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.

Juvenile growth reduces the influence of epithelial sodium channels on myogenic tone in skeletal muscle arterioles.

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days ("weanlings") and 42-49 days ("juveniles"). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG -monomethyl-l-arginine (l-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, ß and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth.

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.

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.

Are sodium transporters in urinary exosomes reliable markers of tubular sodium reabsorption in hypertensive patients?

BACKGROUND: Altered renal sodium handling has a major pathogenic role in salt-sensitive hypertension. Renal sodium transporters are present in urinary exosomes. We hypothesized that sodium transporters would be excreted into the urine in different amounts in response to sodium intake in salt-sensitive versus salt-resistant patients. METHODS: Urinary exosomes were isolated by ultracentrifugation, and their content of Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC) was analyzed by immunoblotting. Animal studies: NKCC2 and NCC excretion was measured in 2 rat models to test whether changes in sodium transporter excretion are indicative of regulated changes in the kidney tissue. Human studies: in hypertensive patients (n = 41), we investigated: (1) a possible correlation between sodium reabsorption and urinary exosomal excretion of sodium transporters, and (2) the profile of sodium transporter excretion related to blood pressure (BP) changes with salt intake. A 24-hour ambulatory BP monitoring and a 24-hour urine collection were performed after 1 week on a low- and 1 week on a high-salt diet. RESULTS: Animal studies: urinary NKCC2 and NCC excretion rates correlated well with their abundance in the kidney. Human studies: 6 patients (15%) were classified as salt sensitive. The NKCC2 and NCC abundance did not decrease after the high-salt period, when the urinary sodium reabsorption decreased from 99.7 to 99.0%. In addition, the changes in BP with salt intake were not associated with a specific profile of exosomal excretion. CONCLUSIONS: Our results do not support the idea that excretion levels of NKCC2 and NCC via urinary exosomes are markers of tubular sodium reabsorption in hypertensive patients.

Aberrant ENaC activation in Dahl salt-sensitive rats.

BACKGROUND: The epithelial sodium channel (ENaC) plays an important role in the regulation of blood pressure by modulating Na reabsorption in the kidney. Dahl salt-sensitive rats on high-salt diet develop severe hypertension, and high-salt diet has been reported to stimulate ENaC mRNA expression in the kidney abnormally in Dahl salt-sensitive rats despite a suppressed plasma aldosterone concentration (PAC). METHODS: We investigated the effect of high-salt diet on ENaC protein expression in Dahl salt-resistant and Dahl salt-sensitive rats, and examined the effect of amiloride (5 mg/kg per day) and eplerenone (0.125% diet) on blood pressure and renal injury in Dahl salt-sensitive rats. RESULTS: Dahl salt-sensitive rats developed hypertension and renal damage following 4 weeks of treatment with high-salt diet. Although PAC and kidney aldosterone content were all suppressed by the high-salt diet in Dahl salt-sensitive rats, both beta and gammaENaC mRNA expression and protein abundance were significantly increased. The molecular weight shift of gammaENaC from 85 to 70 kDa, an indication of ENaC activation, was clearly increased in Dahl salt-sensitive rats on high-salt diet compared with the low-salt group or Dahl salt-resistant rats on high-salt diet. Four weeks of treatment with amiloride, but not eplerenone, significantly ameliorated hypertension and kidney injury in Dahl salt-sensitive rats fed high-salt diet, suggesting aberrant aldosterone-independent activation of ENaC. CONCLUSION: These results suggest that inappropriate expression and activation of ENaC could be one of the underlying mechanisms by which Dahl salt-sensitive rats develop salt-sensitive hypertension and organ damage, and indicate a therapeutic benefit of amiloride in salt-sensitive hypertension where ENaC is excessively activated.

Decoy methods for assessing false positives and false discovery rates in shotgun proteomics.

