Increased phosphorylation of the renal Na+-Cl- cotransporter in male kidney transplant recipient patients with hypertension: a prospective cohort.

Evidence in rodents suggests that tacrolimus-induced posttransplant hypertension is due to upregulation of the thiazide-sensitive Na+-Cl- cotransporter NCC. Here, we analyzed whether a similar mechanism is involved in posttransplant hypertension in humans. From January 2013 to June 2014, all adult kidney transplant recipients receiving a kidney allograft were enrolled in a prospective cohort study. All patients received tacrolimus as part of the immunosuppressive therapy. Six months after surgery, we assessed general clinical and laboratory variables, tacrolimus trough blood levels, and ambulatory 24-h blood pressure monitoring. Urinary exosomes were extracted to perform Western blot analysis using total and phospho-NCC antibodies. A total of 52 patients, including 17 women and 35 men, were followed. At 6 mo after transplantation, of the 35 men, 17 developed hypertension and 18 remained normotensive, while high blood pressure was observed in only 3 of 17 women. The hypertensive patients were significantly older than the normotensive group; however, there were no significant differences in body weight, history of acute rejection, renal function, and tacrolimus trough levels. In urinary exosomes, hypertensive patients showed higher NCC expression (1.7±0.19) than normotensive (1±0.13) (P=0.0096). Also, NCC phosphorylation levels were significantly higher in the hypertensive patients (1.57±0.16 vs. 1±0.07; P=0.0049). Our data show that there is a positive correlation between NCC expression/phosphorylation in urinary exosomes and the development of hypertension in posttransplant male patients treated with tacrolimus. Our results are consistent with the hypothesis that NCC activation plays a major role in tacrolimus-induced hypertension.

Ovarian hormones and prolactin increase renal NaCl cotransporter phosphorylation.

Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

Modulation of NCC activity by low and high K(+) intake: insights into the signaling pathways involved.

Modulation of Na(+)-Cl(-) cotransporter (NCC) activity is essential to adjust K(+) excretion in the face of changes in dietary K(+) intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K(+) diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K(+)-citrate diets for 4 days. The low-K(+) diet decreased and high-K(+) diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr(44)/Thr(48)/Thr(53)) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser(383)/Ser(325)). The effect of the low-K(+) diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K(+) diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K(+) diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K(+)-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K(+) increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K(+) diet.