[Expression and role of the TRPC family in TGF-ß1-induced calcium influx in podocytes].

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-ß1 (TGF-ß1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-ß1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca2+ concentration ([Ca2+]i) in podocytes induced by TGF-ß1. The model of podocyte injury was established by treatment with TGF-ß1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-ß1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca2+ was measured using the fluorescent Ca2+ indicator Fluo-3/AM, and the dynamic change of [Ca2+]i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-ß1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-ß1, respectively. TGF-ß1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-ß1 significantly increased [Ca2+]i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca2+]i induced by TGF-ß1, but TRPC3/6 knockdown significantly decreased the [Ca2+]i. There was no significant difference in the [Ca2+]i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca2+]i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-ß1 increases the expression of the TRPC1/3/6 in podocytes. TGF-ß1 increases [Ca2+]i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca2+]i in podocytes may be greater than that of TRPC3.

Circular RNA-0007059 protects cell viability and reduces inflammation in a nephritis cell model by inhibiting microRNA-1278/SHP-1/STAT3 signaling.

BACKGROUND: Increasing evidence has indicated that circular RNAs (circRNAs) play a role in various diseases. However, the influence of circRNAs in nephritis remains unknown. METHODS: Microarray analysis and RT-qPCR were used to detect the expression of circRNA. Type I IFN were administrated to RMC and HEK293 cells to establish a nephritis cell model. CCK-8, MTT assay, and flow cytometry were used to assess cell proliferation, viability, and apoptosis of cells. Bioinformatics analysis and dual luciferase reporter assay detect the interaction of circ_0007059, miRNA-1278, and SHP-1. Glomerulonephritis was performed in a mouse model by administration of IFNα-expressing adenovirus. IHC staining showed the pathogenic changes. RESULTS: In the present study, the expression of circ_0007059 in type I interferon (IFN)-treated renal mesangial cells (RMCs), lupus nephritis (LN) specimens, and HEK293 cells was downregulated compared with that in normal healthy samples and untreated cells. Circ_0007059 overexpression resulted in increased cell proliferation, cell viability, apoptosis, and inflammation-associated factors (CXCL10, IFIT1, ISG15, and MX1) in RMCs and HEK293 cells. In addition, circ_0007059 overexpression significantly restored cell proliferation and viability and inhibited IFN-induced apoptosis. Further, the increased expression resulted in reduced inflammation and the downregulation of CXCL10, IFIT1, ISG15, and MX1 in RMCs and HEK293 cells. Circ_0007059 serves as a sponge for miR-1278 so that the latter can target the 3'-untranslated region of SHP-1. Overexpressed circ_0007059 inhibited miR-1278 expression and elevated SHP-1 expression, subsequently reducing STAT3 phosphorylation. Meanwhile, miR-1278 was upregulated and SHP-1 was downregulated in LN samples and IFN-treated cells. The restoration of miR-1278 counteracted the effect of circ_0007059 on viability, apoptosis, and inflammation as well as on SHP-1/STAT3 signaling in RMCs and HEK293 cells. We also investigated the role of SHP-1 overexpression in IFN-treated RMCs and HEK293 cells; SHP-1 overexpression resulted in a similar phenotype as that observed with circ_0007059 expression. CONCLUSIONS: The study indicates that circ_0007059 protects RMCs against apoptosis and inflammation during nephritis by attenuating miR-1278/SHP-1/STAT3 signaling.