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

Abstract

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.

a healthy pregnancy requires a large plasma volume expansion, which is necessary to support the growing uterus and fetus. As the fetus has a high metabolic rate and grows rapidly after midpregnancy, it requires a high delivery rate of substrates and O₂. Therefore, a high placental blood flow, which is supported by maternal volume expansion, is essential for substrate supply and fluid balance of the fetus. It has been shown that women with pregnancies complicated by fetal growth restriction have reduced plasma volumes compared with control women with healthy pregnancies (17, 41). This insufficient volume expansion leads to impaired uteroplacental perfusion, which results in an increased incidence of fetal hypoxia and nutrient restriction. Fetal growth restriction increases the risk for adult onset hypertension, metabolic syndrome (4), and reduced nephron number leading to renal disease (7).

The maternal plasma volume expansion is supported by avid renal Na⁺ retention, which is mediated by activation of the renin-angiotensin-aldosterone system. Na⁺ reabsorption is determined by the rate of renal tubular Na⁺ transport, which is set by luminal Na⁺ transporters. We have previously characterized the protein expression of the major apical Na⁺ transporters in pregnancy. We found that the α-subunit of the epithelial Na⁺ channel (αENaC) was increased in mid- and late pregnant rats; however, we did not observe any changes in the Na⁺-Cl⁻ cotransporter (NCC) (53). This was surprising as NCC protein abundance is increased by chronic elevation of aldosterone (22), ANG II (48), and estrogen (50), as occur in pregnancy. Aldosterone (25), ANG II (10, 43), estrogen, and progesterone (40) can increase NCC phosphorylation of the NH₂-terminal tail of the transporter.

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and the related kinase oxidative stress-related kinase (OSR1) are terminal kinases in signaling pathways that regulate NCC (25, 39). Both SPAK and OSR1 can directly phosphorylate NCC at several serine (Ser⁷¹ and possibly Ser⁸⁹) and threonine (Thr⁵³ and Thr⁵⁸) residues in the NH₂-terminal tail of the transporter (39). SPAK and OSR1 are regulated by with no lysine kinases (WNKs), which act as intermediate kinases in NCC signaling cascades (10, 21, 43). WNK1 and WNK3 are activators of SPAK, whereas WNK4 is an inhibitor of SPAK. WNK4 also decreases WNK1- and WNK3-mediated activation of NCC (11). Both WNK3 and serum and glucocorticoid-regulated kinase (SGK)1 interact with the E3 ubiquitin ligase NEDD4-2 to inhibit NEDD4-2-mediated degradation of NCC at the plasma membrane. Since phosphorylation of NCC is critical for activation of the transporter (36, 39), we undertook the present study to examine NCC phosphorylation, the regulatory kinase network, the NCC message, and cellular localization of NCC during pregnancy in the rat.

METHODS

Animals.

Animal experiments were carried out using female Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN). Animals were maintained in the University of Florida animal facility in compliance with institutional guidelines and the National Institutes of Health (NIH) Guide for Animal Care and Use. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida, and experiments were carried out according to institutional guidelines. Female rats destined to become pregnant were placed with a fertile male rat, and day 1 of pregnancy was designated as the day that sperm was present in vaginal smears. Rat gestation is ∼21 days. Rats in group 1 (n = 20) were used for the determination of electrolyte balance and NCC protein abundance by Western blot analysis, and rats in group 2 (n = 9) were used for NCC immunolocalization by immunohistochemistry.

Metabolic cage experiments.

Metabolic cage experiments for the determination of electrolyte balance were performed in group 1 virgin (n = 6), midpregnant (days 12–14, n = 7), and late pregnant (days 19-21, n = 7) rats. Rats were acclimated to the metabolic cages for 24 h before the 48-h collection period. Rats were given ad libitum access to water and powdered rat chow (Harlan Teklad LM-485, 0.3% sodium and 0.8% potassium, Harlan Teklad, Madison, WI). Food intake and urine volume were measured gravimetrically. Urine samples were analyzed by flame photometry for Na⁺ and K⁺. After the collection period, rats were removed from the metabolic cages and sacrificed under isoflurane anesthesia, and kidneys were perfused cell free with cold PBS and then harvested for Western blot analysis.

