CELLULAR AND MOLECULAR MECHANISMS OF AT2R MEDIATED ANTI-PROTEINURIC ACTIVITY
Proteinuria is a sensitive and specific biomarker of and a cause for kidney diseases. Immediate effects upon consumption of high sodium (4%) diet (HSD) on the expression of protein endocytic receptor megalin and its impact on proteinuria, particularly in obesity is not known. There are indications suggesting the involvement of glycogen synthase kinase (GSK)-3β-mediated megalin phosphorylation, and its subsequent endocytosis and lysosomal degradation resulting in proteinuric kidney injury. Present study reports that acute treatment with angiotensin-II type 2 receptor (AT2R) agonist C21 rescues megalin surface expression in the proximal tubules potentially by activating Akt-mediated phosphorylation and inactivation of GSK3β in the kidney of male obese Zucker rats fed HSD for 48 hrs. The HSD intake resulted in proteinuria, which was significantly prevented by C21. Moreover, the expression of glomerular slit diaphragm proteins (nephrin and podocin) the estimated glomerular filtration rate remained unaffected which together suggest that HSD intake caused tubular proteinuria. The in vivo findings in megalin recycling impairment were confirmed by the in vitro studies performed in Opossum Kidney (OK) cells which are the proximal tubule epithelial cells treated with high NaCl (100 mM) without/with C21. Compared to control, high NaCl reduced megalin surface expression, which is protected by C21 treatment. AT2R activation by C21 in OK cells treated with high NaCl increased Akt phosphorylation which was attenuated by AT2R antagonist PD123319 suggesting role of AT2R. Moreover, GSK3 inhibitor SB216763 reversed the effect of high NaCl on megalin surface expression, suggesting role in GSK3-induced disruption in megalin expression. However, Akt and PP2A inhibitors did not inhibit the effects of C21 on megalin rescue. FITC albumin uptake assay, which is a functional measure of megalin, revealed that HS caused reduction in albumin uptake which was prevented by C21 treatment. Moreover, GSK3 inhibitor restored albumin uptake. Akt inhibitor attenuated C21 mediated protection in megalin function. Overall, high NaCl induces proteinuria by disrupting cell surface megalin expression, potentially via Akt-mediated phosphorylation and a decrease in GSK3 phosphorylation. AT2R activation protects megalin cell surface expression and reverses proteinuria, via potentially reducing GSK3 activity.
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