Alterations in key components of the apical endocytic apparatus in the renal proximal tubule accompanied by progression of microalbuminuria has been recently showed in aging SHRs. The present study aimed to investigate podocyte alterations in SHRs, with special attention to the podocyte slit-diaphragm protein nephrin and to the podocyte apoptosis, as possible underlying mechanisms for the development of hypertensive nephrosclerosis.Design and method:
BP was measured in conscious animals using a tail-cuff technique. We compared glomerular tissue from SHRs at 6 (development of HTN), 13 (established HTN) and 20 (beginning of albuminouria) weeks of age and from Wistar Kyoto normotensive rats (WKY) of similar age. Following mechanical separation of renal cortex, isolation and separation of the renal glomeruli from the tubules was performed by infiltration using a filter with a specific pore size. The cells were lysed in a modified RIPA buffer, the cell lysates was centrifuged for 30 min at 10,000 rpm at 4 C and cell proteins were dissolved and quantified using the Bradford method. Western blot analysis was performed after proteins were denatured in SDS-PAGE 7.5%. Membranes were incubated with anti-nephrin and anti-beta-actin antibody in order to normalize the total protein of the samples. Cleaved PARP was used as a marker of apoptosis and was measured by a specific monoclonal antibody.Results:
As expected BP was 30–50% higher in SHRs than in WKY rats, with the difference increasing associated with aging. Increment of nephrin expression was seen in WKY rats over time from 6 to 13 and 20 weeks. In contrast, SHRs failed to upregulate nephrin expression. Downregulation of nephrin in aging SHRs was associated with increased apoptosis of glomerular podocytes. Indeed, although no difference in cleaved PARP was found between WKY rats and SHRs at 6 weeks, cleaved PARP was 38% higher in SHRs than in WKY rats at 13 weeks of age.Conclusions:
Development of hypertension and hypertensive nephrosclerosis in aging SHR rats are associated with increased apoptosis and progressive down-regulation of the vital slit-diaphragm protein nephrin.