Homogenate preparation.

Kidneys were dissected into the cortex (CTX), outer medulla (OM), and inner medulla (IM), snap frozen in liquid nitrogen, and stored at −80°C until homogenization. Tissues were homogenized in ice-cold homogenization buffer [5% sorbitol containing 25 mM histidine-imidazole (pH 7.5), 100 mM Na2EDTA, 20 mg/ml aprotinin, 167 mM PMSF, and a phosphatase inhibitor cocktail (P0044, Sigma)]. After homogenization, samples were centrifuged at 2,000 g for removal of debris. Protein concentration of the homogenates was determined by BCA assay (Pierce Thermo, Rockford, IL). All samples were solubilized at 60°C for 15 min in Laemmli sample buffer and stored at −80°C.

Quantitative immunoblot analysis and reagents.

Protein abundances were detected by Western blot analysis using 60 μg of the kidney CTX, OM, and IM. The linearity of this loading has been previously verified with McDonough and Loffing antibodies to NCC (34). In the present study, we verified 60 μg to be in the linear range of detection for the Masilamani NCC antibody. Samples were loaded on 7.5% polyacrylamide gels and separated by gel electrophoresis at a constant voltage of 100 V for 15 min followed by140 V for 90 min. Membranes were incubated for 36 h with primary antibodies as follows: anti-NCC (1:1,000, Masilamani laboratory), anti-NCC [1:5,000, McDonough laboratory (34)], and three anti-NCC antibodies [phosphorylated at Thr⁵³, Ser⁷¹, and Ser⁸⁹, respectively (NCCpT53, NCCpS71, and NCCpS89, respectively), 1:5,000, Loffing, Zurich, Switzerland (46)]. Blots were then incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:15,000 dilution, sc-2004, Santa Cruz Biotechnology). Bands of interest were visualized using enhanced chemiluminescence reagent (Supersignal West Pico, Thermo Scientific, Rockford, IL) and quantified by densitometry (VersaDoc imaging system and Quantity One Analysis software, Bio-Rad). Densitometry was normalized to Ponceau staining (Sigma) and virgin controls, with the mean for the virgin control group being defined as 100%. The electrophoretic shift in the molecular weight of NCC was quantified by determining the ratio of the density of the whole band (100–180 kDa) to the density of the top region of the band (140–180 kDa).

SPAK phosphorylated at Ser³²⁵ (SPAKpS373) and OSR1 phosphorylated at Ser³²⁵ (OSR1pS325) were detected in samples of the renal CTX run at both 80 and 40 μg/lane (to demonstrate linearity of detection), blotted onto polyvinylidene difluoride membranes, and probed with sheep anti-SPAKpS373/OSR1pS325 antiserum (Division of Signal Transduction Therapy, University of Dundee) after preabsorption against nonphosphorylated SPAK (diluted 1:1,000) and with tagged donkey anti-sheep secondary antibody (DAS680, Invitrogen, diluted 1:5,000). Signals were detected with the Odyssey Infrared Imaging System (Li-COR) and quantified by accompanying software. Arbitrary density units were normalized to the mean intensity of the virgin control group, defined as 1.0. To assess protein loading, 10 μg of protein from each sample were resolved by SDS-PAGE and stained with Coomassie blue, and multiple random bands were quantified as previously described (31). Since samples were run twice (at 40 and 80 μg), the normalized values were averaged and mean values were compiled for statistical analysis after normalization to protein loading.

RNA isolation and quantitative PCR.

