In animals treated with low-dose L-NAME, our results suggest that the initiation of salt-induced hypertension may be mediated by subnormal vasodilation in response to salt loading and not abnormally large increases in blood volume in response to salt loading. Our results further suggest that in the L-NAME-treated animals, the maintenance of hypertension with a high salt diet may require abnormally high renal tubular NCC activity and blood volume. The abnormally high level of NCC activity may be mediated by the effects of oxidative stress on the phosphorylation of NCC.
NO in the kidney could be derived from any isoform of NO synthase 22x22Mount, P.F. and Power, D.A. Nitric oxide in the kidney: Functions and regulation of synthesis. Acta Physiol (Oxf). 2006;
Crossref | PubMed | Scopus (150) | Google ScholarSee all References. It has been reported that NO derived from nNOS plays a role in the regulation of afferent arteriolar diameter and long- and short-term tubule-glomerular feedback (TGF) 23x23Tojo, A., Onozato, M.L., and Fujita, T. Role of macula densa neuronal nitric oxide synthase in renal diseases. Med Mol Morphol. 2006;
Crossref | PubMed | Scopus (22) | Google ScholarSee all References. Whole body eNOS knockout mice showed a high basal mean BP (125±4 mmHg), with a further increase in BP after excess salt intake 24x24Kopkan, L., Hess, A., Husková, Z. et al. High-salt intake enhances superoxide activity in eNOS knockout mice leading to the development of salt sensitivity. Am J Physiol Renal Physiol. 2010;
Crossref | PubMed | Scopus (31) | Google ScholarSee all References. Therefore, we used low-dose L-NAME to systemically inhibit all subunits of NOS. Sergio et al. reported using 25 mg/kg/day of L-NAME (a dose 25 times higher than what we used), whereby the rodents developed salt-sensitive hypertension within 2 weeks of treatment 25x25Sergio, S.Y., Ana, L.S., and Clarice, K.F. Effect of Salt Intake and Inhibitor Dose on Arterial Hypertension and Renal Injury Induced by Chronic Nitric Oxide Blockade. Hypertension. 1996;
Crossref | PubMed | Scopus (79) | Google ScholarSee all References. In that study, the rodents developed hypertension and severe renal fibrosis even when they were given a low salt diet and after cessation of L-NAME, thus suggesting that renal damage and activation of the renin-angiotensin axis 26x26Giani, J.F., Janjulia, T., Kamat, N. et al. Renal Angiotensin-Converting Enzyme Is Essential for the Hypertension Induced by Nitric Oxide Synthesis Inhibition. J Am Soc Nephrol. 2014;
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Abstract | Full Text | Full Text PDF | PubMed | Scopus (4) | Google ScholarSee all References are the causes of salt-sensitive hypertension in that model. In the present study, our model showed no apparent renal parenchyma damage, and the rodents remained normotensive unless a high-salt diet was given (Fig. 2Fig. 2). The L-NAME+HS rats exhibited an increased blood pressure within 24 hours of high salt loading (Fig. 1Fig. 1). The results suggest that this low dose of L-NAME induces salt sensitivity in normotensive animals even in the absence of renal parenchymal damage.
Blood volume expansion was associated with increased blood pressure on day 1 of high salt intake in the L-NAME+HS group, but in the control group, the same blood volume expansion was not associated with an increase in blood pressure on day 1. This finding demonstrated that elevated blood volume is not sufficient for the initiation of hypertension. The results suggest that within the first 24 hours of salt loading, the L-NAME+HS animals failed to normally vasodilate and reduce systemic vascular resistance in response to salt loading and blood volume expansion. In contrast, the normal controls may have responded to the same degree of salt-induced volume expansion by vasodilating and reducing systemic vascular resistance, which prevents the salt-induced increases in blood volume from increasing blood pressure, which is consistent with previous studies5x5Beard, D.A. Assessing the Validity and Utility of the Guyton Model of Arterial Blood Pressure Control. Hypertension. 2018;
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Crossref | PubMed | Scopus (24) | Google ScholarSee all References,13x13Schmidlin, O., Sebastian, A.F., and Morris, R.C. Jr. What initiates the pressor effect of salt in salt-sensitive humans? Observations in normotensive blacks. Hypertension. 2007;
Crossref | PubMed | Scopus (57) | Google ScholarSee all References,14x14Schmidlin, O., Forman, A., Leone, A. et al. Salt sensitivity in blacks: evidence that the initial pressor effect of NaCl involves inhibition of vasodilatation by asymmetrical dimethylarginine. Hypertension. 2011;
Crossref | PubMed | Scopus (40) | Google ScholarSee all References,15x15Kurtz, T.W., DiCarlo, S.E., and Pravenec, M. The pivotal role of renal vasodysfunction in salt sensitivity and the initiation of salt-induced hypertension. Curr OpinNephrol Hypertension. 2018;
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Crossref | PubMed | Scopus (5) | Google ScholarSee all References,17x17Laffer, C.L., Scott, R.C., Titze, J.M. et al. Hemodynamics and Salt-and-Water Balance Link Sodium Storage and Vascular Dysfunction in Salt-Sensitive Subjects. Hypertension. 2016;
Crossref | PubMed | Scopus (33) | Google ScholarSee all References. The decrease in blood volume was associated with an increase in urinary sodium excretion. This finding suggests that to maintain salt-sensitive hypertension, sodium retention and increases in blood volume are necessary. In the current study, we clarified the potential role of NCC in maintaining a high circulating volume after L-NAME treatment both in vivo and in vitro.
