Distal Sodium Transport Research Articles

ClC-Kb pore mutation disrupts glycosylation and triggers distal tubular remodeling.

Mutations in the CLCNKB gene (1p36), encoding a basolateral chloride channel, ClC-Kb, cause type 3 Bartter's syndrome. We identified a family with a mixed Bartter's / Gitelman's phenotype and early-onset kidney failure and employing a candidate gene approach, discovered a homozygous mutation (CLCNKB c.499G>T [p.Gly167Cys]) in exon 6 of CLCNKB in the index patient. We then validated these results with Sanger and whole exome sequencing. Compared to wild-type ClC-Kb, the Gly167Cys mutant conducted less current and impaired, complex N-linked glycosylation in vitro. We demonstrated that loss of Gly-167, rather than gain of a mutant Cys, impairs complex glycosylation but that surface expression remains intact. Moreover, Asn364 was necessary for channel function and complex glycosylation. Morphologic evaluation of human kidney biopsies revealed typical basolateral localization of mutant Gly167Cys ClC-Kb in cortical distal tubular epithelia. However, we detected attenuated expression of distal sodium transport proteins, changes in abundance of distal tubule segments, and hypokalemia-associated intracellular condensates from the index patient compared to control nephrectomy specimens. The present data establish what we believe, are novel regulatory mechanisms of ClC-Kb activity and demonstrate nephron remodeling in man, caused by mutant ClC-Kb, with implications for renal electrolyte handling, blood pressure control, and kidney disease.

Increase in Salt Concentration in the Culture Media Enhances Protein Expression of Tumor Necrosis Factor‐Alpha Receptor Type 1 in Cultured Renal Cortical Collecting Duct Cells But Not in Proximal Tubular Cells

Tumor necrosis factor‐alpha (TNFα) exerts its biological responses via interaction with its’ two cell surface receptors, type 1 (TNFR1) and type 2 (TNFR2) which are differentially expressed and regulated in the kidney. Previously, we have demonstrated that circulating TNFα exerts natriuresis via its action on TNFR1 but not on TNFR2 and such TNFR1 activation exerts inhibitory action on distal tubular sodium transport, particularly tubular ENaC activity. As TNFα level is elevated during dietary intake of high salt (HS), we hypothesized that increases in salt concentration in renal tissue enhances TNFα ‐TNFR1 activity in the renal tubules to induce natriuretic response to HS intake. To test this hypothesis, we examined the protein expression of TNFR1 and TNFR2 in cultured proximal (human kidney [HK‐2] cell line) and distal (murine cortical collecting duct [M‐1] cell line) tubular cells at different concentrations of NaCl in the culture media. Both these HK‐2 and M‐1 cells were incubated for 24 hours in control media with normal NaCl concentration (120 mM) and at media containing higher NaCl (140 mM and 160 mM) concentrations. TNF receptors protein expressions were analyzed by the Western Blot technique using appropriate antibodies for TNFR1 (rabbit anti‐TNFR1; Abcam cat # 19139) and TNFR2 (rabbit anti‐TNFR2; Abcam cat # 109322), which were normalized by expression of β‐actin or GAPDH proteins using appropriate antibodies. The blot image was quantified and measured as band intensity density with Image J software. The quantitative immunoblot analysis in HK‐2 cells showed that there were no significant changes in the band intensity unit (IU) in TNFR1 (120 mM, 70.1±12.8 IU; 140 mM, 73.7±9.9 IU; 160 mM, 57.2±9.8 IU; n=6 in each condition) or TNFR2 protein (120 mM, 83.9 ±6.3 IU; 140 mM, 66.8±14.1 IU; 160 mM, 77.8±2.3 IU; n=6 in each condition) levels. On the other hand, TNFR1 protein expression was significantly higher in M‐1 cells incubated in 160 mM NaCl conc. (135.6 ±7.8 IU, n=7; P< 0.005) than that in 120 mM (109.3±2.8 IU, n=7) and in 140 mM (91.1±10.8 IU, n=7) NaCl conc. However, TNFR2 protein expression in M‐1 cells did not significantly alter with the changes in NaCl conc. in the media (120 mM, 137±20.1 IU; 140 mM, 128±7.2 IU; 160 mM, 143±7.2 IU; n=7 in each condition). These data indicate that TNFR1 activity is upregulated in renal collecting duct cells (CCD) in response increases in tissue NaCl concentration in the kidney. Such upregulation of TNFR1 activity in the CCD induced by high salt concentration, exerts inhibitory action on distal sodium transport and plays a role in enhancing urinary sodium excretion during dietary HS intake.