The potential of getting a significant number of false positives (FPs) in peptide-spectrum matches (PSMs) obtained by proteomic database search has been well-recognized. Among the attempts to assess FPs, the concomitant use of target and decoy databases is widely practiced. By adjusting filtering criteria, FPs and false discovery rate (FDR) can be controlled at a desired level. Although the target-decoy approach is gaining in popularity, subtle differences in decoy construction (e.g., reversing vs stochastic methods), rate calculation (e.g., total vs unique PSMs), or searching (separate vs composite) do exist among various implementations. In the present study, we evaluated the effects of these differences on FP and FDR estimations using a rat kidney protein sample and the SEQUEST search engine as an example. On the effects of decoy construction, we found that, when a single scoring filter (XCorr) was used, stochastic methods generated a higher estimation of FPs and FDR than sequence reversing methods, likely due to an increase in unique peptides. This higher estimation could largely be attenuated by creating decoy databases similar in effective size but not by a simple normalization with a unique-peptide coefficient. When multiple filters were applied, the differences seen between reversing and stochastic methods significantly diminished, suggesting multiple filterings reduce the dependency on how a decoy is constructed. For a fixed set of filtering criteria, FDR and FPs estimated by using unique PSMs were almost twice those using total PSMs. The higher estimation seemed to be dependent on data acquisition setup. As to the differences between performing separate or composite searches, in general, FDR estimated from the separate search was about three times that from the composite search. The degree of difference gradually decreased as the filtering criteria became more stringent. Paradoxically, the estimated true positives in separate search were higher when multiple filters were used. By analyzing a standard protein mixture, we demonstrated that the higher estimation of FDR and FPs in the separate search likely reflected an overestimation, which could be corrected with a simple merging procedure. Our study illustrates the relative merits of different implementations of the target-decoy strategy, which should be worth contemplating when large-scale proteomic biomarker discovery is to be attempted.

Renal tubule sodium transporter abundance profiling in rat kidney: response to aldosterone and variations in NaCl intake.

Based on extensive physiological study of sodium transport mechanisms along the renal tubule, complementary DNAs for all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past decade. There is now a comprehensive set of cDNA and antibody probes that can be used to investigate physiological mechanisms on a molecular level. Using rabbit polyclonal antibodies to all of the major renal Na transport proteins, we have developed profiling methods allowing comprehensive, integrated analysis of sodium transporter protein abundance changes along the renal tubule in response to physiological and pathophysiological perturbations. Here, we review some of our recent findings with this approach, focusing on renal responses to aldosterone and to variations in NaCl intake.

Long-term regulation of ENaC expression in kidney by angiotensin II.

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the alpha subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased alphaENaC protein abundance in rat kidney cortex. The decrease in alphaENaC protein abundance in response to AT1 receptor blockade was associated with a fall in alphaENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on alphaENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of alphaENaC gene expression. Candesartan administration was also found to increase the abundances of the beta and gamma subunits. The increase in beta and gammaENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in beta and gammaENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.

Sodium transporter abundance profiling in kidney: effect of spironolactone.

Renal tubule profiling studies were carried out to investigate the long-term effects of administration of spironolactone, a mineralocorticoid receptor antagonist, on abundances of the major Na transporter and Na channel proteins along the rat renal tubule. Oral administration of spironolactone for 7 days to NaCl-restricted rats did not significantly alter abundances of Na transporters expressed proximal to the macula densa, while substantially decreasing the abundances of the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (ENaC), and the 70-kDa form of the gamma-subunit of ENaC. A dependency of NCC expression on aldosterone was confirmed by showing increased NCC expression in response to aldosterone infusion in adrenalectomized rats. Immunoperoxidase labeling of ENaC in renal cortex confirmed that dietary NaCl restriction causes a redistribution of ENaC to the apical domain of connecting tubule cells and showed that high-dose spironolactone administration does not block this apical redistribution. In contrast, spironolactone completely blocked the increase in alpha-ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC, alpha-ENaC, and the 70-kDa form of gamma-ENaC are regulated via the classical mineralocorticoid receptor, but the subcellular redistribution of ENaC in response to dietary NaCl restriction is not prevented by blockade of the mineralocorticoid receptor.

Regulation of collecting duct AQP3 expression: response to mineralocorticoid.

