gene copy is not sufficient to cause FSGS. We underscore the importance of increased rather than reduced calcium influx through TRPC6 for podocyte cell death.

Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Genetic Risk Factors Associated With Preeclampsia and Hypertensive Disorders of Pregnancy.

Importance: A genetic contribution to preeclampsia susceptibility has been established but is still incompletely understood. Objective: To disentangle the underlying genetic architecture of preeclampsia and preeclampsia or other maternal hypertension during pregnancy with a genome-wide association study (GWAS) of hypertensive disorders of pregnancy. Design, Setting, and Participants: This GWAS included meta-analyses in maternal preeclampsia and a combination phenotype encompassing maternal preeclampsia and preeclampsia or other maternal hypertensive disorders. Two overlapping phenotype groups were selected for examination, namely, preeclampsia and preeclampsia or other maternal hypertension during pregnancy. Data from the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC, 1990-2011), Finnish FinnGen project (1964-2019), Estonian Biobank (1997-2019), and the previously published InterPregGen consortium GWAS were combined. Individuals with preeclampsia or other maternal hypertension during pregnancy and control individuals were selected from the cohorts based on relevant International Classification of Diseases codes. Data were analyzed from July 2020 to February 2023. Exposures: The association of a genome-wide set of genetic variants and clinical risk factors was analyzed for the 2 phenotypes. Results: A total of 16 743 women with prior preeclampsia and 15 200 with preeclampsia or other maternal hypertension during pregnancy were obtained from FINNPEC, FinnGen, Estonian Biobank, and the InterPregGen consortium study (respective mean [SD] ages at diagnosis: 30.3 [5.5], 28.7 [5.6], 29.7 [7.0], and 28 [not available] years). The analysis found 19 genome-wide significant associations, 13 of which were novel. Seven of the novel loci harbor genes previously associated with blood pressure traits (NPPA, NPR3, PLCE1, TNS2, FURIN, RGL3, and PREX1). In line with this, the 2 study phenotypes showed genetic correlation with blood pressure traits. In addition, novel risk loci were identified in the proximity of genes involved in the development of placenta (PGR, TRPC6, ACTN4, and PZP), remodeling of uterine spiral arteries (NPPA, NPPB, NPR3, and ACTN4), kidney function (PLCE1, TNS2, ACTN4, and TRPC6), and maintenance of proteostasis in pregnancy serum (PZP). Conclusions and Relevance: The findings indicate that genes related to blood pressure traits are associated with preeclampsia, but many of these genes have additional pleiotropic effects on cardiometabolic, endothelial, and placental function. Furthermore, several of the associated loci have no known connection with cardiovascular disease but instead harbor genes contributing to maintenance of successful pregnancy, with dysfunctions leading to preeclampsialike symptoms.

p38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

Generation of human induced pluripotent stem cell line (BCRTi006-A) from a patient with focal segmental glomerulosclerosis disease.

Individuals with transient reception potential cation channel 6 (TRPC6) mutation have variable phenotypes, ranging from healthy carriers to focal segmental glomerulosclerosis (FSGS). Human induced pluripotent stem cell (hiPSC) line was generated from the urinary cells of a patient with FSGS with a mutant variant of TRPC6. The cells were reprogrammed with Yamanaka factors (OCT3, SOX2, LIN28, L-MYC, and KLF4) using a commercially available Epi5 Reprogramming Kit. The pluripotency of the hiPSC line was confirmed by the expression of common stem cell markers and by their ability to generate all germ layers in vitro. The line is available and registered in the human pluripotent stem cell registry as BCRTi006-A. The generated line represents a valuable tool for disease modeling and drug development for FSGS.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5.

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

Identification of TRPC6 as a Novel Diagnostic Biomarker of PM-Induced Chronic Obstructive Pulmonary Disease Using Machine Learning Models.

Chronic obstructive pulmonary disease (COPD) was the third most prevalent cause of mortality worldwide in 2010; it results from a progressive and fatal deterioration of lung function because of cigarette smoking and particulate matter (PM). Therefore, it is important to identify molecular biomarkers that can diagnose the COPD phenotype to plan therapeutic efficacy. To identify potential novel biomarkers of COPD, we first obtained COPD and the normal lung tissue gene expression dataset GSE151052 from the NCBI Gene Expression Omnibus (GEO). A total of 250 differentially expressed genes (DEGs) were investigated and analyzed using GEO2R, gene ontology (GO) functional annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) identification. The GEO2R analysis revealed that TRPC6 was the sixth most highly expressed gene in patients with COPD. The GO analysis indicated that the upregulated DEGs were mainly concentrated in the plasma membrane, transcription, and DNA binding. The KEGG pathway analysis indicated that the upregulated DEGs were mainly involved in pathways related to cancer and axon guidance. TRPC6, one of the most abundant genes among the top 10 differentially expressed total RNAs (fold change ≥ 1.5) between the COPD and normal groups, was selected as a novel COPD biomarker based on the results of the GEO dataset and analysis using machine learning models. The upregulation of TRPC6 was verified in PM-stimulated RAW264.7 cells, which mimicked COPD conditions, compared to untreated RAW264.7 cells by a quantitative reverse transcription polymerase chain reaction. In conclusion, our study suggests that TRPC6 can be regarded as a potential novel biomarker for COPD pathogenesis.