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Kidneys continuously filter an enormous amount of sodium and adapt kidney Na+ reabsorption to match Na+ intake to maintain circulatory volume and electrolyte homeostasis. Males (M) respond to high-salt (HS) diet by translocating proximal tubule Na+/H+ exchanger isoform 3 (NHE3) to the base of the microvilli, reducing activated forms of the distal NaCl cotransporter (NCC) and epithelial Na+ channel (ENaC). Males (M) and females (F) on normal-salt (NS) diet present sex-specific profiles of “transporters” (cotransporters, channels, pumps, and claudins) along the nephron, e.g., F exhibit 40% lower NHE3 and 200% higher NCC abundance than M. We tested the hypothesis that adaptations to HS diet along the nephron will, likewise, exhibit sexual dimorphisms. C57BL/6J mice were fed for 15 days with 4% NaCl diet (HS) versus 0.26% NaCl diet (NS). On HS, M and F exhibited normal plasma [Na+] and [K+], similar urine volume, Na+, K+, and osmolal excretion rates normalized to body weight. In F, like M, HS lowered abundance of distal NCC, phosphorylated NCC, and cleaved (activated) forms of ENaC. The adaptations associated with achieving electrolyte homeostasis exhibit sex-dependent and independent mechanisms. Sex differences in baseline “transporters” abundance persist during HS diet, yet the fold changes during HS diet (normalized to NS) are similar along the distal nephron and collecting duct. Sex-dependent differences observed along the proximal tubule during HS show that female kidneys adapt differently from patterns reported in males, yet achieve and maintain fluid and electrolyte homeostasis.

The renal nephron consists of a series of distinct cell types that function in concert to maintain fluid and electrolyte balance and blood pressure. The renin-angiotensin system (RAS) is central to Na(+) and volume balance. We aimed to determine how loss of angiotensin II signaling in the proximal tubule (PT), which reabsorbs the bulk of filtered Na(+) and volume, impacts solute transport throughout the nephron. We hypothesized that PT renin-angiotensin system disruption would not only depress PT Na(+) transporters but also impact downstream Na(+) transporters. Using a mouse model in which the angiotensin type 1a receptor (AT(1a)R) is deleted specifically within the PT (AT(1a)R PTKO), we profiled the abundance of Na(+) transporters, channels, and claudins along the nephron. Absence of PT AT(1a)R signaling was associated with lower abundance of PT transporters (Na(+)/H(+) exchanger isoform 3, electrogenic Na(+)-bicarbonate cotransporter 1, and claudin 2) as well as lower abundance of downstream transporters (total and phosphorylated Na(+)-K(+)-2Cl(−) cotransporter, medullary Na(+)-K(+)-ATPase, phosphorylated NaCl cotransporter, and claudin 7) versus controls. However, transport activities of Na(+)-K(+)-2Cl(−) cotransporter and NaCl cotransporter (assessed with diuretics) were similar between groups in order to maintain electrolyte balance. Together, these results demonstrate the primary impact of angiotensin II regulation on Na(+) reabsorption in the PT at baseline and the associated influence on downstream Na(+) transporters, highlighting the ability of the nephron to integrate Na(+) transport along the nephron to maintain homeostasis. NEW & NOTEWORTHY Our study defines a novel role for proximal tubule angiotensin receptors in regulating the abundance of Na(+) transporters throughout the nephron, thereby contributing to the integrated control of fluid balance in vivo.

Contains fulltext : 238842.pdf (Publisher’s version ) (Closed access) A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT(1)) receptors in the kidney, stimulating Na(+) reabsorption. AT(1) receptors in tubular epithelia control expression and stimulation of Na(+) transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT(1) receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na(+) excretion in SMKO mice, we considered whether alterations in Na(+) transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na(+) transporters and associated proteins. Here, we show that levels of Na(+)-K(+)-2Cl(-) cotransporter isoform 2 (NKCC2) and Na(+)/H(+) exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser(71)), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na(+) channel. However, the increases in α- and γ-epithelial Na(+) channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na(+) excretion observed after cell-specific deletion of AT(1) receptors from smooth muscle cells is associated with altered Na(+) transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na(+) transporters and contributing to the regulation of pressure natriuresis.NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.

Extracellular fluid (ECF) potassium concentration ([K+]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+-deficient diet (0K) versus 1% K+ diet (1K) for 10 days ( n = 5–6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-β2 subunits, decreased K+-Cl− cotransporter isoform 3, and increased phosphorylated (p) Na+,K+,2Cl− cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters’ adaptations, effecting a 98% reduction in K+ excretion, included two- to threefold increased phosphorylated Na+-Cl− cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 μmol K+ into the urine and muscle shifts ~47 μmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency.

Each day, ~1.7 kg of NaCl and 180 liters of water are reabsorbed by nephron segments in humans, with urinary excretion fine tuned to meet homeostatic requirements. These tasks are coordinated by a spectrum of renal Na+transporters and channels. The goal of the present study was to investigate the extent to which inhibitors of transepithelial Na+transport (TNa) along the nephron alter urinary solute excretion and how those effects may vary between male and female subjects. To accomplish that goal, we developed sex-specific multinephron models that represent detailed transcellular and paracellular transport processes along the nephrons of male and female rat kidneys. We simulated inhibition of Na+/H+exchanger 3 (NHE3), bumetanide-sensitive Na+-K+-2Cl−cotransporter (NKCC2), Na+-Cl−cotransporter (NCC), and amiloride-sensitive epithelial Na+channel (ENaC). NHE3 inhibition simulations predicted a substantially reduced proximal tubule TNa, and NKCC2 inhibition substantially reduced thick ascending limb TNa. Both gave rise to diuresis, natriuresis, and kaliuresis, with those effects stronger in female rats. While NCC inhibition was predicted to have only minor impact on renal TNa, it nonetheless had a notable effect of enhancing excretion of Na+, K+, and Cl−, particularly in female rats. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na+(increased) and K+(decreased) and to have only a minor impact on whole kidney TNa. Unlike inhibition of other transporters, ENaC inhibition induced stronger natriuresis and diuresis in male rats than female rats. Overall, model predictions agreed well with measured changes in Na+and K+excretion in response to diuretics and Na+transporter mutations.

