Tissue-specific KO of the NOS1 in the macula densa enhanced tubuloglomerular feedback responsiveness in the isolated perfused JGAin vitroand at the single-nephron levelin vivo. After infusion of angiotensin II, mean arterial pressure increased by 61. 6 Rabbit Polyclonal to OR4D1 mmHg in MD-NOS1KO mice versus 32. 0 mmHg in WT mice (P <0. 01) fed a high-salt diet. These results indicate that NOS1is a primary NOS1 isoform expressed in the macula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP. Keywords: NOS1, macula densa, tubuloglomerular feedback, salt-sensitive, hypertension The existence of the tubuloglomerular feedback system was first predicted in the 1930s by Goormaghtigh1and Zimmerman2and later confirmed by Thurau and Schnermann. 3Tubuloglomerular feedback operates at the level of the juxtaglomerular apparatus (JGA) in each nephron, causing single-nephron GFR (SNGFR) to be inversely dependent on the tubular flow to the macula densa. Increases in NaCl delivery to the macula densa initiate a tubuloglomerular feedback response that constricts the afferent arteriole (Af-Art) and decreases SNGFR. This unfavorable feedback loop prevents acute fluctuations in Af-Art pressure from altering SNGFR and stabilizes NaCl delivery to the distal nephron. 414 We recently reported that the macula densa expresses-, -, and-splice variants of nitric oxide synthase 1 (NOS1) and that expression of NOS1increases in animals fed a high-salt diet. 15Therefore, NOS1may be a salt-sensitive isoform in the macula densa that modulates tubuloglomerular feedback response, promotes sodium excretion, and protects against the development of salt-sensitive hypertension. To test this hypothesis, we developed a tissuespecific knockout (KO) strain, in which all of the NOS1 splice variants were deleted from the macula densa. We also measured the expression of splice variants of NOS1 of the macula densa isolated with laser capture microdissection (LCM). The results indicate that NOS1is a primary splice variant of NOS1 expressed in the macula densa and that deletion of NOS1 from the macula densa enhances tubuloglomerular feedback response, blunts the natriuretic response to salt weight, and promotes the development of salt-sensitive hypertension. These findings not only provide a novel mechanism for salt-sensitive hypertension but also, show the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP. == Results == == NOS1Expression in the Macula Densa == We first compared the expression of splice variants of NOS1 mRNA in RG2833 (RGFP109) the macula densa using LCM and real-time PCR. We found that levels of NOS1mRNA were 322. 7-fold higher than NOS1in the macula densa of C57BL/6 mice (P <0. 01; The Jackson Laboratory, Bar Harbor, ME) (Figure 1A). == Figure 1 RG2833 (RGFP109) . == Expression of NOS1 splice variants in the macula densa of C57BL/6 mice. The macula densa cells were isolated with LCM, and mRNA was measured with real-time PCR. (A) NOS1level was about 32-fold higher than NOS1(n=7). Protein levels of NOS1 splice variants RG2833 (RGFP109) were measured with Western blot. (B and C) In the brain, NOS1is <5% of NOS1; however , in renal cortex, NOS1is more than fivefold higher than NOS1(n=5). *P <0. 01 versus NOS1. The expression of splice variants of NOS1 protein in renal cortex was measured by Western blot using a Cterminal NOS1 antibody that can detect all of the splice variants of NOS1. 15, 16We used brain as a positive control for NOS1, which is the predominant splice variant of NOS1 expressed in brain. 1719As shown inFigure 1, B and C, NOS1was the primary splice variant expressed in the brain, and.