KELCH-3 (KLHL3): A Potential Regulator of Insulin-Induced Renal Haemodynamic Alterations in Obesity

Background: The contribution of elevated insulin levels to renal disease in pre-diabetic obesity remains underappreciated, as glomerular damage is typically attributed to hyperglycemia and high blood pressure (BP). However, impaired renal autoregulation—characterized by increased glomerular capillary pressure (P GC ) and renal blood flow (RBF)—plays a pivotal role in obesity-induced kidney damage (OIKD) when insulin levels are high while blood glucose (BG) levels remain normal. Renal autoregulation is maintained through two intrinsic feedback mechanisms: 1.) Tubuloglomerular feedback (TGF), a vasoconstrictor mechanism, and 2.) Distal convoluted tubule-2/connecting tubule-2 (DCT-2/CNT)-mediated afferent arteriole (Af-Art) dilation, driven by epithelial sodium channels (ENaC) located in the DCT-2/CNT. Previous studies in hyperinsulinemic obese Zucker rats (OZR) from our lab identified an upregulated DCT-2/CNT-mediated Af-Art dilatory mechanism, increased P GC , and enhanced renal blood flow (RBF), while no changes were observed in TGF.Interestingly, Insulin is a known activator of ENaC and ENaC expression is reduced in renal cortical tissue of KLHL3 null mice. Objective of this study was to to investigate the role of insulin in regulating KLHL3 expression in the DCT-2/CNT and its contribution to ENaC-mediated afferent arteriole dilation, increased RBF, elevated P GC , and subsequent renal damage in pre-diabetic obesity. Hypothesis: Insulin-induced KLHL3 expression in the DCT-2/CNT contributes to ENaC-mediated Af-Art dilation, resulting in increased RBF and subsequent renal damage in obesity. Methods and Results/Data: First, we examined whether insulin directly induces Af-Art dilation via ENaC. Rabbit CNTs with attached Af-Art were perfused with insulin in an ex-vivo settings, which caused significant Af-Art dilation. Co-administration of the ENaC inhibitor benzamil reversed this effect, indicating that ENaC mediates insulin-induced vasodilation. Next, to evaluate whether insulin increases RBF independently of blood glucose and BP in-vivo, we performed hyperinsulinemic-euglycemic (HI-EUG) clamp experiments in OZR. Insulin infusion significantly increased cortical blood flow (CBF) by 20.8±4.9% when blood glucose levels were normal. This effect was reversed by benzamil, confirming the role of ENaC in mediating insulin-induced CBF changes.These alterations occurred independently of BP, which remained unaffected throughout the experiments. To identify the molecular mechanisms underlying these hemodynamic changes, we performed RNA sequencing of the DCT-2/CNT segment in OZR kidneys. KLHL3 emerged as the most upregulated transcript. To confirm insulin-induced KLHL3 expression, insulin infusion into one kidney of lean rats (renal subcapsular method) increased KLHL3 by significant 65% in calbindin-1-positive DCT-2/CNT compared to the contralateral kidney, as measured by immunohistochemistry on renal cortex serial sections. Finally, to determine if insulin induces renal damage, insulin (1.8 IU/kg/day) was infused into one kidney of lean rats for six weeks.The insulin-infused kidney exhibited significantly thicker glomerular basement membranes (GBM), a marker of renal damage (199.4±17.0 nm vs. vehicle: 145.5±3.6 nm, p<0.05), indicating insulin-induced damage independent of blood glucose and BP. Conclusion: This study is the first to demonstrate that insulin critically regulates KLHL3 upregulation in the DCT-2/CNT, enhancing ENaC-mediated Af-Art dilation, increasing CBF and contributing to glomerular damage in obesity. These findings suggest that KLHL3 may serve as a potential therapeutic target for mitigating obesity-induced renal damage. Career Development Award (24CDA1269174). American Heart Association Henry Ford Hospital Internal Funding This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.