This Exploratory/Developmental Research Proposal (R21) is submitted in response to the NIDDK Program Announcement PA-06-148 """"""""Pilot and Feasibility Program Related to the Kidney"""""""". Rationale: This proposal rests on four key elements: (1) Recent evidence stresses the functional significance of the connecting tubule (CNT) in electrolyte and fluid homeostasis; (2) With the observation of inducible renin expression in CNT, we have proposed that a paracrine tubular renin-angiotensin system (RAS) participates in the coordinated regulation of sodium reabsorption at proximal and distal sites along the nephron; (3) CNT renin mRNA and protein, not detected under euvolemic conditions, can be markedly increased in parallel or independently of renin at the juxtaglomerular apparatus (JGA); (4) There is now convincing experimental evidence that luminal angiotensin II can affect sodium reabsorption through the amiloride-sensitive epithelial sodium channel in distal nephron. Hypothesis: Renin expression at the merging points of multiple nephrons is strategically positioned to act on angiotensinogen originating in proximal tubule and thereby to modulate tubular A-II formation and electrolyte transactions in distal nephron.
Research Aims. In this Exploratory/Developmental R21, we will perform the following pilot studies: (1) To begin the development of mouse models with targeted alterations of CNT renin expression (1.1) by generating an animal with targeted overexpression of renin in distal nephron (knock-in, KI) by homologous recombination using a Cre/Lox strategy and (1.2) by developing the targeting vectors and the embryonal stem cell clones necessary to ablate expression of renin in CNT (knock-out, KO). (2) To perform preliminary characterization of the KI animal, we will: (2.1) Test and quantitate heterologous and endogenous renin mRNA and protein in KI and control animals in response to a step increase in sodium intake, both in situ and in pools of segments of distal nephron; (2.2) Test the effects of sodium loading on acute and chronic changes in blood pressure, sodium excretion, plasma renin and aldosterone in KI and control animals; (2.3) Test and quantitate gene expression of key regulators and effectors of electrolyte transport in distal nephron using nephron segments isolated in (2.1). Perspective. Achieving these limited and focused objectives will demonstrate and validate experimental models and protocols for a subsequent R01 research proposal involving complementary expertise in the field. Animal models and protocols will be shared with other scientists. The work should help defining the physiological role of CNT renin in sodium balance, plasma volume and arterial pressure regulation. Summary Over 50% of the population of the United States have hypertension, or high blood pressure of unknown cause, by the time they reach 65 years of age, and only a fraction of these patients have their blood pressure under control. The renin-angiotensin system (RAS) is a major controller of blood pressure and of its relationship to dietary salt, but its function is complex because it involves overlapping circulating and tissue systems. We have delineated a tubular RAS operating along the nephron, the filtering unit of the kidney that controls sodium reabsorption, that includes renin expression at a site that controls the final rate of sodium excretion. We propose to investigate the consequences of genetic manipulations of the expression renin, the rate limiting enzyme of the RAS, at this strategic site to probe its significance in the regulation of plasma volume and blood pressure in response to dietary salt. The work should advance diagnosis and treatment of hypertension. ? ? ?
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