RNA was isolated using a Direct-zol RNA MiniPrep kit with on column DNA digestion (Zymo Research) according to the manufacturer's instructions. RNA (2 μg) samples were used as a template for reverse transcription with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNAs (20 ng) were then used as a template in quantitative real-time PCR reactions (Applied Biosystems) to evaluate changes in NCC (Rn00571074_m1), SGK1 (Rn01537468_g1), total WNK1 (Rn00671521_m1), WNK3 (Rn01409051_m1), and WNK4 (Rn00598070_m1) mRNA levels. The WNK1 Taqman primer/probe set used in the present study targets the exon 25–26 boundary, which recognizes both the long kinase active WNK1 isoform and short kinase defective WNK1 isoform. Furthermore, exon 26 of the WNK1 gene undergoes tissue-specific alternative splicing. Therefore, the transcript analysis in the present study excluded WNK1 isoforms that lack exon 26. However, since exon 26 is highly represented in the mammalian kidney (52), the contribution of these excluded isoforms to total renal WNK1 transcript abundance should be small. Cycle threshold (C_t) values were normalized against GAPDH, and relative quantification was performed using the ΔΔC_t method (28). Fold change values were calculated as the change in mRNA expression levels relative to the control. TaqMan primer/probe sets were purchased from Applied Biosystems.

Tissue preparation for immunohistochemistry.

Rats in group 2 [virgin, 11- to 13-day midpregnant, and 18- to 20-day late pregnant rats (all n = 3)] were anesthetized with inhalant isoflurane. Kidneys were perfused via the abdominal aortic cannula, first blood free with PBS (pH 7.4) followed by periodate-lysine-2% paraformaldehyde for 6 min, at a controlled pressure of 100 mmHg. The perfused kidneys were then cut transversely into several 2- to 4-mm-thick slices, immersed ∼24 h at 4°C in the same fixative, and placed in periodate-lysine buffer at 4°C. Kidney samples from each animal were embedded in polyester wax [polyethylene glycol 400 distearate (Polysciences, Warrington, PA) and 10% 1-hexadecanol], and 3-μm-thick sections were cut and mounted on triple chrome-alum-gelatin-coated glass slides (51).

Immunohistochemistry.

Immunolocalization was accomplished using immunoperoxidase as follows: sections were dewaxed in ethanol, rehydrated, heated in Trilogy (Cell Marque, Rocklin, CA) to 88°C for 30 min and then to 96°C for 30 min, cooled for 30 min, and rinsed in PBS. Endogenous peroxidase activity was inhibited by incubating the sections in 3% H₂O₂ in distilled water for 45 min. Sections were blocked for 15 min with Serum-Free Protein Block (DakoCytomation) and then incubated at 4°C overnight with primary antibody. Sections were washed in PBS, incubated for 30 min with polymer-linked, peroxidase-conjugated goat anti-rabbit IgG (MACH2, Biocare Medical, Concord, CA), washed again with PBS, and then exposed to diaminobenzidine for 5 min. Sections were washed in distilled water, dehydrated with xylene, mounted, and observed by light microscopy. Comparisons of labeling were made only between sections of the same thickness from the same immunohistochemistry experiment. Sections were examined on a Leica DM2000 microscope and photographed using a Leica DFC425 digital camera and Leica DFC Twain Software and the LAS application suite (Leica Microsystems, Buffalo Grove, IL).

Quantitative analysis of immunohistochemisty.

Quantification was determined as previously described (23). All tissues were prepared and stained using identical procedures and were viewed at the same time using identical settings. Briefly, high-resolution digital micrographs were taken of defined tubular segments using a Leica DM2000 microscope equipped with a DFC425 digital camera and DFC Twain Software and the LAS application suite. Freely available software (NIH ImageJ, version 1.48v) quantified pixel intensity across a line drawn from the tubule lumen through the center of an individual cell. In cells with visible nuclei, the line was drawn to the side of the nucleus, i.e., excluding the nucleus. These data were then analyzed using custom software. Pixel intensity at each point of the line was displayed graphically. The apical and basolateral edges were determined by the user. Total cellular expression was determined by integrating net pixel intensity through the entire cell. Cell height was determined as the distance in pixels between the apical and basolateral edges of the cells. Immunoreactivity expression in the apical 20% of the cell was determined by integrating pixel intensity in this region of the cell. The individual performing the microscopy, photography, and quantitative analysis was blinded to the treatment status of the animal.

Statistics.

Results are presented as means ± SE. Statistical analyses were performed using an unpaired t-test or one-way ANOVA, and P < 0.05 was considered statistically significant.

RESULTS

Electrolyte measurements.