Extracellular potassium (K+) homeostasis is achieved by a concerted effort of multiple organs and tissues. A limitation in studies of K+ homeostasis is inadequate techniques to quantify K+ fluxes into and out of organs and tissues in vivo. The goal of the present study was to test the feasibility of a novel approach to estimate K+ distribution and fluxes in vivo using stable K+ isotopes. 41K was infused as KCl into rats consuming control or K+-deficient chow ( n = 4 each), 41K-to-39K ratios in plasma and red blood cells (RBCs) were measured by inductively coupled plasma mass spectrometry, and results were subjected to compartmental modeling. The plasma 41K/39K increased during 41K infusion and decreased upon infusion cessation, without altering plasma total K+ concentration ([K+], i.e., 41K + 39K). The time course of changes was analyzed with a two-compartmental model of K+ distribution and elimination. Model parameters, representing transport into and out of the intracellular pool and renal excretion, were identified in each rat, accurately predicting decreased renal K+ excretion in rats fed K+-deficient vs. control diet ( P < 0.05). To estimate rate constants of K+ transport into and out of RBCs, 41K/39K were subjected to a simple model, indicating no effects of the K+-deficient diet. The findings support the feasibility of the novel stable isotope approach to quantify K+ fluxes in vivo and sets a foundation for experimental protocols using more complex models to identify heterogeneous intracellular K+ pools and to answer questions pertaining to K+ homeostatic mechanisms in vivo.

Angiotensin II (ANG II) raises blood pressure partly by stimulating tubular Na+ reabsorption. The effects of ANG II on tubular Na+ transporters (i.e., channels, pumps, cotransporters, and exchangers) vary between short-term and long-term exposure. To better understand the physiological impact, we used a computational model of transport along the rat nephron to predict the effects of short- and long-term ANG II-induced transporter activation on Na+ and K+ reabsorption/secretion, and to compare measured and calculated excretion rates. Three days of ANG II infusion at 200 ng·kg−1·min−1 is nonpressor, yet stimulates transporter accumulation. The increase in abundance of Na+/H+ exchanger 3 (NHE3) or activated Na+-K+-2Cl− cotransporter-2 (NKCC2-P) predicted significant reductions in urinary Na+ excretion, yet there was no observed change in urine Na+. The lack of antinatriuresis, despite Na+ transporter accumulation, was supported by Li+ and creatinine clearance measurements, leading to the conclusion that 3-day nonpressor ANG II increases transporter abundance without proportional activation. Fourteen days of ANG II infusion at 400 ng·kg−1·min−1 raises blood pressure and increases Na+ transporter abundance along the distal nephron; proximal tubule and medullary loop transporters are decreased and urine Na+ and volume output are increased, evidence for pressure natriuresis. Simulations indicate that decreases in NHE3 and NKCC2-P contribute significantly to reducing Na+ reabsorption along the nephron and to pressure natriuresis. Our results also suggest that differential regulation of medullary (decrease) and cortical (increase) NKCC2-P is important to preserve K+ while minimizing Na+ retention during ANG II infusion. Lastly, our model indicates that accumulation of active Na+-Cl− cotransporter counteracts epithelial Na+ channel-induced urinary K+ loss.

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients.

The World Health Organization ranks hypertension the leading global risk factor for disease, specifically, cardiovascular disease. Blood pressure (BP) is higher in Westernized populations consuming Na+-rich processed foods than in isolated societies consuming K+-rich natural foods. Evidence suggests that lowering dietary Na+ is particularly beneficial in hypertensive individuals who consume a high-Na+ diet. Nonetheless, numerous population studies demonstrate a relationship between higher dietary K+, estimated from urinary excretion or dietary recall, and lower BP, regardless of Na+ intake. Interventional studies with K+ supplementation suggest that it provides a direct benefit; K+ may also be a marker for other beneficial components of a “natural” diet. Recent studies in rodent models indicate mechanisms for the K+ benefit: the distal tubule Na+-Cl− cotransporter (NCC) controls Na+ delivery downstream to the collecting duct, where Na+ reabsorbed by epithelial Na+ channels drives K+ secretion and excretion through K+ channels in the same region. High dietary K+ provokes a decrease in NCC activity to drive more K+ secretion (and Na+ excretion, analogous to the actions of a thiazide diuretic) whether Na+ intake is high or low; low dietary K+ provokes an increase in NCC activity and Na+ retention, also independent of dietary Na+. Together, the findings suggest that public health efforts directed toward increasing consumption of K+-rich natural foods would reduce BP and, thus, cardiovascular and kidney disease.

The goal of the present study was to investigate the functional implications of sexual dimorphism in the pattern of transporters along the rodent nephron as reported by Veiras et al. ( J Am Soc Nephrol 28: 3504–3517, 2017). To do so, we developed sex-specific computational models of water and solute transport along the superficial nephrons from male and female rat kidneys. The models account for the sex differences in the abundance of apical and basolateral transporters, single nephron glomerular filtration rate, and tubular dimensions. Model simulations predict that ~70% and 60% of filtered Na+ is reabsorbed by the proximal tubule of male and female rat kidneys, respectively. The lower fractional Na+ reabsorption in female kidneys is due primarily to their smaller transport area, lower Na+/H+ exchanger activity, and lower claudin-2 abundance, culminating in significantly larger fractional delivery of water and Na+ to the downstream nephron segments in female kidneys. Conversely, the female distal nephron exhibits a higher abundance of key Na+ transporters, including Na+-K+-Cl− cotransporters, Na+-Cl− cotransporters, and epithelial Na+ channels. The higher abundance of transporters accounts for the enhanced water and Na+ transport along the female, relative to male, distal nephron, resulting in similar urine excretion between the sexes. Consequently, in response to a saline load, the Na+ load delivered distally is greater in female rats than male rats, overwhelming transport capacity and resulting in higher natriuresis in female rats.

The goal of this study is to investigate the functional implications of the sexual dimorphism in transporter patterns along the proximal tubule. To do so, we have developed sex-specific computational models of solute and water transport in the proximal convoluted tubule of the rat kidney. The models account for the sex differences in expression levels of the apical and basolateral transporters, in single-nephron glomerular filtration rate, and in tubular dimensions. Model simulations predict that 70.6 and 38.7% of the filtered volume is reabsorbed by the proximal tubule of the male and female rat kidneys, respectively. The lower fractional volume reabsorption in females can be attributed to their smaller transport area and lower aquaporin-1 expression level. The latter also results in a larger contribution of the paracellular pathway to water transport. Correspondingly similar fractions (70.9 and 39.2%) of the filtered Na+ are reabsorbed by the male and female proximal tubule models, respectively. The lower fractional Na+ reabsorption in females is due primarily to their smaller transport area and lower Na+/H+ exchanger isoform 3 and claudin-2 expression levels. Notably, unlike most Na+ transporters, whose expression levels are lower in females, Na+-glucose cotransporter 2 (SGLT2) expression levels are 2.5-fold higher in females. Model simulations suggest that the higher SGLT2 expression in females may compensate for their lower tubular transport area to achieve a hyperglycemic tolerance similar to that of males.

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.

The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

Potassium homeostasis has a very high priority because of its importance for membrane potential. Although extracellular K+ is only 2% of total body K+, our physiology was evolutionarily tuned for a high-K+, low-Na+ diet. We review how multiple systems interface to accomplish fine K+ balance and the consequences for health and disease.

The proximal tubule Na+/H+exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na+reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution.

Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na+-Cl− cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na+ reabsorption and shifts Na+ downstream for reabsorption by epithelial Na+ channels (ENaC), which can drive K+ secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K+ concentration ([K+]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K+] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K+] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K+, Na+, and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na+-K+-2Cl− cotransporter (NKCC) phosphorylation 25% ( P = 0.055). When plasma [K+] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% ( P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na+/H+ exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K+ channel. In summary, raising plasma [K+] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K+ ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na+ reabsorption and flow drive K+ secretion.

The renal distal tubule Na-Cl cotransporter (NCC) reabsorbs +but is a key control point for blood pressure regulation by angiotensin II (ANG II), angiotensin-converting enzyme inhibitors (ACEI), and thiazide diuretics. This study aimed to determine whether NCC phosphorylation (NCCp) was regulated by acute (20–30 min) treatment with the ACEI captopril (12 μg/min × 20 min) or by a sub-pressor dose of ANG II (20 ng·kg−1·min−1) in Inactin-anesthetized rats. By immuno-EM, NCCp was detected exclusively in or adjacent to apical plama membranes (APM) in controls and after ACEI or ANG II treatment, while NCC total was detected in both APM and subapical cytoplasmic vesicles (SCV) in all conditions. In renal homogenates, neither ACEI nor ANG II treatment altered NCCp abundance, assayed by immunoblot. However, by density gradient fractionation we identified a pool of low-density APM in which NCCp decreased 50% in response to captopril and was restored during ANG II infusion, and another pool of higher-density APM that responded reciprocally, indicative of regulated redistribution between two APM pools. In both pools, NCCp was preferentially localized to Triton-soluble membranes. Blue Native gel electrophoresis established that APM NCCp localized to ∼700 kDa complexes (containing γ-adducin) while unphosphorylated NCC in intracellular membranes primarily localized to ∼400 kDa complexes: there was no evidence for native monomeric or dimeric NCC or NCCp. In summary, this study demonstrates that phosphorylated NCC, localized to multimeric complexes in the APM, redistributes in a regulated manner within the APM in response to ACEI and ANG II.

Hypertension provokes differential trafficking of the renal proximal tubule Na+/H+ exchanger 3 (NHE3) to the base of the apical microvilli and Na+-Pi cotransporter 2 (NaPi2) to endosomes. The resultant diuresis and natriuresis are key to blood pressure control. We tested the hypothesis that this differential trafficking of NHE3 vs. NaPi2 was associated with partitioning to distinct membrane domains. In anesthetized rats, arterial pressure was increased (104 ± 2 to 142 ± 4 mmHg, 15 min) by arterial constriction and urine output increased 23-fold. Renal membranes were fractionated by cold 1% Triton X-100 extraction then centrifugation through OptiPrep flotation gradients. In controls, 84 ± 9% of NHE3 localized to flotillin-enriched lipid raft domains and 69 ± 5% of NaPi2 localized to transferrin receptor-enriched nonrafts. MyosinVI and dipeptidyl peptidase IV, associated with NHE3 regulation, coenriched in lipid rafts with NHE3, while NHE regulatory factor-1 coenriched in nonrafts with NaPi2. Partitioning was not altered by hypertension. Detergent insoluble membranes were pelleted after detergent extraction. NHE3 detergent insolubility decreased as it redistributed from body (80 ± 10% detergent insoluble) to base (75 ± 3%) of the apical microvilli, while NaPi2 partitioned into more insoluble domains as it moved from the microvilli (45 ± 7% detergent insoluble) to endosomes (82 ± 1%). In conclusion, NHE3 and NaPi2, while both localized to apical microvilli, are segregated into domains: NHE3 to lipid rafts and NaPi2 to nonrafts. These domain properties may play a role in the distinct trafficking patterns observed during elevated pressures: NHE3 remains in rafts and settles to the base of the microvilli while NaPi2 is freely endocytosed.

The potassium homeostatic system is very tightly regulated. Recent studies have shed light on the sensing and molecular mechanisms responsible for this tight control. In addition to classic feedback regulation mediated by a rise in extracellular fluid (ECF) [K+], there is evidence for a feedforward mechanism: Dietary K+ intake is sensed in the gut, and an unidentified gut factor is activated to stimulate renal K+ excretion. This pathway may explain renal and extrarenal responses to altered K+ intake that occur independently of changes in ECF [K+]. Mechanisms for conserving ECF K+ during fasting or K+ deprivation have been described: Kidney NADPH oxidase activation initiates a cascade that provokes the retraction of K+ channels from the cell membrane, and muscle becomes resistant to insulin stimulation of cellular K+ uptake. How these mechanisms are triggered by K+ deprivation remains unclear. Cellular AMP kinase–dependent protein kinase activity provokes the acute transfer of K+ from the ECF to the ICF, which may be important in exercise or ischemia. These recent advances may shed light on the beneficial effects of a high-K+ diet for the cardiovascular system.

During high-salt (HS) diet the kidney increases urinary Na+ and volume excretion to match intake. We recently reported that HS provokes a redistribution of distal convoluted tubule Na+-Cl− cotransporter (NCC) from apical to subapical vesicles and decreases NCC abundance. This study aimed to test the hypothesis that the other renal Na+ transporters' abundance and or subcellular distribution is decreased by HS diet. Six-week-old Sprague-Dawley rats were fed a normal (NS) 0.4% NaCl diet or a HS 4% NaCl diet for 3 wk or overnight. Kidneys excised from anesthetized rats were fractionated on density gradients or analyzed by microscopy; transporters and associated regulators were detected with specific antibodies. Three-week HS doubled Na+/H+ exchanger (NHE)3 phosphorylation at serine 552 and provoked a redistribution of NHE3, dipeptidyl peptidase IV (DPPIV), myosin VI, Na+-Pi cotransporter (NaPi)-2, ANG II type 2 receptor (AT2R), aminopeptidase N (APN), Na+-K+-2Cl− cotransporter (NKCC2), epithelial Na+ channel (ENaC) β-subunit, and Na+-K+-ATPase (NKA) α1- and β1-subunits from low-density plasma membrane-enriched fractions to higher-density intracellular membrane-enriched fractions. NHE3, myosin VI, and AT2R retraction to the base of the microvilli (MV) during HS was evident by confocal microscopy. HS did not change abundance of NHE3, NKCC, or NKA α1- or β1-subunits but increased ENaC-β in high-density intracellular enriched membranes. Responses to HS were fully apparent after just 18 h. We propose that retraction of NHE3 to the base of the MV, driven by myosin VI and NHE3 phosphorylation and accompanied by redistribution of the NHE3 regulator DPPIV, contributes to a decrease in proximal tubule Na+ reabsorption during HS and that redistribution of transporters out of low-density plasma membrane-enriched fractions in the thick ascending limb of the loop of Henle and distal nephron may also contribute to the homeostatic natriuretic response to HS diet.

