Abstract 044: Ddah-1 And Adma Regulate Proximal Tubular Fluid Reabsorption: Comparison Of Pharmacological Blockade Of Ddah-1 With Gene Knockdown

Abstract

Background: Proximal tubule (PT) fluid reabsorption (Jv) is diminished by knockout of NOS-I or III. ADMA is an endogenous inhibitor of NOS, but its effects on tubular function are unknown. ADMA is degradated by DDAH whose type 1 isoform (DDAH-1) is richly expressed in the PT. SNIPs of DDAH-1 predict the decline of GFR in chronic kidney disease (CKD). Hypothesis: that DDAH-1 metabolizes ADMA in the PT thereby reducing its plasma concentration and enhancing Jv. Methods: Jv was measured in anesthetized rats and mice in S2 segments of the PT by direct in vivo micropuncture and microperfusion of artificial tubular fluid (ATF). Results: Addition of L-257 (10 -4 M; DDAH-1 inhibitor) vs vehicle to ATF perfusing the PT of rats for 10 mins did not affect Jv (3.4 ± 0.20 vs 3.1±0.39 nl/min/mm). However, a bolus i.v. injection (60mg/kg) of L-257 2hrs before and 10 mins of PT perfusion significantly (P< 0.005) reduced Jv by 33% to 2.3±0.17 nl/min/mm and increased plasma ADMA (systemic Veh: 0.46 ± 0.030 vs. systemic L-257: 0.67 ± 0.029 μmol/l; P < 0.0001) without changing SDMA. Microperfusion of ADMA (10 -4 M) or L-NAME (10 -4 M) in rats both reduced Jv significantly by >40% to 2.0±0.24 and 1.8±0.22 nl/min/mm, respectively (P<0.05). An IV 1 ml bolus of saline containing Transit and siRNA targeted to DDAH-1 (hydrodynamic in vivo gene silencing in mice) three days later reduced Jv significantly by 36% compared to scrambled siRNA (2.2 ± 0.16 vs 1.4 ± 0.26 nl/min/mm; P<0.05). Surprisingly, PT cell-specific DDAH-1 knockdown in mice did not affect Jv significantly compared to wild type (PTC-D1WT: 1.7 ± 0.21 vs PTC-D1KO: 2.0 ± 0.15). Conclusions: PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two-hour delay for L-257 to diminish Jv likely was required for accumulation of sufficient tubular ADMA to impair PT reabsorption. Whereas systemic DDAH-1 gene knockdown in mice was as effective as systemic DDAH-1 blockade with L-257 in rats in reducing Jv in the PT, lifelong deletion of PT DDAH-1 was ineffective, suggesting robust adaptive mechanisms that restore PT reabsorption. Thus, L-257 is a novel regulator of PT function. SNIPs that alter DDAH-1 in the PT may change tubular function and thereby contribute to CKD progression.