As shown in Table 1, Na⁺ and K⁺ intake increased progressively during pregnancy, and despite a small increase in urinary excretion of Na⁺ at midterm, the difference between Na⁺ intake and urinary excretion also increased progressively, suggesting significant Na⁺ retention compared with virgin control rats. Hematocrit also fell progressively, demonstrating the expected cumulative plasma volume expansion of normal pregnancy. In addition, the usual gestational falls in plasma Na⁺ concentration and plasma osmolality were observed (5, 13). In contrast to the progressive renal Na⁺ retention, K⁺ was retained only in late pregnant rats, in association with a significant rise in plasma K⁺ concentration compared with virgin rats (Table 1).

Table 1.

Urine and plasma measurements

	Virgin Rats	Midpregnant Rats	Late Pregnant Rats
Body weight, g	269 ± 8	292 ± 3*	384 ± 7*†
Na+ intake, meq/24 h	2.13 ± 0.09	2.91 ± 0.04*	3.30 ± 0.09*†
Urine volume, ml/24 h	16.5 ± 2.0	29.4 ± 2.3*	20.9 ± 1.3†
UNaV, meq/24 h	1.70 ± 0.12	2.27 ± 0.05*	1.59 ± 0.09†
Na+ intake − UNaV, meq/24 h	0.43 ± 0.04	0.65 ± 0.04*	1.72 ± 0.08*†
Hematocrit, %	38 ± 1	34 ± 1*	32 ± 1*†
Plasma Na+ concentration, meq/l	145 ± 2	140 ± 2*	139 ± 1*
Plasma osmolality, mosm/kg	292 ± 3	283 ± 3	281 ± 1*
K+ intake, meq/24 h	3.34 ± 0.14	4.57 ± 0.07*	5.18 ± 0.14*†
UKV, mEq/24 h	3.04 ± 0.17	4.32 ± 0.09*	4.61 ± 0.12*
K+ intake − UKV, meq/24 h	0.30 ± 0.04	0.25 ± 0.07	0.57 ± 0.12†
Plasma K+ concentration, meq/l	3.4 ± 0.2	4.1 ± 0.3	4.3 ± 0.2*

Values are means ± SE.

U_NaV, urinary excretion of Na⁺; U_KV, urinary excretion of K⁺.

^*P < 0.05 vs. virgin rats;

^†P < 0.05 vs. midpregnant rats (by ANOVA).

Renal NCC protein abundance and phosphorylation.

Protein abundance of total NCC (using two different primary antibodies, SM and McD) as well as phosphorylated NCC (NCCpThr53, NCCpSer71, and NCCpSer89) was determined in renal cortical tissue of pregnant and virgin rats, as shown in Figs. 1 and 2. There were no differences detected in total or phosphorylated NCC abundance in midpregnant versus virgin kidneys (Fig. 1). However, in late pregnant rats, both total NCC and phosphorylated NCC were decreased compared with virgin control rats (Fig. 2). A shift in electrophoretic mobility was also detected in late pregnant rats with the SM antibody. In virgin rats, 83 ± 2% of the total NCC density was in high-molecular-weight forms (140–180 kDa) compared with 47 ± 2% in late pregnant rats (P ≤ 0.001 by unpaired t-test).

Fig. 1.

Protein abundance of total Na⁺-Cl⁻ cotransporter (NCC; using two different primary antibodies, SM and McD) and phosphorylated NCC (NCCpThr53, NCCpSer71, and NCCpSer89) in renal cortical tissue of midpregnant (n = 7) and virgin (n = 6) rats. Band densities were normalized to virgin control rats, with virgin rats set at 100%, and shown as bar graphs. An unpaired t-test was performed. Data are presented as means ± standard error.

Fig. 2.

Protein abundance of total NCC (using two different primary antibodies, SM and McD) and phosphorylated NCC (NCCpThr53, NCCpSer71, and NCCpSer89) in renal cortical tissue of late pregnant (n = 7) and virgin (n = 6) rats. Band densities were normalized to virgin control rats, with virgin rats set at 100%, and shown as bar graphs. An unpaired t-test was performed. Data are presented as means ± SE. *P < 0.05 vs. virgin rats.

SPAK and OSR1 phosphorylation.