In vivo experiments showed that HCTZ but not amiloride induced a strong natriuretic effect in the L-NAME plus HS group, which suggests that NCC, but not ENaC, may be responsible. This result is partly consistent with a previous study on nephron-specific disruption of NOS3 in mice, in which phosphorylated and total NCC, but not ENaC, were elevated in the KO mice after 4 hours of an acute 3.2% Na+ load 28x28Gao, Y., Stuart, D., Takahashi, T. et al. Nephron-Specific Disruption of Nitric Oxide Synthase 3 Causes Hypertension and Impaired Salt Excretion. J Am Heart Assoc. 2018;
Crossref | PubMed | Scopus (5) | Google ScholarSee all References. This study disrupted NOS3 in the whole nephron, and NOS1 and 2 were preserved. In our study, whole NOSs were inhibited by L-NAME and could induce sustained high oxidative stress to activate NCC phosphorylation by the SPAK/OSR pathway. Corresponding to the response to HCTZ, phosphorylated NCC was reduced after HS but was sustained in the L-NAME plus HS group. This finding may be due to the inappropriate activation of NCC by L-NAME plus HS, which reduces sodium delivery to the collecting duct, and thus, ENaC may not be activated 29x29Mu, S., Shimosawa, T., Ogura, S. et al. Epigenetic modulation of the renal β-adrenergic-WNK4 pathway in salt sensitive hypertension. Nat Med. 2011;
Crossref | PubMed | Scopus (139) | Google ScholarSee all References. Moreover, the results from the NCC-deficient mice indicate that NCC is indispensable for L-NAME-induced salt sensitivity.
L-NAME changes renal blood flow 30x30Dobrowolski, L. and Kuczeriszka, M. Role of atrial natriuretic peptide in mediating the blood pressure-independent natriuresis elicited by systemic inhibition of nitric oxide. Pflugers Arch. 2015;
Crossref | Scopus (5) | Google ScholarSee all References and to exclude microenvironmental changes in the kidney, we examined the effect of L-NAME and NO in vitro using mDCT cells in which eNOS is expressed. Blockade of NO in the mDCT cells with L-NAME and treatment with SNP altered the phosphorylation of NCC, and these data suggest that NO interacts with the phosphorylation of NCC. It is well known that L-NAME results in high oxidative stress 31x31Amaral, T.A.S., Ognibene, D.T., Carvalho, L.C.R.M. et al. Differential responses of mesenteric arterial bed to vasoactive substances in L-NAME-induced preeclampsia: Role of oxidative stress and endothelial dysfunction. Clin Exp Hypertens. 2017;
Google ScholarSee all References, a result that was also confirmed by analyzing superoxide levels in the present study. After cotreatment with the SOD mimetic TEMPO, the L-NAME-induced increases in p-NCC were downregulated. pSPAK, a classic activator of NCC, phosphorylates NCC at conserved Ser/Thr residues in the cytoplasmic N-terminal domain 32x32Mercier-zuber, A. and Shaughnessy, K.M.O. Role of SPAK and OSR1 signaling in the regulation of NaCl cotransporters. Curr Opin Nephrol Hypertens. 2011;
Crossref | PubMed | Scopus (40) | Google ScholarSee all References. In this study, the expression of pSPAK was also increased by L-NAME stimulation and downregulated by TEMPO + L-NAME cotreatment in mDCT cells. Furthermore, the NO donor decreased the superoxide levels and pSPAK expression in mDCT cells. To confirm the role of pSPAK, we inhibited pSPAK and subsequently demonstrated the failure of L-NAME to activate NCC in the presence of a pSPAK inhibitor. Furthermore, we demonstrated the role of ROS in NO-induced salt-sensitive hypertension in vivo. After 4 weeks of treatment, TEMPO attenuated the L-NAME- and salt-induced increases in superoxide levels and the mean BP and p-NCC expression in C57BL/6J mice. These results indicate that oxidative stress and pSPAK play a role in the interaction between NO and NCC. Moreover, we formerly reported that sympathetic overactivity induces salt-sensitive hypertension by changing WNK4 transcription and NCC phosphorylation 29x29Mu, S., Shimosawa, T., Ogura, S. et al. Epigenetic modulation of the renal β-adrenergic-WNK4 pathway in salt sensitive hypertension. Nat Med. 2011;
Crossref | PubMed | Scopus (139) | Google ScholarSee all References. In the low-dose L-NAME and high oxidative stress models, we did not observe changes in WNK4 protein (Supplementary Fig. 4).