Dynamics of proximal and distal transport of sodium in case of bilateral gonadectomy and the efficacy of hormonal replacement therapy

Introduction. The characteristic feature of current trauma in Ukraine and in the world is the prevalence of highly energetic skeletal trauma which is followed by the appearance of combined and multiple injuries with the formation of multiorgan dysfunction and failure. Because of the increase of life duration, the high interest of current traumatology is the investigation process of trauma course in older people. Among them the high interest is given to females in postmenopausal period. The decrease of estrogen level causes very unfavorable ground for the trauma course. However, the influence of severe trauma on changes in proximal and distal transport of sodium in case of hypoestrogenic state is not fully investigated. There is no available data concerning the effectiveness of hormonal replacement therapy in this condition.The goal of the study: is to investigate the influence of cranioskeletal injury on the dynamics of proximal and distal sodium transport in rats with bilateral ovariectomy in the period of late changes of traumatic disease and evaluate the effectiveness of hormonal replacement therapy. Materials and methods. Experiments were performed on 42 non-linear white female rats with the weight of 200-220 g. The model of hypoestrogenic state was performed via surgical removal of the ovaries. 1 month after the gonadectomy in rats skeletal trauma was performed. As a treatment option in one subgroup of rats with gonadectomy and cranioskeletal injury we performed hormonal replacement therapy. The control groups were formed by intact rats and osteoporotic rats 1 month after gonadectomy which were not injured. In control groups and after 1 and 2 months of posttraumatic period we were investigating the functional state of kidneys by performing the water upload method. Afterwards we performed the laboratory checkup of creatinine and sodium levels in serum and urine and additionally calculated the values of proximal and distal sodium transport. Results and its discussion. It was found that the level of proximal and distal sodium transport depends on the estrogen level, the decrease of its secretion leads to the decrease of sodium reabsorption in tubules and the increase in secretion. These changes were more prevalent in proximal tubules. In the course of trauma 1 month after posttraumatic period the value was decreasing in both research groups. However, in rats with gonadectomy the changes were much bigger and stayed at the same level until the 2nd month of the experiment. In rats without gonadectomy these values were achieving the same levels as control group until 2nd month. The use of combined hormonal replacement therapy caused the increase of proximal and distal sodium transport in comparison to the group without receiving treatment. The results that we got though was not achieving the level of rats without gonadectomy, but it showed the perspective for the use of combined hormonal replacement therapy in case of hypoestrogenic state for the decrease of renal dysfunction. Conclusions: Bilateral ovariectomy in female rats after 1 month shows the decrease of proximal and distal sodium levels comparing to rats with preserved gonads. The cranioskeletal injury that happened 1 month after bilateral ovariectomy causes the much bigger decrease of proximal and distal sodium transport comparing to rats with preserved gonads. The use of combined hormonal replacement therapy caused lower changes in proximal and distal sodium transport comparing to rats without this treatment.

The Influence of Peptides on the Morphofunctional State of Kidneys in Old Rats

More than a quarter of the elderly and senile age population suffers from kidney pathology. For this reason, a prophylaxis of kidney diseases with safe and effective nephroprotectors is a priority of gerontology. This work studies the influence of the polypeptide kidney complex (PKC) and peptides AED, EDL, and AEDG, on the functional state of kidneys in old rats. The administration of the PKC and peptides AED and EDL increased diuresis by 1.2–1.4 times. The PKC and peptide AED reduced the urine protein level and protein excretion by 1.5–2.8 times. The PKC and peptides AED and EDL increased distal sodium transport by 1.2–1.3 times. The peptides AED and EDL increased sodium excretion by 1.3 and 1.6 times respectively. The renal effects of the peptide AEDG resulted in a 21% reduction of glomerular filtration rate, a 3.1-fold decrease in the urine protein level, and a 2.5-fold decrease in protein excretion. The peptide AEDG reduced absolute sodium reabsorption by 1.3 times and increased distal sodium transport by 1.4 times. The realization of glomerular–tubular and tubular–tubular balances is verified by the correlation between the glomerular filtration rate (GFR) and absolute, proximal, and distal sodium reabsorption. In kidney tissue, stimulation of the antioxidant enzyme activity on the background of inhibition of the intensity peroxidation processes was observed, which, together with morphological data, reflects the absence of nephrotoxic effects. PKC and peptides AED, EDL, and AEDG may be considered nephroprotective agents in kidney aging.

Aldosterone Modulates the Association between NCC and ENaC

Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption.

Renal tubular angiotensin converting enzyme is responsible for nitro-L-arginine methyl ester (L-NAME)-induced salt sensitivity

Renal parenchymal injury predisposes to salt-sensitive hypertension, but how this occurs is not known. Here we tested whether renal tubular angiotensin converting enzyme (ACE), the main site of kidney ACE expression, iscentral to the development of salt sensitivity in this setting. Two mouse models were used: it-ACE mice in whichACE expression is selectively eliminated from renal tubular epithelial cells; and ACE 3/9 mice, a compound heterozygous mouse model that makes ACE only in renal tubular epithelium from the ACE 9 allele, and in liver hepatocytes from the ACE 3 allele. Salt sensitivity was induced using a post L-NAME salt challenge. While bothwild-type and ACE 3/9 mice developed arterial hypertension following three weeks of high salt administration, it-ACE mice remained normotensive with low levels of renal angiotensin II. These mice displayed increased sodium excretion, lower sodium accumulation, and an exaggerated reduction in distal sodium transporters. Thus, in mice with renal injury induced by L-NAME pretreatment, renal tubular epithelial ACE, and not ACE expression by renal endothelium, lung, brain, or plasma, isessential for renal angiotensin II accumulation and salt-sensitive hypertension.