Protein abundance of phosphorylated SPAK (pS373) and OSR1 (pS325) was determined in renal cortical tissue of pregnant and virgin rats, as shown in Fig. 3. There were no differences detected in phosphorylated SPAK and OSR1 abundance in pregnant versus virgin kidneys (by one-way ANOVA). However, values of both phosphorylated SPAK and OSR1 were both significantly lower in late pregnant versus midpregnant rats (P = 0.014 and 0.0018 by unpaired t-test, respectively).

Fig. 3.

Protein abundance of phosphorylated STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK; pSer³⁷³) and phosphorylated oxidative stress-related kinase 1 (OSR1; pSer325) in renal cortical tissue of virgin, midpregnant, and late pregnant rats. Band densities were normalized to virgin control rats, with virgin rats set at 100%, and shown as bar graphs. An unpaired t-test was performed. Data are presented as means ± SE; n = 7 rats/group. †P < 0.05 vs. midpregnant rats.

Transcript expression of NCC and regulatory kinases.

mRNA expression of NCC, SGK1, total WNK1, WNK3, and WNK4 was determined in renal cortical tissue of pregnant and virgin rats. No differences were detected in transcript expression for the genes of interest (Fig. 4).

Fig. 4.

NCC, serum and glucocorticoid-regulated kinase 1 (SGK1), total with no lysine kinase (WNK)1, WNK3, and WNK4 mRNA expressions in the renal cortex of virgin, midpregnant, and late pregnant rats. Total RNA was isolated from the dissected cortex and converted to cDNA. Real-time quantitative RT-PCR was performed to evaluate mRNA expressions. One-way ANOVA with a Tukey post hoc test was performed. Data are presented as means ± SE; n = 6 rats/group.

NCC immunolocalization.

As shown in Fig. 5, expression of NCC was not significantly reduced in midpregnant rats but decreased in late pregnant rats compared with virgin control rats (virgin rats: 1,820 ± 96 pixel intensity in apical 20% of cells; midpregnant rats: 1,475 ± 42 pixel intensity, not significant; late pregnant rats: 1,343 ± 145 pixel intensity, P < 0.05 by ANOVA).

Fig. 5.

Immunolocalization of NCC. Immunoreactivity expression as determined by pixel intensity was determined in the apical 20% of the cell and shown as bar graphs. One-way ANOVA with a Tukey post hoc test was performed. Data are presented as means ± SE; n = 3 rats/group. *P < 0.05 vs. virgin rats.

DISCUSSION

The main findings of the present study are that renal NCC protein abundance is not increased in pregnancy and, in fact, by late pregnancy NCC abundance falls, despite continued renal Na⁺ retention. In addition to NCC total protein abundance, phosphorylation and apical localization were unchanged in midpregnancy, and in late pregnancy, both NCC phosphorylation and apical localization were decreased.

The assessment of the NCC protein abundance in this study was exhaustive, since we used two different and well-characterized primary antibodies to total NCC (34) as well as three different phosphorylated NCC antibodies (46). There was remarkable accordance with these five different antibodies by Western blot analysis, indicating no change in NCC abundance at midterm and that NCC abundance falls in late pregnancy. In accordance with this, by quantitative immunohistochemistry, the density of NCC in the apical region of the distal convoluted tubule is unchanged at midterm and decreased in late pregnancy compared with virgin rats. The fall in late pregnancy was particularly unexpected as this is when renal Na⁺ reabsorption is highest, as indicated in the present study and by earlier observations (1, 3). Late pregnancy is also when plasma aldosterone levels are highest (8, 18), and NCC protein is a known target for aldosterone stimulation (22) in situations where K⁺ balance is normal (see below) (16). This decrease in NCC abundance in late pregnancy is posttranslational as NCC transcript levels were the same in virgin, midpregnant, and late pregnant rats.