Injection of 50 μl 10% phenol into rat renal cortex activates renal sympathetic nerve activity which provokes acute hypertension that persists for weeks. We have previously shown with membrane fractionation that phenol injury caused a redistribution of the main proximal tubule (PT) apical transporter NHE3 (Na+/H+ exchanger isoform 3) to low density membranes enriched in apical microvilli. The aim of this study was to determine whether phenol injury increases PT apical Na+/H+ exchanger (NHE) activity. NHE activity was measured in vivo as the initial rate of change in intracellular pH (dpHi/d t) during luminal Na+ removal in PT preloaded with the pH-sensitive fluorescence dye BCECF. Injection of 50 μl 10% phenol increased blood pressure from 113 ± 5.2 to 130 ± 4.6 mmHg without changing glomerular filtration rate or urine output. NHE activity increased 2.6-fold by 70 min after phenol injury. The increase of NHE activity was accompanied with an increase of tubular reabsorption. Total NHE activity/NHE3 protein in cortical brush-border membrane (BBM) vesicles, measured by acridine orange quench and immunoblot, respectively, was unchanged by phenol injury. In conclusion, acute phenol injury provokes coincident increases in PT apical NHE activity, redistribution of NHE3 into low density apical membranes, and hypertension. The increase in NHE activity may contribute to the lack of pressure-diuresis and the maintenance of chronic hypertension in this model.

We previously reported that Na+/H+exchanger type 3 (NHE3) and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension [high blood pressure (BP)] or parathyroid hormone (PTH) treatment. By subcellular membrane fractionation, NHE3 and NaPi2 show indistinguishable redistribution patterns out of light-density into heavy-density membranes in response to either treatment consistent with a retraction from the apical MV to the intermicrovillar cleft region. This study aimed to examine the redistribution of PT NHE3 vs. NaPi2 by confocal and electron microscopy during high BP and during PTH treatment to determine whether their respective destinations overlap or are distinct. High-BP protocol: systolic BP was increased 50–60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused with 6.6 μg/kg iv of PTH followed by 0.1 μg·kg−1·min−1infusion for 1 h. For light microscopy, rats were infused with 25 mg of horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2 and either clathrin-coated vesicle adaptor protein AP2 or endosome marker HRP. The results demonstrate retraction of NHE3 from the MV to the base of MV during either high-BP or PTH treatment: NHE3 staining did not retract below the AP2-stained domain or to HRP-labeled endosomes in either model. In comparison, NaPi2 was retracted from MV to below the AP2-stained region in both models, a little colocalizing with HRP staining. At the electron microscopic level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the MV while NaPi2 moved to endosomes. The results demonstrate that there are divergent routes of retraction of PT NHE3 and NaPi2 from the MV during acute hypertension or PTH treatment: NHE3 is not internalized but remains at the base of the MV while NaPi2 is internalized.

Renal parathyroid hormone (PTH) action is often studied at high doses (100 μg PTH/kg) that lower mean arterial pressure significantly, albeit transiently, complicating interpretation of studies. Little is known about the effect of acute hypotension on proximal tubule Na+transporters. This study aimed to determine the effects of acute hypotension, induced by aortic clamp or by high-dose PTH (100 μg PTH/kg), on renal hemodynamics and proximal tubule Na/H exchanger isoform 3 (NHE3) and type IIa Na-Picotransporter protein (NaPi2) distribution. Subcellular distribution was analyzed in renal cortical membranes fractionated on sorbitol density gradients. Aortic clamp-induced acute hypotension (from 100 ± 3 to 78 ± 2 mmHg) provoked a 62% decrease in urine output and a significant decrease in volume flow from the proximal tubule detected as a 66% decrease in endogenous lithium clearance. There was, however, no significant change in glomerular filtration rate (GFR) or subcellular distribution of NHE3 and NaPi2. In contrast, high-dose PTH rapidly (−1·h−1) did not change blood pressure and was diuretic. In conclusion, acute hypotension per se increases proximal tubule Na+reabsorption without changing NHE3 or NaPi2 subcellular distribution, indicating that trafficking of transporters to the surface is not the likely mechanism; in comparison, hypotension secondary to high-dose PTH blocks the primary diuretic effect of PTH but does not inhibit the PTH-stimulated redistribution of NHE3 and NaPi2 to the base of the microvilli.

Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na+/H+exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. We found that 10−7M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization.

Acute hypertension inhibits proximal tubule (PT) sodium reabsorption. The resultant increase in NaCl delivery to the macula densa suppresses renin release. We tested whether the sustained pressure-induced inhibition of PT sodium reabsorption requires a renin-mediated decrease in ANG II levels. Plasma ANG II concentration of anesthesized Sprague-Dawley rats was clamped by simultaneous infusion of the ANG I-converting enzyme inhibitor captopril (12 μg/min) and ANG II (20 ng · kg−1· min−1). Blood pressure was increased 50 mmHg for 20 min by arterial constriction ± ANG II clamp or by sham operation. This acute hypertension increased urine output and endogenous Li+clearance, and these responses were blunted 40–50% in ANG II clamped rats. Acute hypertension provoked a rapid redistribution of Na+/H+exchanger isoform 3 (NHE3) out of apical brush-border membranes (21 ± 4% decrease of total NHE3 abundance) to endosomal/lysosomal membranes (16 ± 6% increase of total). In ANG II-clamped rats, acute hypertension also provoked disappearance of NHE3 from the apical membranes (27 ± 2% decrease of total), but NHE3 was shifted to membranes enriched in intermicrovillar cleft and dense apical tubules ( step 1) rather than endosomal/lysosomal membranes ( step 2). This difference was independently confirmed by confocal analysis. In contrast, the pressure-induced redistribution of Na+-Picotransporter type 2 was not altered by ANG II clamp. We conclude that the responses to acute hypertension, including diuresis and redistribution of PT NHE3 into intracellular membranes, require a responsive renin-angiotensin system and that the responses may be induced by the sustained increase in NaCl delivery to the macula densa during acute hypertension.

Maintaining extracellular fluid (ECF) K+concentration ([K+]) within a narrow range is accomplished by the concerted responses of the kidney, which matches K+excretion to K+intake, and skeletal muscle, the main intracellular fluid (ICF) store of K+, which can rapidly buffer ECF [K+]. In both systems, homologous P-type ATPase isoforms are key effectors of this homeostasis. During dietary K+deprivation, these P-type ATPases are regulated in opposite directions: increased abundance of the H,K-ATPase “colonic” isoform in the renal collecting duct drives active K+conservation while decreased abundance of the plasma membrane Na,K-ATPase α2-isoform leads to the specific shift of K+from muscle ICF to ECF. The skeletal muscle response is isoform and muscle specific: α2and β2, not α1and β1, levels are depressed, and fast glycolytic muscles lose >90% α2, whereas slow oxidative muscles lose ∼50%; however, both muscle types have the same fall in cellular [K+]. To understand the physiological impact, we developed the “K+clamp” to assess insulin-stimulated cellular K+uptake in vivo in the conscious rat by measuring the exogenous K+infusion rate needed to maintain constant plasma [K+] during insulin infusion. Using the K+clamp, we established that K+deprivation leads to near-complete insulin resistance of cellular K+uptake and that this insulin resistance can occur before any decrease in plasma [K+] or muscle Na+pump expression. These studies establish the advantage of combining molecular analyses of P-type ATPase expression with in vivo analyses of cellular K+uptake and excretion to determine mechanisms in models of disrupted K+homeostasis.

