Protective Role of Angiotensin II type 2 receptor (AT2R) in High Salt Diet‐Induced Low Molecular Weight Proteinuria and Loss of Megalin in the Kidney
Authors: Kulkarni Kalyani , Patel Sanket , Ali Riyasat , Hussain Tahir 
Published: May 2022

Proteinuria is a cause and a risk factor for tubulointerstitial injury with the progression of end‐stage kidney disease. Proteinuria is a function of the glomerular filtration of protein and the reabsorption of protein from the post‐glomerular filtrate by the endocytic receptors, megalin and cubilin, localized in the renal proximal tubules. We have shown that treatment with the angiotensin‐II type 2 receptor (AT2R) agonist compound 21 (C21) for 2 weeks reduces proteinuria in obese Zucker rats (OZR) fed with high salt diet (HSD). OZR is a salt‐sensitive model but has no effect on blood pressure in the first 3 days of HSD treatment. However, the early cellular and molecular mechanism of proteinuria in response to HSD and the protective effect of AT2R activation is unknown. Therefore, the present study is designed to determine the molecular/cellular mechanism of AT2R‐mediated protection against HSD‐induced proteinuria, independent of glomerular injury in obesity. We hypothesize that AT2R activation prevents the loss of megalin in proximal tubule epithelial cells and thereby decreases the urinary excretion of low molecular weight (LMW) proteins in HSD fed obese rats. Male OZR (11–14 weeks of age) were fed on normal salt diet (NSD) (0.4%) or HSD (4%) for 2 days with and without treatment with C21. The animals were placed in metabolic cages for collecting urine at various time intervals namely 6, 12, 24 and 48 hours. We observed that HSD caused proteinuria at 24 hrs. and was prevented by C21 treatment. Further analysis by gel electrophoresis revealed that proteinuria was based on LMW protein (<60 kDa) excretion in the urine. This observation was confirmed by no change in albuminuria by HSD and C21. Dot blot analysis and confocal immunofluorescence microscopy revealed that megalin and not cubilin, was significantly decreased in HSD treatment group and the remaining megalin was mostly present in the cytosolic compartment with negligible expression on plasma membrane, suggesting megalin recycling impairment. The C21 treatment restored megalin back to the plasma membrane. FcRn is an exclusive receptor for albumin transport, which remained unaltered by HSD and C21. Western blot of nephrin and podocin, indices of glomerular filtration apparatus, remained unchanged suggesting structural integrity of the glomerular filtration assembly. Consistent to this notion, GFR measured by creatinine clearance method remained same in all the three groups. HSD caused a significant increase in the urinary sodium excretion, which was not affected by C21 treatment and the plasma sodium concentration in all the groups remained the same. Overall, the data suggest that acute feeding of HSD causes LMW proteinuria due to reduction as well as cytosolic expression of megalin in proximal tubule and not due to the change in the glomerular filtration. AT2R activation restores megalin expression and prevents proteinuria with a potential of long‐term protection against tubule‐interstitial injury and kidney disease.