High salt intake induces sympathetic activation in Dahl salt‐sensitive rats through activation of orexin‐TNF signaling in the hypothalamic paraventricular nucleus (PVN)

Increased sympathetic nerve activity (SNA) plays an important role in the development of salt‐sensitive hypertension (SSHTN). However, the detailed mechanisms underlying high salt (HS) induced increase in SNA are not well understood. In this study, we tested the hypothesis that HS diet upregulates activity of the orexin system in the hypothalamic paraventricular nucleus (PVN) thereby contributes to SNA increase in SSHTN. Eight‐week‐old male Dahl salt‐sensitive (Dahl S) rats, and age as well as sex matched Sprague Dawley (SD) rats were divided into two groups and were fed either a HS (8% NaCl) or normal salt (NS, 0.4% NaCl). Four weeks following different diets treatment, animals were euthanized, and PVN tissues were punched out and subjected to real time PCR to assay orexin, orexin receptors and other genes of interest. Consistent with previous studies, HS intake induced hypertension in Dahl S rats but not in SD rats. HS diet also resulted in significant increase in mRNA levels of prepro‐orexin (2.6‐fold), the precursor of orexin A and B; orexin receptor I (OX1R, 1.5‐fold); TNFα (3.5‐fold), a proinflammatory cytokine; and Fosl1 (2.5‐fold), a marker for neuronal activation, in Dahl S rats but not in SD rats. We further tested whether there was a difference in SNA response to orexin receptor activation in the PVN between NS and HS Dahl S and SD rats. Acute microinjection of orexin A (25 pmol) into the PVN showed increases in splanchnic SNA (SSNA) and renal SNA (RSNA) in both Dahl S and SD rats with either HS or NS diet, but this increase was greater in Dahl S rats with a HS diet (n=3) compared to Dahl S rats with a NS diet (n=4) (SSNA, HS: 132 ± 32% vs. NS: 59 ± 9%, P<0.05; RSNA, HS: 71 ± 15% vs. NS: 32 ± 6%, P<0.05); But no significant difference was observed in the increases in SSNA (HS, 76 ± 15% vs. NS, 73 ± 16%, n=5, P>0.05) and RSNA (HS: 58 ± 19% vs. NS: 72 ± 11%, P>0.05, n=5) in response to orexin A PVN microinjection between HS and LS intake SD rats. We also investigated whether the increase in the expression of TNFa and Fra1 was induced by orexin receptor activation using primary neuronal cultures from the hypothalamus of neonatal SD rats. Incubation of neurons with orexin A (1μM) resulted in a significant increase in the mRNA levels of TNFα (1.5‐fold) and Fra1 (4‐fold). Finally, we tested which receptor mediates orexin‐induced increase in the expression of TNFa and Fra1. PC12 cells without expression of orexin receptors, PC12‐OX1 with expression of human OX1R, and PC12‐OX2 with expression of human orexin receptor II were used in this experiment. The results showed that incubation of PC12‐OX1 with orexin A induced dramatic increases in the mRNA levels of TNFa (maximal 16‐fold) and fosl1 (maximal 6‐fold) in a dose‐and time‐dependent manner, but these increases were not observed in PC12 and PC12‐OX2 cells, indicating that orexin A mediated upregulation of TNFa and Fra1 may through activation of OX1R. These results, combined with the importance of TNFa in the modulation of neuronal activity, suggest that a HS diet triggers orexin‐OX1R‐TNFa signaling in the PVN which may contribute to increased SNA and development of SSHTN. Support or Funding Information NIH 1R15HL129213 (Shan) and 1R15HL122952 (Chen)