eNOS Mediates l -Arginine–Induced Inhibition of Thick Ascending Limb Chloride Flux

Abstract

Abstract-We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoformsϪendothelial (eNOS), inducible (iNOS), and neuronal (nNOS)Ϫhave been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J Cl ) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0.5 mmol/L L-Arg to the bath decreased J Cl from 105.8Ϯ17.5 to 79.2Ϯ15.8 pmol/mm per minute (PϽ0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J Cl. In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J Cl of THALs from eNOS knockout mice. When 10 mol/L SPM was added to the bath of eNOS knockout THALs, J Cl decreased from 89.1Ϯ8.6 to 74.8Ϯ7.5 pmol/mm/min (PϽ0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J Cl in THALs from iNOS and nNOS knockout mice by 37.7Ϯ6.4% (PϽ0.05) and 31.8Ϯ8.3% (PϽ0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J Cl . Key Words: nitric oxide Ⅲ kidney Ⅲ mice R enal nitric oxide (NO) is an important controller of urinary sodium excretion. NO may enhance natriuresis by inhibiting transport along the nephron as well as altering renal hemodynamics and glomerular filtration rate. 1 Several in vivo studies support the hypothesis that NO directly inhibits tubular sodium reabsorption. Urinary sodium excretion increases when NO production is stimulated 2 and decreases when NO synthase (NOS) is inhibited 3 in the absence of altered renal hemodynamics.In vitro studies indicate that NO directly affects tubular absorption. 4 -6 We recently reported that L-Arginine (L-Arg) inhibits chloride flux through the thick ascending limb of the loop of Henle (THAL) of rats via an L-NAME-sensitive mechanism. These data indicate that endogenously produced NO inhibits THAL transport. 7 However, these studies did not address the NOS isoform(s) mediating tubular NO synthesis.mRNA for each of the NOS isoforms has been detected in THALs. With the use of reverse transcription polymerase chain reaction (RT-PCR) of microdissected nephrons, Ujiie et al 8 detected endothelial (eNOS) mRNA in rat THALs, whereas Mohaupt et al 9 used com...

Luminal flow induces eNOS activation and translocation in the rat thick ascending limb. II. Role of PI3-kinase and Hsp90

“…However, in some cells Very little is known about the role of PI3-kinase-stimulated eNOS activity along the nephron and its role in regulating salt and water reabsorption. Previous data from our laboratory showed that eNOS-derived NO inhibits NaCl absorption by the THAL (35,38) and that NO produced in this nephron segment acts in a paracrine manner to modulate tubuloglomerular feedback (TGF) (52). We have also reported that hormones that inhibit NaCl absorption by the THAL may do so by activating the PI3-kinase/eNOS pathway (37).…”

“…Hsp90 is necessary for eNOS translocation to the apical membrane in response to increased luminal flow. Given the importance of eNOS-derived NO in the regulation of THAL NaCl absorption (35,38) and the role of eNOS in various physiological processes in epithelial cells of the respiratory tract, brain, and testis (14,29,55,57,60), this mechanism is likely to play an essential role in the regulation of NO levels in these cells.…”

“…We previously reported that NO acts as an autacoid to inhibit NaCl and bicarbonate absorption by the thick ascending limb of the loop of Henle (THAL) (31,32,39). More recently, we identified eNOS as the NOS isoform responsible for the regulation of NaCl absorption by the THAL (35,38). We recently observed that increasing luminal flow acutely stimulated eNOS activity and induced translocation of eNOS from the basolateral membrane and cytoplasm to the apical membrane of isolated THALs (34a).…”

Recent Advances in the Regulation of Nitric Oxide in the Kidney

“…27 NOS 3 is responsible for autocrine inhibition of NaCl absorption in the thick ascending limb. 28 We showed that NOS 3 function could be restored selectively to this segment of NOS 3 knockout mice using an adenovirus with a tissue-specific promoter. 13 Because we were able to transduce Ϸ75% to 95% of thick ascending limbs in vivo, we are now studying the specific role of the medullary thick ascending limb NOS 3 in the regulation of salt and water excretion in vivo.…”

Nephron‐Specific Disruption of Nitric Oxide Synthase 3 Causes Hypertension and Impaired Salt Excretion

“…However, other studies, albeit not involving renal cell‐specific gene targeting, have suggested that nephron NOS3 may be an important modulator of renal salt handling and BP. l ‐arginine‐induced inhibition of transepithelial Cl − flux was abolished in TAL from global NOS3 KO mice, but not from global NOS1 or NOS2 KO mice 9. Further, NOS3 gene transfer into TAL of NOS3 KO mice restored l ‐arginine‐induced inhibition of NaCl reabsorption 10.…”