Acute hypertension rapidly decreases proximal tubule (PT) Na+reabsorption, facilitated by a redistribution of PT Na+/H+exchangers (NHE3) out of the apical brush border, increasing NaCl at the macula densa, the signal for autoregulation of renal blood flow and GFR. This study aimed to determine whether NHE3 activity per transporter decreases during acute hypertension and the time dependence of the response. Blood pressure was elevated by 50–60 mmHg in male Sprague-Dawley rats for 5 or 30 min by constricting arteries. Renal cortical membranes were fractionated by density gradient centrifugation. NHE3 transport activity was assayed as the rate of appearance of acridine orange (AO) from AO-loaded vesicles in response to an inwardly directed Na+gradient. After 5-min hypertension, 20% of total NHE3 protein, assayed by immunoblot, redistributed from low-density apical membranes to middensity membranes enriched in intermicrovillar cleft markers; by 30 min, a similar percentage shifted to heavier density membranes containing markers of endosomes. NHE3 activity shifted to higher density membranes along with NHE3 protein, that is, no change in activity/transporter during acute hypertension. Confocal analysis of NHE3 distribution also verified removal from apical microvilli and appearance in subapical vesicles. We conclude that the decrease in renal PT Na+transport during acute hypertension is mediated by removal of transport-competent NHE3 from the apical brush border to subapical and internal reserves.

Na,K‐ATPase generates the driving force for sodium reabsorption in the kidney. Na,K‐ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na+ retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K‐ATPase activity was documented in the Fxyd2−/− mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K‐ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide‐sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na+ reabsorption in this segment. Specifically, NCC total increased 30% and NCC phosphorylated at T53 and S71, associated with activation, increased 4‐6 fold. The phosphorylation of the closely related thick ascending limb (TAL) apical NKCC2 (Slc12a1) increased at least twofold. Abundance of the total and cleaved (activated) forms of ENaC α‐subunit was not different between genotypes. Nonetheless, no elevation of blood pressure was evident despite the fact that NCC and NKCC2 are in states permissive for Na+ retention. Activation of NCC and NKCC2 may reflect an intracellular linkage to elevated Na,K‐ATPase activity or a compensatory response to Na+ loss proximal to the TAL and DCT., We discovered a substantial activation of renal NCC cotransporter in mice genetically depleted for the regulatory inhibitory subunit of Na,K‐ATPase, FXYD2. Surprisingly, no significant changes in urine output as well as elevation of blood pressure were detected suggesting compensatory adaptation elsewhere in nephron

Human Na+-K+-ATPase α1β1, α2β1, and α3β1heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13–17 nM for α1β1and α3β1at 37°C and 32 nM for α2β1, indicating that the human α-subunit isoforms have a similar high affinity for cardiac glycosides. K0.5values for antagonism of ouabain binding by K+were ranked in order as follows: α2(6.3 ± 2.4 mM) > α3(1.6 ± 0.5 mM) ≈ α1(0.9 ± 0.6 mM), and K0.5values for Na+antagonism of ouabain binding to all heterodimers were 9.5–13.8 mM. The molecular turnover for ATP hydrolysis by α1β1(6,652 min−1) was about twice as high as that by α3β1(3,145 min−1). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem275: 1976–1986, 2000). In contrast to Na+pumps expressed in Xenopus oocytes, the α2β1complex in yeast membranes was significantly less stable than α1β1or α3β1, resulting in a lower functional expression level. The α2β1complex was also more easily denatured by SDS than was the α1β1or the α3β1complex.

Three α-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant ( Kd) for all samples calculated using the single-site model was 18 ± 6 nM (mean ± SD). No difference in Kdwas found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only ∼50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human α-isoforms are similar to each other, although calculated Kdvalues were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human α-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt–treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II–infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.

We aimed to test the feasibility of quantifying insulin action on cellular K+uptake in vivo in the conscious rat by measuring the exogenous K+infusion rate needed to maintain constant plasma K+concentration ([K+]) during insulin infusion. In this “K+clamp” the K+infusion rate required to clamp plasma [K+] is a measure of insulin action to increase net plasma K+disappearance. K+infusion rate required to clamp plasma [K+] was insulin dose dependent. Renal K+excretion was not significantly affected by insulin at a physiological concentration (∼90 μU/ml, P > 0.05), indicating that most of insulin-mediated plasma K+disappearance was due to K+uptake by extrarenal tissues. In rats deprived of K+for 2 days, plasma [K+] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K+disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K+uptake route. After 10 days K+deprivation, plasma [K+] fell to 2.9 mM, insulin-mediated K+disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and α2-subunit levels. In conclusion, the present study proves the feasibility of the K+clamp technique and demonstrates that short-term K+deprivation leads to a near complete insulin resistance of cellular K+uptake that precedes changes in muscle sodium pump expression.

Redistribution of apical Na+/H+exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20–40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 ± 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 ± 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 ± 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis.

We measured Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity and subunit abundance in renal cortical homogenates and basolateral membranes (BLM) from fetal, newborn, and adult guinea pigs. Pump specific activity increased four- to fivefold in cortical homogenates and BLM during the transition from fetus to newborn. Immunoblots of BLM showed that alpha- and beta-subunit abundance increased four- to seven- and fourfold, respectively, during the transition from fetus to newborn. Immunoblots of cortical homogenates revealed similar developmental patterns, with newborns having 3.5-fold (alpha) and 2.3-fold (beta) greater subunit abundances than fetuses. Therefore, the bulk of the postnatal increase in BLM-Na(+)-K(+)-ATPase abundance resulted from increased pump production or decreased pump degradation, rather than from redistribution of pumps from intracellular pools. Despite the developmental increase in alpha- and beta-subunit protein levels, newborn guinea pig kidneys had only 1.4- to 2.1-fold greater alpha 1-subunit mRNA abundance and only a 1.5-fold greater beta 1-subunit mRNA abundance than fetal kidneys. These results demonstrate large increases in renal cortical Na(+)-K(+)-ATPase specific activity and protein abundance immediately after birth. These increases, which appear to result largely from posttranscriptional upregulation, may play an important role in mediating the rapid postnatal increase in tubular NaCl reabsorption.

