Role of endothelin-1 (ET-1) in glomerular inflammation and glomerular permeability in normal and diabetic kidneys

Endothelin-1 (ET-1) is a potent vasoactive peptide implicated in the pathogenesis of hypertension and renal disease. The overall specific aim of this dissertation is to investigate the role of ET-1 in mediating glomerular inflammation and permeability, especially in diseases characterized by high activity of the ET-1 system, such as diabetic nephropathy. The first study was designed to test the hypotheses that ET-1 increases albumin permeability of glomeruli isolated from normal rats and that chronic ET-1 infusion will increase glomerular permeability and inflammation independent of blood pressure. Glomerular permeability to albumin (Palb) was determined from the change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients. Incubation of glomeruli taken from normal rats with ET-1 at a concentration that did not produce direct glomerular contraction (1 nM) significantly increased Palb, reaching a maximum after 4 hrs. Chronic ET-1 infusion for 2 weeks in Sprague-Dawley (SD) rats significantly increased Palb and nephrin excretion rate, effects that were attenuated in rats given an ETA receptor antagonist, ABT-627. Urinary protein and albumin excretion and mean arterial pressure (telemetry) were not changed by ET-1 infusion. Acute incubation of glomeruli isolated from ET-1-infused rats with the selective ETA antagonist significantly reduced Palb, an effect not observed with acute treatment with a selective ETB antagonist. Chronic ET-1 infusion increased glomerular and plasma sICAM-1 and MCP-1 and elevated the number of macrophages and lymphocytes in renal cortices (CD68- and CD3-positive staining, respectively). These effects were all attenuated in rats given an ETA selective antagonist. These data support the hypothesis that ET-1 directly increases glomerular permeability to albumin and renal inflammation via ETA receptor activation independent of changes in arterial pressure. The second study was designed to test the hypothesis that ETA receptor activation increases P alb and elevates pro-inflammatory markers in hyperglycemic rats. Male SD rats were given streptozotocin (STZ) or saline (sham). Half of the animals in each group received ABT-627 beginning immediately after hyperglycemia had been confirmed. Glomeruli were isolated by sieving and Palb determined from the change in glomerular volume induced by exposing glomeruli to oncotic gradients of albumin. Glomerular nephrin expression was assessed by immunofluorescence, whereas urinary nephrin was measured by enzyme-linked immunosorbent assay. Three and 6 weeks after STZ injection, proteinuria was significantly increased compared to sham controls and was significantly reduced by ABT-627 treatment. Palb was also increased at 3 and 6 wk post-STZ; ABT-627 had no effect on Palb or protein excretion in sham rats. Glomerular and plasma content of sICAM-1 and MCP-1 were significantly increased 6 wk after STZ. ABT-627 attenuated these increases. After 6 weeks of hyperglycemia, glomerular nephrin expression was decreased with a concurrent increase in urinary nephrin excretion; ABT-627 prevented glomerular nephrin loss in the hyperglycemic rats. These observations support the hypothesis that ET-1, via the ETA receptor, mediates the increase in proteinuria and P alb, possibly via nephrin loss, as well as early inflammation in the hyperglycemic rat. In the third study, we determined the actions of ETA and ETB receptors on measures of glomerular function and renal inflammation in the early stages of diabetic renal injury in rats. Six weeks after STZ-induced hyperglycemia, rats were given ABT-627 (5 mg/kg/d) a selective ETA antagonist; A-182086 (10 mg/kg/d), a combined ET A/B antagonist; or vehicle for 1 week. Sham controls received STZ vehicle (saline). Hyperglycemia led to significant proteinuria, increased Palb , nephrinuria, and an increase in total matrix metalloprotease (MMPs) and transforming growth factor-beta 1 (TGF-beta1) activities in glomeruli. Plasma and glomerular sICAM-1 and MCP-1 were elevated after 7 weeks of hyperglycemia. Daily administration of both ABT-627 and A-182086 for 1 week significantly attenuated proteinuria, the increase in Palb, nephrinuria, and total MMPs and TGF-beta1 activity. However, glomerular sICAM-1 and MCP-1 expression was attenuated with ABT-627, but not A-182086 treatment. In summary, both selective ETA and combined ETA/B antagonists reduced proteinuria, glomerular permeability and restored glomerular filtration barrier components integrity, but only ETA selective blockade had anti-inflammatory and anti-fibrotic effects. We conclude that selective ETA antagonists are more likely to be preferred for treatment of diabetic kidney disease.;KEY WORDS: endothelin-1, glomerular permeability, glomerular inflammation, nephrin, sICAM-1, MCP-1, kidney, diabetes mellitus...