Effect of Down-regulation of TRPC6 on the Puromycin Aminonucleoside-induced Apoptosis of Mouse Podocytes

Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

Effect of TRPC6 Knockdown on Puromycin Aminonucleoside-induced Podocyte Injury

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

Fluvastatin protects against puromycin aminonucleoside-induced podocyte injury by inhibiting TRPC6

In the present study,we aimed to investigate the effect of puromycin aminonucleoside(PAN)on the expression and distribution of transient receptor potential canonical 6(TRPC6)of murine podocytes and further study the protective mechanism of fluvastatin on podocyte injury by TRPC6 in vitro.Podocytes were treated by PAN at different doses and at different time points.The expressions of TRPC6 at mRNA and protein levels were assessed.An immunofluorescent assay was used to observe the distribution of TRPC6.Cultured podocytes were then divided into four groups.The expressions of TRPC6 at mRNA and protein levels were measured.The intracellular calcium concentration of podocytes was measured with a laser-scanning confocal microscope.The paracellular permeability to BSA was evaluated using Millicell-PCF Inserts.The expressions of TRPC6 at mRNA and protein levels were increased in a dose and time-dependent manner after exposure to PAN.Immunofluorescence showed that the expression intensity of TRPC6 was significantly increased,and the distribution of TRPC6 was changed after PAN was applied to podocytes.With fluvastatin intervention,PAN-induced up-regulation of TRPC6 was significantly reversed.The ratio of the peak value of intracellular calcium to the basic calcium value in the PAN group was significantly higher after TRPC6 was activated by OAG,while it is obviously reversed under the action of fluvastatin.The podocyte permeability was significantly increased after 48 h of PAN treatment,while the above situation was effectively improved after fluvastatin intervention.The changes of distribution and expression of TRPC6 were related to podocyte injury induced by PAN.Fluvastatin could exert its protective effects on podocytes by down-regulating TRPC6.

Sinkihwan-gamibang ameliorates puromycin amino nucleoside-induced nephrotic syndrome

Nephrotic syndrome(NS)is a kidney disease characterized by hypertriglyceridemia,massive proteinuria,hypo-albuminemia and peripheral edema.Sinkihwan-gamibang(SKHGMB)was recorded in a traditional Chinese medical book named“Bangyakhappyeon(方藥合編)”and its three prescriptions Sinkihwan,Geumgwe-sinkihwan,and Jesaeng-sinkihwan belong to Gamibang.This study confirmed the effect of SKHGMB on renal dysfunction in an NS model induced by puromycin aminonucleoside(PAN).The experimental NS model was induced in male Sprague Dawley(SD)rats through injection of PAN(50 mg·kg^(-1))via the femoral vein.SKHGMB not only reduced the size of the kidneys increased due to PAN-induced NS,but also decreased proteinuria and ascites.In addition,SKHGMB significantly ameliorated creatinine clearance,creatinine,and blood urea nitrogen.SKHGMB relieved glomeruli dilation and tubules fibrosis in the glomeruli of the NS model.SKHGMB inhibited the protein and mRNA levels of the NLRP3 inflammasome including NLRP3,ASC,and pro-caspase-1 in NS rats.SKHGMB reduced the protein and mRNA levels of fibrosis regulators in NS rats.The results indicated that SKHGMB exerts protective effects against renal dysfunction by inhibiting of renal inflammation and fibrosis in NS rats.