During angiotensin II (ANG II)-dependent hypertension, ANG II stimulates, while hypertension inhibits, Na+transporter activity to balance Na+output to input. This study tests the hypothesis that ANG II infusion activates Na+transporters in the distal nephron while inhibiting transporters along the proximal nephron. Male Sprague-Dawley rats were infused with ANG II (400 ng·kg−1·min−1) or vehicle for 2 wk. Kidneys were dissected (cortex vs. medulla) or fixed for immunohistochemistry (IHC). ANG II increased mean arterial pressure by 40 mmHg, urine Na+by 1.67-fold, and urine volume by 3-fold, evidence for hypertension and pressure natriuresis. Na+transporters' abundance and activation [assessed by phosphorylation (-P) or proteolytic cleavage] were measured by immunoblot. During ANG II infusion Na+/H+exchanger 3 (NHE3) abundance decreased in both cortex and medulla; Na-K-2Cl cotransporter 2 (NKCC2) decreased in medullary thick ascending loop of Henle (TALH) and increased, along with NKCC2-P, in cortical TALH; Na-Cl cotransporter (NCC) and NCC-P increased in the distal convoluted tubule; and epithelial Na+channel subunits and their cleaved forms were increased in both cortex and medulla. Like NKCC2, STE20/SPS1-related proline alanine-rich kinase (SPAK) and SPAK-P were decreased in medulla and increased in cortex. By IHC, during ANG II NHE3 remained localized to proximal tubule microvilli at lower abundance, and the differential regulation of NKCC2 and NKCC2-P in cortex versus medulla was evident. In summary, ANG II infusion increases Na+transporter abundance and activation from cortical TALH to medullary collecting duct while the hypertension drives a natriuresis response evident as decreased Na+transporter abundance and activation from proximal tubule through medullary TALH.

Acute arterial hypertension provokes a rapid decrease in proximal tubule (PT) Na+ reabsorption, increasing flow to the macula densa, the signal for tubuloglomerular feedback. We tested the hypothesis, in rats, that Na+ transport is decreased due to rapid redistribution of apical Na+/H+ exchangers and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either natriuretic stimuli, cortex was removed, and membranes were fractionated by density gradient centrifugation. Urine output and endogenous lithium clearance increased threefold in response to either stimuli. Acute hypertension provoked a redistribution of apical Na+/H+ exchanger NHE3, alkaline phosphatase, and dipeptidyl peptidase IV to higher density membranes enriched in the intracellular membrane markers. Basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity decreased 50%, 25-30% of the alpha 1-and beta 1-subunits redistributed to higher density membranes, and the remainder is attributed to decreased activity of the transporters. Benzolamide did not alter Na+ transporter activity or distribution, implying that decreasing apical Na+ uptake does not initiate redistribution or inhibition of basolateral Na(+)-K(+)-ATPase. We conclude that PT natriuresis provoked by acute arterial pressure is mediated by both endocytic removal of apical Na+/H+ exchangers and basolateral Na+ pumps as well as decreased total Na+ pump activity.

The cardiac Na+ pump (Na+ -K+ -ATPase) provides the driving force for the Na+/Ca2+ exchanger, a determinant of intracellular Ca2+ stores. Three Na+ pump alpha-catalytic subunit isoforms are expressed in human heart, alpha1 and alpha2 are expressed in rat heart, and only alpha1 is expressed in guinea pig heart. The objective of this study was to determine whether there are isoform-specific patterns of expression in the transverse tubules (T tubules) vs. the peripheral sarcolemma. In adult rat cardiomyocytes, anti-alpha1-specific antibodies labeled the T tubules more intensely than the peripheral sarcolemma, in which labeling was patchy, the same pattern reported for distribution of the Na+/Ca2+ exchanger (J. S. Frank, G. Mottino, D. Reid, R. S. Molday, and K. D. Philipson, J. Cell Biol. 117: 337-345, 1992), whereas anti-alpha2- and anti-beta1-antibodies uniformly labeled T tubules and peripheral sarcolemma. In guinea pig cardiomyocytes, an anti-alpha-antibody against an extracellular epitope evenly labeled the peripheral sarcolemma and T tubules, and immunogold labeling demonstrated coincidence of alpha-subunits and intramembranous particles in sarcolemma. In summary, Na+ pumps are located in both peripheral sarcolemma and T tubules of cardiomyocytes expressing either multiple or single Na+ pump isoforms.

Inhibiting cardiac Na pump activity decreases the driving force for the Na/Ca exchanger transport that increases cellular Ca stores and contractility. Decreased abundance of Na pumps would be expected to have the same effect as decreased activity unless there was reciprocal regulation of Na/Ca exchanger expression to oppose the effects of depressed Na pump activity on intracellular Ca stores. The aim of this study was to test the hypothesis that cardiac Na/Ca exchanger abundance is regulated in a reciprocal fashion to Na-K-ATPase abundance in a number of models known to have altered Na-K-ATPase abundance. In renovascular hypertension, cardiac ventricular Na-K-ATPase alpha 1-levels are unaltered, alpha 2-isoform subunit mRNA and protein levels decrease to 0.76 +/- 0.06 and 0.56 +/- 0.07 of control, respectively, and the Na/Ca exchanger protein (not mRNA) increased 1.35 +/- 0.11-fold. In the transition from hypothyroid to hyperthyroid cardiac alpha 1 doubles, alpha 2-protein increases 8.83 +/- 1.06-fold, and the Na/Ca exchanger protein decreases to 0.64 +/- 0.11. A similar pattern was seen during cardiac development in the preweaning rat heart. Treatment with the antiarrhythymic amiodarone has no effect on alpha 1, decreases alpha 2-protein expression to 0.51 +/- 0.08 of control, and increases exchanger expression 1.42 +/- 0.16-fold. In conclusion, the reciprocal regulation of the Na/Ca exchanger and of Na-K-ATPase alpha 2-expression provides evidence for a homeostatic mechanism that would oppose the changes in cellular Ca stores driven by the changes in Na-K-ATPase activity.

