[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.

[Platycodin D improves pulmonary fibrosis in mice by down-regulating TRPC6 expression and reducing ROS production in lung fibroblasts].

OBJECTIVE: To assess the effect of platycodin D (PD) for alleviating pulmonary fibrosis in mice and explore the underlying mechanism. METHODS: C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days. The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6 (TRPC6) were evaluated with immunohistochemistry, HE staining and Sirius Red staining. Western blotting was used to detect α-SMA expression in the lung tissues of the mice. Primary cultures of mouse lung fibroblasts were pretreated with PD (2.5, 5.0, and 10 µmol/L) or larixyl acetate (LA; 10 µmol/L) before exposure to 10 ng/mL transforming growth factor-ß1 (TGF-ß1), and the changes in cell survival rate, expressions of collagen Ⅰ, α-SMA and TRPC6, reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell proliferation capacity were assessed. Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis. RESULTS: PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models (P<0.05). In TGF-ß1-induced primary mouse lung fibroblasts, PD effectively inhibited the cell proliferation, reduced ROS production (P<0.0001), rescued the reduction of mitochondrial membrane potential (P<0.001), and inhibited the expressions of α-SMA and collagen Ⅰ (P<0.05). Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis. Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice. In primary mouse lung fibroblasts, PD significantly inhibited TGF-ß1-induced TRPC6 expression (P<0.05), and LA treatment obviously lowered the expression levels of TRPC6, α-SMA and collagenⅠ (P<0.05). CONCLUSION: PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

The synthesis and effects of a novel TRPC6 inhibitor, BP3112, on hepatocellular carcinoma.

Aims: Transient receptor potential canonical-6 (TRPC6) is a therapeutic target for hepatocellular carcinoma. The authors aimed to synthesize and determine whether indole-2-carboxamide derivatives have anti-hepatocellular carcinoma activities targeting TRPC6. Materials & methods: Molecular docking was carried out to design these derivatives. The top five compounds were synthesized for activity validation using microscale thermophoresis. Cell cytotoxicity, flow cytometry, western blotting and cell transfection were used to investigate the anti-hepatocellular carcinoma activities and mechanisms in vitro. Xenografts of nude mice were used for in vivo evaluation. Results: The indole-2-carboxamide derivative, BP3112, promoted apoptosis and G1-phase arrest in HCCs via inhibiting TRPC6, and dose-dependently inhibit tumor growth in vivo. Conclusion: BP3112 as a specific inhibitor of TRPC6 is a potential therapeutic agent for hepatocellular carcinoma.

Probing the therapeutic potential of TRPC6 for Alzheimer's disease in live neurons from patient-specific iPSCs.

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer's disease (AD) under patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher levels of AD pathological markers Aß and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neurons with TRPC6 protein fragment or agonist inhibited the elevation of Aß and phospho-tau. Our results in live AD neurons manifest that the compromised expression of TRPC6 substantially contributed to Aß pathology of sporadic AD, suggesting that targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.