ClC-5 with Megalin (LRP2), Cubilin, Disabled 2 (Dab2) and Amnionless (AMN) is part of the molecular complex involved at proximal tubular level in albumin endocytosis. LRP2, Cubilin and ClC-5 expression in podocytes of human renal biopsies was been already reported. Moreover, it was demonstrated that podocytes are able to perform albumin endocytosis.
Aims of this study were to explore the presence of the tubular endocytic machinery components in human podocytes in vitro and to evaluate whether albumin modulates this system.
By immunofluorescence we evaluated the presence of ClC-5, LRP2, Cubilin, Dab2 and AMN in human podocytes cultured in standard condition and stimulated with FITC-BSA (10µg/ml).
By time lapse experiments with FITC-BSA (10µg/ml) we verified the presence of an uptake mechanism.
We evaluated the uptake kinetic stimulating podocytes with BSA at different time (30 min, 2h) and doses (10µg/ml, 100µg/ml and 1mg/ml) at 4 and 37°C.
Finally, we stimulated human podocytes with increasing concentrations of BSA (range 10µg/ml - 30mg/ml) and evaluated the expression of the endocytic machinery components at different time points (2, 4, 8, 24, 48 and 72 hours) by Real Time PCR.
We confirmed the presence of the endocytic machinery in human podocytes and observed the co-localization of LRP2 and Cubilin with FITC-BSA.
Albumin internalization in podocytes was detected from 1 to 16 hours with an uptake kinetic typically receptor-mediated.
In proteinuric environment (10 and 30mg/ml of albumin) a significant time and dose dependent increase in CLCN5, CUBILIN and AMN expression was found.
In conclusion, we confirmed in human podocytes the presence of albumin uptake process mediated by the typical tubular endocytic machinery.
Moreover, we demonstrated that protein overload upregulates CLCN5, CUBILIN and AMN.
Taken together these results support the idea of a partnership between tubular and glomerular cells in albumin uptake, via the same mechanism of internalization.