identification of the genes involved in polycystic kidney disease (PKD) suggests that defects in ciliary function are a common feature (4). In renal tubular epithelia, primary cilia on the apical surface normally protrude into the tubular lumen where they can detect fluid flow, likely due to the bending of the cilium and the triggering of calcium transients. How the lack of a central cilium and the disruption of this sensing and signaling could lead to the development of a pathologic cystic phenotype remain a very active area of investigation. The Oak Ridge polycystic kidney mouse (ORPK), first described through collaborative efforts of Avner and Woychik (7), displays renal cystic lesions that resemble cysts found in human autosomal recessive polycystic kidney disease (ARPKD). The phenotype arose from an insertional mutation in the Ift88 gene encoding the protein IFT88 (Polaris), a mutation that leads to the lack of a well-formed central cilium in multiple tissues. The study by Olteanu et al. (8) in this issue, a collaboration between a number of labs with complementary strengths in renal and epithelial physiology, utilizes both conditionally immortalized cortical collecting duct (CCD) principal cells (PCs) isolated from the ORPK mouse, as well as a kidney-specific conditional cilium knockout mouse generated from the ORPK mouse to assess altered renal transporter activity and polarity in these cilia-deficient cells. A previous study from this group in cilium-deficient CCD PCs reported a fourfold increase in apical epithelial Na+ channel (ENaC)-mediated sodium reabsorption (9), suggesting that the cilium-sensing pathway controls ENaC activity, a potential clinically relevant finding given the early onset of hypertension that is observed in the ARPKD population (3). The current study provides evidence that Na/H exchanger 1 (NHE1), normally expressed in a polarized fashion in the basolateral membrane of CCD PCs, is expressed in both the apical and the basolateral membranes of the ORPK CCD cells. Another recent study reports that vasopressin receptors are also mislocated to the apical membrane in CD cells from the ORPK mouse (10). These studies indicate that the lack of Polaris expression alters the polarized trafficking of membrane proteins which may lead to, directly or indirectly, elevated Na+ and water reabsorption in these cilium-deficient cells. The major positive features of the Olteanu study include: 1) the use of a chamber that allowed precise independent control of the cultured CCD cells' apical and basolateral bathing fluids compartments coupled to, 2) recording of Na+/H+ exchanger activity in real time as changes in intracellular pH using a pH-sensitive dye and, 3) implementation of multiple Na+/H+ exchanger inhibitors and specific antibodies to identify apical NHE1, as well as, 4) the generation of a complementary HoxB7 cre-lox kidney-specific conditional cilium knockout mouse (ift88flox/Δ), which allowed the study of isolated perfused, intact cortical collecting tubular function with and without ciliated PCs. These genetic studies confirmed the results of the cellular studies and went on to show that knockout mice exhibited metabolic alkalosis consistent with excessive acid excretion via apical NHE1 in the cilium-deficient PC. PKDs are the most common primary renal genetic disorders, affecting between 1 in 400–1,000 (over 13 million people worldwide), and producing hypertension and end-stage renal disease in children as well as adults (5). Despite major advances over the past decade in delineating the genetics and pathophysiology of PKD, therapeutic efforts are limited to treating the complications of the disease, as there is still no proven therapy for PKD (12). The studies of Olteanu et al. (8, 9) are relevant to the growing body of literature on the relationships between ciliary structure/function and renal epithelial transport, and may be relevant to hypertension via control of ENaC abundance. However, careful evaluation is needed before concluding that the authors' meticulous studies in the ORPK model are applicable to human PKD. We summarize central issues for the readers' consideration: 1) Data from many laboratories demonstrate that a secretory event is critical for cystic enlargement in PKD (Fig. 1). PKD epithelia are characterized by proliferative, extracellular matrix and secretory abnormalities. In ARPKD, although cysts remain open to tubular lumens, secretion may contribute to cyst enlargement with low flow states, distal obstruction, or by as-yet unknown mechanisms (5). It is unclear from the authors' study how increased Na+ reabsorption (via apical NHE and ENaC) leads to a secretory phenotype. In an era of orthologous models of PKD, the value of nonorthologous models is related to their overall phenotypic similarities, specifically the well-described, characteristic cystic epithelial cellular phenotype of PKD (5, 11, 12). However, the overall phenotype of the ORPK mouse may be more consistent with syndromic renal disease or “renal ciliopathies” (6), but further comment is difficult due to the heterogeneity and diverse pathophysiology of these disorders. Fig. 1. This figure illustrates how a normal tubule becomes cystic in genetic polycystic kidney disease (PKD). A primary factor in both autosomal dominant (ADPKD) and autosomal recessive (ARPKD) is tubular cell proliferation. Such proliferation must occur with ... 2) Neither hypertension nor left ventricular hypertrophy (a pathological marker of hypertension) has been documented in the ORPK mouse on any genetic background (to our knowledge). In addition, increased activity of the renin-angiotensin system (RAS) is implicated as the major cause of hypertension in PKD, rather than elevated sodium reabsorption (2). Another critical point is that the results collected in immortalized cystic PCs from another nonorthologous model report that Na+ absorption via ENaC is reduced (rather than stimulated) when compared with cells from age-matched wild-type littermates (11, 13). Reduced ENaC is consistent with both the secretory phenotype seen in PKD and elevated RAS. 3) Neither autosomal dominant (ADPKD) nor ARPKD patients have been reported to be alkalotic at any stage of their disease. Indeed, PKD patients have systemic acidosis in direct proportion to their degree of renal failure. In sum, the authors have presented valuable new data regarding the relationship between renal cellular cilia expression and renal cellular transport. The major limitation of the study is the authors' extrapolation of data from a nonorthologous model of PKD in which neither the animal model nor the cellular phenotype is consistent with findings in human ARPKD. This collaborative group of authors is in an excellent position to take the steps to apply their elegant methodologies to characterize the transport abnormalities of PKD in orthologous models or fresh renal tissue obtained from patients with ADPKD and/or ARPKD.

Dietary potassium (K+) restriction and hypokalemia have been reported to change the abundance of most renal Na+and K+transporters and aquaporin-2 isoform, but results have not been consistent. The aim of this study was to reexamine Na+, K+and H2O transporters' pool size regulation in response to removing K+from a diet containing 0.74% NaCl, as well as from a diet containing 2% NaCl (as found in American diets) to blunt reducing total diet electrolytes. Sprague-Dawley rats ( n = 5–6) were fed for 6 days with one of these diets: 2% KCl, 0.74% NaCl (2K1Na, control chow) compared with 0.03% KCl, 0.74% NaCl (0K1Na); or 2% KCl, 2%NaCl (2K2Na) compared with 0.03% KCl, 2% NaCl (0K2Na, Na+replete). In both 0K1Na and 0K2Na there were significant decreases in: 1) plasma [K+] (+excretion (+/H+exchanger isoform 3; Na+-K+-Cl−cotransporter; Na+-Cl−cotransporter, oxidative stress response kinase-1; renal outer medullary K+channel; autosomal recessive hypercholesterolemia; c-Src, aquaporin 2 isoform; or renin. Thus, despite profound hypokalemia and renal K+conservation, we did not confirm many of the changes that were previously reported. We predict that changes in transporter distribution and activity are likely more important for conserving K+than changes in total abundance.