In addition to total protein abundance, NCC activity can be increased by phosphorylation at many sites in NCC protein (32, 36, 39). We used three antibodies, which recognize the phosphorylated Thr⁵³, Ser⁷¹, and Ser⁸⁹ sites, respectively (46), as these do not cross react with homologous sites in the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC) (27). Despite the fact that increased aldosterone (25), ANG II (10, 43), estrogen, and progesterone (40) have been reported to promote phosphorylation and NCC activation, in the pregnant rat kidney CTX, we found that the quantity of phosphorylated NCC remained proportional to total NCC, with no change in phosphorylated NCC protein at midterm, and with falls in late pregnancy.

We also measured phosphorylated SPAK and phosphorylated OSR1, since these kinases are known to phosphorylate NCC at Thr⁵³ and Ser⁷¹ and may phosphorylate NCC at Ser⁸⁹ (21, 39). Compared with virgin rats, there was no difference in the abundance of phosphorylated SPAK and phosphorylated OSR1 compared to either midpregnant or late pregnant rats, although values were lower in late pregnancy versus midterm, which is in accordace with the decreased phosphorylation of NCC in the late pregnant rat kidney (Fig. 3). However, WNK1 and WNK4 are serine/threonine kinases that interact in a complex cascade with SPAK to regulate NCC phosphorylation (21, 39), and these were unchanged at the transcript level in pregnancy. Overall, it seems likely that the decrease in phosphorylated NCC in late pregnant rats compared with virgin rats is a reflection of decreased total NCC and not of alterations in SPAK/OSR1 kinase activity. Another mechanism for NCC regulation is by the ubiquitin ligase NEDD4-2. Since WNK3 (26) and SGK1 (2) both activate NCC by independent mechanisms involving interactions with NEDD4-2, we measured transcript levels of SGK1 and WNK3 in pregnancy. Neither WNK3 nor SGK1 were changed in pregnant rats compared with virgin rats, which suggests that this degradation pathway is also unchanged in pregnancy.

NCC is also a target for endoplasmic reticulum-associated degradation (33), and changes in NCC glycosylation status may reflect sluggish trafficking in the biosynthetic pathway, which could lead to enhanced endoplasmic reticulum-associated degradation. Here, we show a shift in the electrophoretic mobility of NCC in late pregnancy, which was most notable using the Masilamani antibody. This shift represents an increase in lower-molecular-weight, immature forms of NCC in late pregnancy.

Thus, in normal late pregnancy, we have the aldosterone- and ANG II-sensitive renal Na⁺ transporter NCC decreased in abundance and activity despite high circulating aldosterone and ANG II levels and clear evidence of marked renal Na⁺ and K⁺ retention.

In earlier work, we have reported that the α₁-subunit of renal cortical Na⁺-K⁺-ATPase as well as ATPase activity were reduced in both midpregnant and late pregnant rats, whereas medullary α₁-Na⁺-K⁺-ATPase and enzyme activity increased in midpregnancy and remain unchanged close to term (29). Na⁺-K⁺-ATPase is also a well-known target of aldosterone stimulation (15). Taken together, these observations raise the possibility of regional differences in the ANG II/aldosterone-dependent regulation of renal Na⁺ transporters in normal pregnancy, with the renal CTX being, for some reason, rendered resistant.

Phosphodiesterase (PDE)5 is another protein whose localization is regionally regulated in the kidney during pregnancy. There is a selective increase in PDE5 activity and protein abundance in the medulla with no change in the renal CTX during pregnancy in the rat (35). We have shown that this selective medullary increase in PDE5 plays an important role to blunt the cGMP-mediated natriuretic responses to atrial natriuretic peptide and nitric oxide (NO) during pregnancy (24, 44), thereby allowing antinatriuretic influences (aldosterone and ANG II) to predominate. Since this increased PDE5 is confined to the medulla, we anticipate that the actions of cGMP continue unabated in the CTX. Indeed, there is considerable evidence that the increased renal plasma flow and glomerular filtration rate seen in normal pregnancy reflects cGMP-dependent vasodilation in response to the increased cortical NO (6, 12, 14, 45).

