Renal NCC is unchanged in the midpregnant rat and decreased in the late pregnant rat despite avid renal Na+ retention.

Pregnancy is characterized by plasma volume expansion due to Na(+) retention, driven by aldosterone. The aldosterone-responsive epithelial Na(+) channel is activated in the kidney in pregnancy. In the present study, we investigated the aldosterone-responsive Na(+)-Cl(-) cotransporter (NCC) in mid- and late pregnant rats compared with virgin rats. We determined the abundance of total NCC, phosphorylated NCC (pNCC; pT53, pS71 and pS89), phosphorylated STE20/SPS-1-related proline-alanine-rich protein kinase (pSPAK; pS373), and phosphorylated oxidative stress-related kinase (pOSR1; pS325) in the kidney cortex. We also measured mRNA expression of NCC and members of the SPAK/NCC regulatory kinase network, serum and glucocorticoid-regulated kinase (SGK)1, total with no lysine kinase (WNK)1, WNK3, and WNK4. Additionally, we performed immunohistochemistry for NCC kidneys from virgin and pregnant rats. Total NCC, pNCC, and pSPAK/OSR1 abundance were unchanged in midpregnant versus virgin rats. In late pregnant versus virgin rats, total NCC and pNCC were decreased; however, pSPAK/OSR1 was unchanged. We detected no differences in mRNA expression of NCC, SGK1, total WNK1, WNK3, and WNK4. By immunohistochemistry, NCC was mainly localized to the apical region in virgin rats, and density in the apical region was reduced in late pregnancy. Therefore, despite high circulating aldosterone levels in pregnancy, the aldosterone-responsive transporter NCC is not increased in total or activated (phosphorylated) abundance or in apical localization in midpregnant rats, and all are reduced in late pregnancy. This contrasts to the mineralocorticoid-mediated activation of the epithelial Na(+) channel, which we have previously reported. Why and how NCC escapes aldosterone activation in pregnancy is not clear but may relate to regional differences in aldosterone sensitivity the increased K(+) intake or other undefined mechanisms.

Renal epithelial sodium channel is critical for blood pressure maintenance and sodium balance in the normal late pregnant rat.

Normal pregnancy is a state marked by avid sodium retention and plasma volume expansion. Insufficient plasma volume expansion results in the compromised maternal state of intrauterine growth restriction, which afflicts ∼5% of all human pregnancies. We have recently shown that renal epithelial sodium channel (ENaC) activity in vivo in the late pregnant (LP) rat is increased. To determine the importance of the renal versus extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy, we have chronically blocked the ENaC pharmacologically with daily subcutaneous injections of benzamil and genetically using intrarenal transfection of αENaC short hairpin RNA. Compared with untreated LP control animals, LP rats treated with benzamil retain less sodium and have reduced mean arterial blood pressure. Furthermore, LP rats treated with benzamil had lower maternal body weight gain. Intrarenal transfection of αENaC short hairpin RNA versus scrambled small RNA successfully decreased renal αENaC mRNA expression in LP rats. Intrarenal transfection of αENaC short hairpin RNA reduced maternal sodium retention, body weight gain and pup weight. Redundant physiological systems that protect blood pressure and sodium homeostasis were unable to compensate for the loss of ENaC activity in the pregnant rat. These findings demonstrate that the renal ENaC is necessary for maintaining pregnancy-mediated sodium retention, volume expansion and blood pressure regulation.

Chronic vasodilation increases renal medullary PDE5A and α-ENaC through independent renin-angiotensin-aldosterone system pathways.

