Abstract P032: High Glucose Increases Prorenin Receptor (PRR) at the Cell Plasma Membrane in the Collecting Duct Which Promotes the Induction of Downstream Fibrotic Factors

Abstract

In streptozotocin (STZ)-induced Type-1 diabetic (T1D) rats, the collecting duct is the main source of intrarenal prorenin. The fact that binding of prorenin to the PRR triggers intracellular signals linked to tissue fibrosis has raised the expectation that prorenin might be directly responsible for diabetic nephropathy (DN). We showed that STZ-induced T1D rats exhibit greater apical distribution of PRR in the collecting duct. Therefore, we hypothesize that high glucose increases PRR abundance at the cell plasma membrane (PM) of collecting duct cells, which contributes to the stimulation of downstream fibrotic factors. To test this, we used male Sprague-Dawley rats (14w of age) with STZ-induced hyperglycemia for 7d (ip, single injection; 60 ng/kg), and cultures of collecting duct M-1 cells treated for 0, 5 min, and 1, 6, 12 and 24h with normal glucose (NG; 5mM glucose + 20mM mannitol) and high glucose (HG; 25mM). After 7d, STZ-induced rats (N=9) showed plasma levels of glucose as 428±13 vs.138±9 gr/dL and insulin as 0.07±0.02 vs. 2.4±0.01 ng/mL; compared to controls (N=7). Although PRR mRNA levels did not differ between groups; PRR protein levels and its downstream target TGF-β mRNA levels were augmented in the renal medulla of STZ-induced rats (0.55±0.03 vs.0.44±0.02 PRR/β-actin protein ratio; P<0.01; 1.22±0.06 vs.0.97±0.03 TGF-β/β-actin mRNA ratio; P<0.01). Interestingly, PRR protein levels were maximum elevated at 1h in PM extracts from M-1 cells treated with HG (0.95±0.04 vs. 0.33±0.2 PRR/E-cadherin protein ratio) but not in NG-treated cells. HG also unregulated downstream fibrotic factor fibronectin (1.9±0.2 vs.0.46±0.05 fold change) compared with NG-treated cells. Immunofluorescence studies of M-1 cells treated with NG for 1h showed that PRR is mainly localized in the perinuclear areas, whereas in HG-treated cells it was predominantly localized toward the cell surface, as it was more clearly observed in de-convoluted images. These data indicate that hyperglycemia leads to PRR trafficking alterations by inducing PRR translocation towards PM in the collecting duct cells and suggest that the activation of PRR during hyperglycemia conditions might be a novel mechanism underlying the development of DN, particularly tubulointerstitial fibrosis in DM.