The renal brush border has high ACE activity, which contributes to intrarenal angiotensin II formation in vivo, and local ACE in the kidney was reported to play a pivotal role in L-NAME-induced salt sensitivity 28x28Gao, Y., Stuart, D., Takahashi, T. et al. Nephron-Specific Disruption of Nitric Oxide Synthase 3 Causes Hypertension and Impaired Salt Excretion. J Am Heart Assoc. 2018;
Crossref | PubMed | Scopus (5) | Google ScholarSee all References,29x29Mu, S., Shimosawa, T., Ogura, S. et al. Epigenetic modulation of the renal β-adrenergic-WNK4 pathway in salt sensitive hypertension. Nat Med. 2011;
Crossref | PubMed | Scopus (139) | Google ScholarSee all References. In the current study, we showed that the angiotensin receptor blocker losartan did not block the phosphorylation of NCC induced by L-NAME in vitro (Supplementary Fig. 5), suggesting that angiotensin AT1 receptor signaling may not be involved in the L-NAME-induced NCC activation in vitro. However, because we did not test the active metabolite of losartan, these in vitro studies should be interpreted with caution.
TEMPO did not completely reduce the BP level back to the control level, thus suggesting that in addition to oxidative stress, other pathways might be involved in L-NAME-induced salt sensitivity. It has been shown that NKCC2 plays a role in the NO-related sodium balance 33x33Ramseyer, V.D., Ortiz, P.A. et al. Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs. Am J Physiol Renal Physiol. 2016;
Crossref | PubMed | Scopus (1) | Google ScholarSee all References. Gene transfer of eNOS to the thick ascending limb of the loop of Henle in eNOS KO mice restored the effects of L-arginine on NaCl absorption via NKCC2 34x34Ortiz, P.A., Hong, N.J., and Wang, D. Gene Transfer of eNOS to the Thick Ascending Limb of eNOS-KO Mice Restores the Effects of l-Arginine on NaCl Absorption. Hypertension. 2003;
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There are limitations in our study. The major limitation of the study is that we did not monitor cardiac output and vascular resistance during initiation or maintenance of salt-induced hypertension; therefore, we can only speculate on the hemodynamic mechanisms through which low-dose L-NAME treatment enables a high-salt diet to initiate and maintain hypertension. We did not directly examine NCC activity by measuring sodium transport but by pharmacological examination in response to HCTZ and amiloride. Although we could not change the local NO levels solely in the kidney using kidney-specific knockout of three subtypes of NOS, our model is closer to the clinical condition than other models. This study used a rodent model, and it is unclear if the results are applicable in humans; however, in human studies, it has been reported that NOS polymorphisms and decreased NOS activity are common in black or aging people who have a high prevalence of salt-sensitive hypertension 35x35Stephen, T., Chapman, A.B., and Schwartz, G.L. Effects of Endothelial Nitric Oxide Synthase, a-Adducin, and Other Candidate Gene Polymorphisms on Blood Pressure Response to Hydrochlorothiazide. AJH. 2003;
Google ScholarSee all References,36x36Elijovich, F., Weinberger, M.H., Anderson, C.A.M. et al. Salt Sensitivity of Blood Pressure A Scientific Statement From the American Heart Association. Hypertension. 2016;
Crossref | Scopus (121) | Google ScholarSee all References,37x37Svetkey, L.P., McKeown, S.P., and Wilson, A.F. Heritability of salt sensitivity in black Americans. Hypertension. 1996;
Crossref | PubMed | Scopus (83) | Google ScholarSee all References,38x38Sverdlov, A.L., Ngo, D.T., Chan, W.P., Chirkov, Y.Y., and Horowitz, J.D. Aging of the nitric oxide system: are we as old as our NO?. J Am Heart Assoc. 2014;
Google ScholarSee all References, and salt-sensitive humans release less NO during NO agonist administration than patients with salt-resistant essential hypertension 39x39Ghiadoni, L., Virdis, A., Taddei, S. et al. Defective nitric oxide pathway in salt-sensitive essential hypertensive patients. Am J Hypertens. 1997;
Crossref | Google ScholarSee all References. Our study showed that even a slight decline in NO in the whole body induced vascular hypodilation together with inappropriate sodium resorption to cause salt-sensitive hypertension.
In conclusion, the initiation of salt sensitivity in normotensive rodents could be due to hyporeactivity of the vasculature, and maintaining blood pressure could results in a high circulating volume due to inappropriate NCC activity in the low-dose L-NAME model. L-NAME impairs natriuresis by activating NCC via the oxidative stress/pSPAK pathway and induces blood volume expansion, finally leading to established salt-sensitive hypertension.