Expression Evaluation of TRPC6 and PODXL Genes in Podocyte Cell Culture After Albumin Overload with and Without Puromycin-Aminoglycoside Damage

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Background: Proteinuria is a prognostic marker for kidney disease and one of the main symptoms of podocytopathies. This study aimed to evaluate the relative expression of TRPC6 and PODXL genes after albumin overload in podocytes in vitro. Methods: Human podocyte cultures were exposed to progressive 0, 3, 20 and 40 mg/mL of albumin for 24h without and with 15 ug/uL exposure to puromycin aminoglycoside (PAN) for 12h. qPCR was performed with RPLP0 as endogenous control. The phenotype recovery was evaluated, either with gradual (removing 25% every 24h of total overload) or abrupt (removing all albumin in the first 24h) regression conditions. SDS-page and Western Blot were performed to evaluate protein expression. Statistical analysis was performed with ANOVA and Tukey’s a posteriori test (p<0.05) (n=5). Results: The rate of PODXL gene transcript was higher than control in 40 mg/mL of albumin without PAN and in 0, 20 and 40 mg/mL of albumin with PAN (p<0.0001). Phenotype recovery was observed after gradual removal of albumin overload at 20 mg/mL with PAN. Similar results were observed for protein expression, both in progressive and regressive conditions (p<0.05). For TRPC6 gene, there was a decrease in expression after progressive albumin exposure with 3, 20 and 40 mg/mL without PAN (p<0.0001) and an increase expression in 20 and 40 mg/mL with PAN treatment (p<0.0001). Phenotype recovery was observed in gradual and abrupt regressive conditions. Conclusions: When podocytes are injured, podocalyxin is released in vesicles that are excreted in the urine and used as an early glomerular disease biomarker. Therefore, we believe that this mechanism might explain why the phenotype was not recovered in the 40 mg/mL condition. Regarding TRPC6’s increased expression after PAN exposure, it might increase Ca+2 influx leading to apoptosis, aggravating podocyte damage after proteinuria insult. Funding: Government Support - Non-U.S.

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