We have previously observed that many of the renal and hemodynamic adaptations seen in normal pregnancy can be induced in virgin female rats by chronic systemic vasodilation. Fourteen-day vasodilation with sodium nitrite or nifedipine (NIF) produced plasma volume expansion (PVE), hemodilution, and increased renal medullary phosphodiesterase 5A (PDE5A) protein. The present study examined the role of the renin-angiotensin-aldosterone system (RAAS) in this mechanism. Virgin females were treated for 14 days with NIF (10 mg·kg(-1)·day(-1) via diet), NIF with spironolactone [SPR; mineralocorticoid receptor (MR) blocker, 200-300 mg·kg(-1)·day(-1) via diet], NIF with losartan [LOS; angiotensin type 1 (AT1) receptor blocker, 20 mg·kg(-1)·day(-1) via diet], enalapril (ENAL; angiotensin-converting enzyme inhibitor, 62.5 mg/l via water), or vehicle (CON). Mean arterial pressure (MAP) was reduced 7.4 ± 0.5% with NIF, 6.33 ± 0.5% with NIF + SPR, 13.3 ± 0.9% with NIF + LOS, and 12.0 ± 0.4% with ENAL vs. baseline MAP. Compared with CON (3.6 ± 0.3%), plasma volume factored for body weight was increased by NIF (5.2 ± 0.4%) treatment but not by NIF + SPR (4.3 ± 0.3%), NIF + LOS (3.6 ± 0.1%), or ENAL (4.0 ± 0.3%). NIF increased PDE5A protein abundance in the renal inner medulla, and SPR did not prevent this increase (188 ± 16 and 204 ± 22% of CON, respectively). NIF increased the α-subunit of the epithelial sodium channel (α-ENaC) protein in renal outer (365 ± 44%) and inner (526 ± 83%) medulla, and SPR prevented these changes. There was no change in either PDE5A or α-ENaC abundance vs. CON in rats treated with NIF + LOS or ENAL. These data indicate that the PVE and renal medullary adaptations in response to chronic vasodilation result from RAAS signaling, with increases in PDE5A mediated through AT1 receptor and α-ENaC through the MR.

Time course of renal sodium transport in the pregnant rat.

Progressive sodium retention and cumulative plasma volume expansion occur to support the developing fetus during pregnancy. Sodium retention is regulated by individual tubular transporters and channels. An increase or decrease in any single transporter could cause a change in sodium balance. Understanding the time-course for changes in each sodium transporter during pregnancy will enable us to understand progressive sodium retention seen in pregnancy. Here, we examined the activity of the major apical sodium transporters found in the nephron using natriuretic response tests in virgin, early pregnant, mid-pregnant, and late pregnant rats. We also measured renal and serum aldosterone levels. We found that furosemide sensitive sodium transport (NKCC2) is only increased during late pregnancy, thiazide sensitive sodium transport (NDCBE/pendrin) is increased in all stages of pregnancy, and that benzamil sensitive sodium transport (ENaC) is increased beginning in mid-pregnancy. We also found that serum aldosterone levels progressively increased throughout gestation and kidney tissue aldosterone levels increased only during late pregnancy. Here we have shown progressive turning on of specific sodium transport mechanisms to help support progressive sodium retention through the course of gestation. These mechanisms contribute to the renal sodium retention and plasma volume expansion required for an optimal pregnancy.

Increased renal alpha-epithelial sodium channel (ENAC) protein and increased ENAC activity in normal pregnancy.

Pregnancy-mediated sodium (Na) retention is required to provide an increase in plasma volume for the growing fetus. The mechanisms responsible for this Na retention are not clear. We first used a targeted proteomics approach and found that there were no changes in the protein abundance compared with virgin rats of the ß or γ ENaC, type 3 Na(+)/H(+) exchanger (NHE3), bumetanide-sensitive cotransporter (NKCC2), or NaCl cotransporter (NCC) in mid- or late pregnancy. In contrast, we observed marked increases in the abundance of the α-ENaC subunit. The plasma volume increased progressively during pregnancy with the greatest plasma volume being evident in late pregnancy. ENaC inhibition abolished the difference in plasma volume status between virgin and pregnant rats. To determine the in vivo activity of ENaC, we conducted in vivo studies of rats in late pregnancy (days 18-20) and virgin rats to measure the natriuretic response to ENaC blockade (with benzamil). The in vivo activity of ENaC (U(Na)V postbenzamil-U(Na)V postvehicle) was markedly increased in late pregnancy, and this difference was abolished by pretreatment with the mineralocorticoid receptor antagonist, eplerenone. These findings demonstrate that the increased α-ENaC subunit of pregnancy is associated with an mineralocorticoid-dependent increase in ENaC activity. Further, we show that ENaC activity is a major contributor of plasma volume status in late pregnancy. These changes are likely to contribute to the renal sodium retention and plasma volume expansion required for an optimal pregnancy.

Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones.

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-beta estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334+/-29%), cortex (219+/-20%) and outer medulla (133+/-9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-beta estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.