Nitric oxide and ANG II are natural antagonists, and one possible link explaining the regional differences in Na⁺ transporters in pregnancy may relate to the effect of NO on Na⁺ transport. NO is known to have natriuretic actions, mediated by cGMP, which include inhibition of several renal Na⁺ transporters (19). In the proximal tubule, the NO effect is variable, but most studies have shown that NO inhibits both Na⁺/H⁺ exchanger (NHE)3 and Na⁺-K⁺-ATPase (19). There is a well-established inhibitory effect of NO on both NHE3 and NKCC2 in the thick ascending limb of the loop of Henle, and there is also evidence that NO inhibits ENaC activity by cGMP-dependent mechanisms (19, 20, 37). While there is no direct evidence of an inhibitory interaction between NO and NCC, Garvin and colleagues (19) concluded that such an interaction is likely. Thus, the elevated NO production together with the lack of activation of PDE5 that occurs in the renal CTX during pregnancy may contribute to the failure of NCC to increase.

NCC abundance and activity are also regulated by K⁺ balance. In the nonpregnant state, volume depletion activates NCC, which reduces Na⁺ delivery to the collecting duct. This uncoupling of aldosterone's action on the collecting duct to promote electrogenic K⁺ secretion permits Na⁺ retention without renal K⁺ wasting in volume-depleted states (9, 16, 32, 47). In contrast, when K⁺ intake is high and plasma K⁺ increases, NCC abundance and phosphorylation (activation) are reduced (9, 21, 38). However, there have been reports that K⁺ loading causes both increases (49) and decreases in phosphorylated SPAK (9, 38), and these differences could be dependent on the accompanying anion (9). In the context of this discussion, K⁺ balance may predominate over changes in extracellular fluid volume in the regulation of NCC (16, 47); in fact, a high-K⁺ diet prevents the mineralocorticoid-induced increases in NCC (38, 49). As shown here, K⁺ intake is increased ∼55% during late pregnancy and is associated with an increase in plasma K⁺ compared with virgin rats together with a marked K⁺ retention (Table 1). This increased K⁺ intake and elevated plasma K⁺ could be in part mediating the reduction in NCC and its phosphorylation seen in late pregnancy. However, it is of note that there was no difference in plasma K⁺ between midpregnant and late pregnant rats and yet their protein profiles for NCC and phosphorylated SPAK/phosphorylated OSR1 were strikingly different. This suggests that unlike high K⁺ feeding, pregnancy is a unique state in which plasma K⁺ alone cannot account for the late pregnancy-mediated changes in total and phosphorylated NCC. The fact that both Na⁺ and K⁺ retention occur in normal late pregnancy, in the face of decreased NCC activity, which would promote K⁺ secretion in the normal nonpregnant individual, further suggests that unique adaptations must be occurring in the pregnant collecting duct. Indeed, women with Gitelman's syndrome experience relatively uncomplicated pregnancies, with appropriate “for gestational age babies.” This suggests that in these women, with K⁺ supplementation, maternal K⁺ and Na⁺ retention continue despite nonfunctioning NCCs (30). Future studies should investigate this further.

In conclusion, aldosterone/ANG II signaling in the renal CTX may be disrupted in pregnancy, as evidenced by the failure of NCC to increase in midpregnancy and fall close to term. This may be related to the increased plasma K⁺ of late pregnancy, activation of the renal cortical NO system, which occurs throughout pregnancy, and/or other mechanisms. While NCC activation cannot contribute to the gestational renal Na⁺ retention, it is clear that other compensatory changes are sufficient to permit marked, cumulative Na⁺ retention and plasma volume expansion.

GRANTS

This work was supported by the Robert and Mary Cade Professorship of Physiology (to C. Baylis) and National Institutes of Health Grants R01-DK-083785 (to A. McDonough) and T32-HL-083810, R01-HD-041571, and R01-DK-56843 (to C. Baylis).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: C.A.W., S.M.M., and C.B. conception and design of research; C.A.W. and A.A.M. performed experiments; C.A.W., A.A.M., J.W.V., and C.B. analyzed data; C.A.W., J.W.V., and C.B. interpreted results of experiments; C.A.W. and A.A.M. prepared figures; C.A.W. and C.B. drafted manuscript; C.A.W., A.A.M., S.M.M., J.W.V., and C.B. edited and revised manuscript; C.A.W., A.A.M., S.M.M., J.W.V., and C.B. approved final version of manuscript.