Adrenocortical steroid hormones are importantly involved in the regulation of extracellular fluid volume. The present study was aimed at examining whether aldosterone and/or glucocorticoid regulates the abundance of aquaporin-3 (AQP3), -2, and -1 in rat kidney. In protocol 1, rats were adrenalectomized, followed by aldosterone replacement, dexamethasone replacement, or combined aldosterone and dexamethasone replacement (rats had free access to water but received a fixed amount of food). Protocol 2 was identical to protocol 1, except that all groups received fixed daily food and water intake. In both protocols 1 and 2, aldosterone deficiency was associated with increased fractional Na excretion and severe hyperkalemia. Semiquantitative immunoblotting revealed that aldosterone deficiency was associated with a dramatic downregulation of AQP3 abundance. Consistent with this, immunocytochemistry and immunoelectron microscopy revealed a marked decrease in AQP3 labeling in the basolateral plasma membranes of collecting duct principal cells. In contrast, AQP1 and AQP2 abundance and distribution were unchanged. Glucocorticoid deficiency revealed no changes in AQP3, -2, or -1 abundance. In protocol 3, Na restriction (to increase endogenous aldosterone levels) or exogenous aldosterone infusion in either normal rats or vasopressin-deficient Brattleboro rats was associated with a major increase in AQP3 abundance. In protocol 4, aldosterone levels were clamped by infusion of aldosterone, while Na intake was altered from a low to a high level. Under these circumstances, there were no changes in AQP3 or AQP2 abundance, although the level of the thiazide-sensitive Na-Cl cotransporter was decreased. In conclusion, the results uniformly demonstrate that aldosterone regulates AQP3 abundance independently of Na intake. In contrast, changes in glucocorticoid levels in these models do not influence AQP3 or AQP2 abundance. Therefore, in the collecting duct aldosterone may regulate, at least in part, AQP3 expression in addition to regulating Na and K transport.

Time course of renal Na-K-ATPase, NHE3, NKCC2, NCC, and ENaC abundance changes with dietary NaCl restriction.

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq. 200 g body wt(-1). day(-1)), rats were switched to a NaCl-deficient diet (0.02 meq. 200 g body wt(-1). day(-1)). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease <4% of baseline levels on day 3, of NaCl restriction. The decreased Na excretion at day 1 occurred despite the absence of a significant increase in plasma aldosterone level or in the abundance of any of the major renal Na transporters. However, after a 1-day lag, plasma aldosterone levels increased in association with increases in abundances of three aldosterone-regulated Na transporter proteins: the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (alpha-ENaC), and the 70-kDa form of gamma-ENaC. RNase protection assays of transporter mRNA levels revealed an increase in renal alpha-ENaC mRNA coincident with the increase in alpha-ENaC protein abundance. However, there was no change in NCC mRNA abundance, suggesting that the increase in NCC protein in response to dietary NaCl restriction was not a result of altered gene transcription. These results point to early regulatory processes that decrease renal Na excretion without an increase in the abundance of any Na transporter, followed by a late aldosterone-dependent response associated with upregulation of NCC and ENaC.

Decreased abundance of collecting duct urea transporters UT-A1 and UT-A3 with ECF volume expansion.

Clinical disorders of extracellular fluid (ECF) volume regulation are often associated with changes in plasma urea concentration. To investigate possible renal causes, we measured the relative abundance of the urea transporters UT-A1, UT-A2, and UT-A3 in renal medulla of rats with aldosterone-induced NaCl retention. ECF volume-expanded rats received aldosterone by osmotic minipump plus a diet containing a high level of NaCl. Control rats received the same infusion of aldosterone plus a virtually NaCl-free diet, which prevented ECF volume expansion. Preliminary measurements demonstrated transient positive Na and water balance, decreased serum urea concentration, and increased urea clearance, but no change in creatinine clearance. Immunoblotting of homogenates from inner medulla showed a marked decrease in the abundance of the collecting duct urea transporters UT-A1 and UT-A3. There were no differences in the abundance of UT-A2, aquaporin (AQP)-2, AQP-3, or AQP-4 in ECF volume-expanded rats vs. controls. Time course experiments demonstrated that changes in UT-A1 abundance paralleled the fall in serum urea concentration after the switch from a low-NaCl to a high-NaCl diet, whereas the fall in UT-A3 abundance was delayed. Candesartan administration markedly decreased the abundance of UT-A1 and UT-A3 in the renal inner medulla, which is consistent with a role for the angiotensin II type 1 receptor in urea transport regulation. The results support the view that ECF-related changes in serum urea concentration are mediated, at least in part, through altered urea transporter abundance.