The activity of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), the sodium pump, which drives active Na+ reabsorption along the nephron, varies over an order of magnitude, depending on the nephron segment, and activity is increased in the outer medullary collecting tubule (MCT) during hypokalemia. The aims of the present study were to assess abundance of sodium pump alpha 1- and beta 1-subunits in dissected nephron segments of the rat by immunoblotting, to determine if alpha 2- or alpha 3-protein could be detected in the collecting tubules, as suggested by Barlet-Bas et al. (C. Barlet-Bas, E. Arystarkhova, L. Cheval, S. Marsy, K. Sweadner, N. Modyanov, and A. Doucet. J. Biol. Chem. 268: 11512-11515, 1993) for rabbit, and to determine if alpha 1 and beta 1 were increased in MCT by hypokalemia. Tubules from the rat were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12-100 mm/lane), blotted, and probed with subunit-specific antisera. alpha 1 and beta 1, detected in all tubule segments assayed, were highest in cortical and medullary thick ascending limbs and proximal convoluted tubule (PCT), lower in the MCT and barely detectable in proximal straight tubule. In the cortical collecting tubule (CCT), alpha 1 abundance was equivalent to that in PCT, whereas beta 1 and enzymatic activity were both less than one-half of that in PCT. After 2 wk of a K(+)-deficient diet, alpha 1- and beta 1-subunit levels in MCT increased 3.4 +/- 0.6- and 11.7 +/- 4.0-fold, respectively, associated with a 5-fold increase in activity. alpha 2 and alpha 3 were not detected in the CCT or MCT.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryHypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and nonredundant role in determining the level of BP. Abrogation of AT1 angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.

Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein consisting of an alpha-catalytic and a beta-glycoprotein subunit. Because these two subunits are derived from two separate genes, they may not be synthesized with stoichiometric equivalence. The aim of this study was to estimate relative rates of synthesis and degradation of nascent and mature Na(+)-K(+)-ATPase alpha- and beta-subunits to determine whether either of the nascent subunits accumulates in excess and, if so, the fate of the excess subunits. We studied a pig kidney cell line (LLC-PK1/Cl4) that expresses only alpha 1- and beta 1-subunits. Relative synthesis and degradation rates of nascent subunits were first estimated by pulsing cells for 10 min with [35S]methionine followed by chase periods of up to 120 min and by immunoprecipitation. We found that directly after labeling, beta-subunits were present in threefold excess over alpha-subunits and that nearly 50% of this beta-subunit pool was degraded by 60 min. Nascent alpha-subunits were not degraded during the chase period. In a second strategy to examine relative rates of nascent alpha- vs. beta-subunit accumulation, cells were pulsed for 5-60 min and immunoprecipitated directly (without chase). The rate of accumulation of labeled alpha was greater than that of beta between 5 and 60 min, consistent with the results of the pulse-chase strategy, demonstrating a significant component of degradation of beta during this period. Despite the very different degradation rates of newly synthesized alpha- vs. beta-subunits, the degradation rates of alpha- and beta-subunits beyond 4 h after synthesis were indistinguishable (t0.5 = 10-12 h).(ABSTRACT TRUNCATED AT 250 WORDS)

the numbers are grim. According to the American Diabetes Association, in the United States, ∼29 million individuals have diabetes and an additional 86 million are prediabetic. In 2010, diabetes was listed as the underlying cause of ∼70,000 deaths and a contributing factor to an additional >230

One-hundred years ago, Starling articulated the interdependence of renal control of circulating blood volume and effective cardiac performance. During the past 25 years, the molecular mechanisms responsible for the interdependence of blood pressure (BP), extracellular fluid volume (ECFV), the renin-angiotensin system (RAS), and sympathetic nervous system (SNS) have begun to be revealed. These variables all converge on regulation of renal proximal tubule (PT) sodium transport. The PT reabsorbs two-thirds of the filtered Na+and volume at baseline. This fraction is decreased when BP or perfusion pressure is increased, during a high-salt diet (elevated ECFV), and during inhibition of the production of ANG II; conversely, this fraction is increased by ANG II, SNS activation, and a low-salt diet. These variables all regulate the distribution of the Na+/H+exchanger isoform 3 (NHE3) and the Na+-phosphate cotransporter (NaPi2), along the apical microvilli of the PT. Natriuretic stimuli provoke the dynamic redistribution of these transporters along with associated regulators, molecular motors, and cytoskeleton-associated proteins to the base of the microvilli. The lipid raft-associated NHE3 remains at the base, and the nonraft-associated NaPi2 is endocytosed, culminating in decreased Na+transport and increased PT flow rate. Antinatriuretic stimuli return the same transporters and regulators to the body of the microvilli associated with an increase in transport activity and decrease in PT flow rate. In summary, ECFV and BP homeostasis are, at least in part, maintained by continuous and acute redistribution of transporter complexes up and down the PT microvilli, which affect regulation of PT sodium reabsorption in response to fluctuations in ECFV, BP, SNS, and RAS.

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na+/H+exchanger isoform 3 (NHE3), Na+/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 μg/min for 20 min) that increased PT flow rate ∼20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng·kg−1·min−1for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H+-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.

In this study, we measured the time courses of change in Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and beta 1-subunit mRNA and protein abundance in cardiac myocytes isolated from euthyroid, hypothyroid, and hyperthyroid (hypothyroids injected daily with 1 microgram T3/g body wt) rats. In hypothyroids, alpha 1-, alpha 2-, and beta 1-protein levels were decreased to 0.55, 0.42, and 0.57 of euthyroids, predicting the decrease in Na(+)-K(+)-ATPase activity to 0.53 of control. There was no change in these subunits' mRNA levels, indicating that the decreases in protein levels were not due to decreased subunit transcription rates. In hyperthyroids, the alpha 1-mRNA increased to a steady state of threefold over hypothyroid by 1 day of T3 treatment, and the alpha 1-protein levels increased to twofold over hypothyroid by 4 days of T3. alpha 2-mRNA increased to 5-fold over hypothyroid by 2 days, whereas the alpha 2-protein levels increased to 14-fold above hypothyroid by 4 days of T3. Beta 1-mRNA increased to 12-fold above hypothyroid by 1 day of T3 treatment, whereas beta 1-protein increased to only 2.5-fold over hypothyroid by 4 days of T3. The discoordinate changes in alpha 2- and beta 1-mRNA vs. protein can be reconciled with the hypothesis that beta 1 is rate limiting for assembly in eu- and hypothyroids, and favors assembly with alpha 1, while excess unassembled alpha 2 is degraded. In the hyperthyroids we predict beta 1 is not rate limiting and there is increased alpha 2 beta 1-assembly. We calculate that T3 decreases the alpha 1-to-alpha 2 ratio from 24:1 in hypothyroid to 3.4:1 in hyperthyroid cardiomyocytes.

Parathyroid hormone (PTH) plays a critical role in the regulation of renal phosphorous homeostasis by altering the levels of the sodium-phosphate cotransporter NaPi2a in the brush border membrane (BBM) of renal proximal tubular cells. While details of the molecular events of PTH-induced internalization of NaPi2a are emerging, the precise events governing NaPi2a removal from brush border microvilli in response to PTH remain to be fully determined. Here we use a novel application of total internal reflection fluorescence microscopy to examine how PTH induces movement of NaPi2a out of brush border microvilli in living cells in real time. We show that a dynamic actin cytoskeleton is required for NaPi2a removal from the BBM in response to PTH. In addition, we demonstrate that a myosin motor that has previously been shown to be coregulated with NaPi2a, myosin VI, is necessary for PTH-induced removal of NaPi2a from BBM microvilli.