Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney

Abstract

NG-monomethylarginine (L-NMA) and asymmetric NG, NG-dimethylarginines (ADMA) are endogenous inhibitors of cellular L-arginine uptake and/or nitric oxide (NO) synthesis that are implicated in renal parenchymal and Dahl salt-sensitive hypertension. Since the L-arginine:(L-NMA + ADMA) ratio determines NO synthase (NOS) activity, we compared the immunohistochemical distribution of NOS with NG, NG-dimethylarginine dimethylaminohydrolase (DDAH), which inactivates dimethylarginines (DMA) and L-NMA by hydrolysis to L-citrulline. Neuronal NOS (nNOS) was expressed predominantly in tubular epithelial cells of macula densa (MD), endothelial NOS (eNOS) in vascular endothelial cells (EC), and inducible NOS (iNOS) quite widely in tubular epithelium, including proximal tubules (PT), thick ascending limbs of Henle (TAL), distal convoluted tubule and intercalated cells (IC) of the collecting duct. Immunostaining for DDAH was present in PT, TAL, MD, and IC, and was also present in the glomerulus, Bowman's capsule, and endothelium of blood vessels. DDAH was detected in small vesicles of TAL and PT by electron microscopic (EM) immunocytochemistry. To study the effects of methylarginines on tubuloglomerular feedback (TGF) response, vehicle or methylarginines (10(-3) M) were added to artificial tubular fluid (ATF) perfused orthogradely from the late PT at 40 nl. min-1 while assessing changes in glomerular capillary pressure from proximal stop flow pressure (PSF). Whereas the maximal TGF responses were unchanged by vehicle (delta TGF 0 +/- 0%) or symmetric DMA (SDMA; +1 +/- 2%, NS), they were enhanced by L-NMA (+22 +/- 4%, P < 0.001) and asymmetric DMA (ADMA; +28 +/- 3%, P < 0.001). Since L-arginine transport can regulate renal epithelial NO generation, methylarginines (10(-3) M) or vehicle were co-perfused orthogradely with [3H]-L-arginine from the late PT and collected at the early distal tubule to study arginine uptake from the perfused loop of Henle. All methylarginines reduced fractional loop [3H] absorption significantly (P < 0.001; vehicle, 84 +/- 6; ADMA, 49 +/- 6; SDMA, 56 +/- 6; L-NMA, 41 +/- 6%). In conclusion, sites of DDAH expression in the vasculature or nephron are all sites of expression of an isoform of NOS. L-NMA, ADMA, and SDMA all inhibit renal tubular L-arginine uptake, whereas L-NMA and ADMA, but not SDMA, enhance TGF responses. Therefore, DDAH may regulate the cellular L-arginine: methylarginine levels in specific renal cells, thereby governing cell-specific L-arginine uptake and NO generation in renal tubular epithelium.