Role of stimulated intrarenal angiotensinogen in hypertension.

Experimental models of hypertension and patients with inappropriately increased renin formation due to a stenotic kidney, arteriosclerotic narrowing of the renal arterioles or a rare juxtaglomerular cell tumor have shown a progressive augmentation of the intrarenal/intratubular renin-angiotensin system (RAS). The increased intrarenal angiotensin II (Ang II) elicits renal vasoconstriction and enhanced tubular sodium reabsorption in proximal and distal nephron segments. The enhanced intrarenal Ang II levels are due to both increased Ang II type 1 (AT1) receptor mediated Ang II uptake and AT1 receptor dependent stimulation of renal angiotensinogen (AGT) mRNA and augmented AGT production. The increased AGT formation and secretion into the proximal tubular lumen leads to local formation of Ang II, which stimulates proximal transporters such as the sodium/hydrogen exchanger. Enhanced AGT production also leads to spillover of AGT into the distal nephron segments as reflected by AGT in the urine, which provides an index of intrarenal RAS activity. There is also increased Ang II concentration in distal nephron with stimulation of distal sodium transport. Increased urinary excretion of AGT has been demonstrated in patients with hypertension, type 1 and type 2 diabetes mellitus, and several types of chronic kidney diseases indicating an upregulation of intrarenal RAS activity.

Epoxyeicosatrienoic Acids (EETs) Regulate Epithelial Sodium Channel Activity by Extracellular Signal-regulated Kinase 1/2 (ERK1/2)-mediated Phosphorylation

The epithelial sodium channel (ENaC) participates in the regulation of plasma sodium and volume, and gain of function mutations in the human channel cause salt-sensitive hypertension. Roles for the arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), in ENaC activity have been identified; however, their mechanisms of action remain unknown. In polarized M1 cells, 14,15-EET inhibited amiloride-sensitive apical to basolateral sodium transport as effectively as epidermal growth factor (EGF). The EET effects were associated with increased threonine phosphorylation of the ENaC β and γ subunits and abolished by inhibitors of (a) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal regulated kinases 1 and 2 (MEK/ERK1/2) and (b) EGF receptor signaling. CYP2C44 epoxygenase knockdown blunted the sodium transport effects of EGF, and its 14,15-EET metabolite rescued the knockdown phenotype. The relevance of these findings is indicated by (a) the hypertension that results in mice administered cetuximab, an inhibitor of EGF receptor binding, and (b) immunological data showing an association between the pressure effects of cetuximab and reductions in ENaCγ phosphorylation. These studies (a) identify an ERK1/2-dependent mechanism for ENaC inhibition by 14,15-EET, (b) point to ENaC as a proximal target for EET-activated ERK1/2 mitogenic kinases, (c) characterize a mechanistic commonality between EGF and epoxygenase metabolites as ENaC inhibitors, and (d) suggest a CYP2C epoxygenase-mediated pathway for the regulation of distal sodium transport.

Candesartan differentially regulates distal sodium transporters and channel subunits in cortex versus medulla in streptozotocin‐induced diabetic rats.

Diabetes is associated with increased activity of the renal renin angiotensin system (RAS). Previously, we demonstrated that streptozotocin (STZ)-induced diabetic rats have significantly increased abundances of renal distal sodium transporters and channels, e.g., the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). To determine the role of elevated RAS activity in these changes, male Sprague-Dawley rats (100–150 g) were made diabetic by intravenous injection of STZ (65 mg/kg·bw) or treated with vehicle (n = 12/group). Rats were fed standard 1% NaCl rodent chow and watered ad libitum. After 14 days, rats were further divided (n = 6/group) to receive candesartan in the drinking water (2 mg/kg/day) or plain water for 7 additional days. Immunoblotting revealed, in cortex, diabetes caused a modest increase in NCC, β- and γ-ENaC (85-kDa band) abundances (band density ~ 150% of non-diabetic controls). Candesartan not only reversed these increases, but significantly reduced abundance of NCC and ENaC subunits to ~50% of non-candesartan-treated rats in both diabetic and non-diabetic rats. In contrast, in the medulla, diabetes caused a much greater increase in ENaC subunits and α-1-Na-K-ATPase (band densities 200–600% of non-diabetic controls) and these increases were irreversible by candesartan. Moreover, candesartan increased β- and γ-ENaC in the non-diabetic rats, in the medulla tip. Thus the increased abundances of sodium transporters in the cortex, but not the medulla, with diabetes, are candesartan-sensitive, and therefore, likely due to enhanced AT1-mediated signaling.

Sex and Age Differences in Renal Ability to Conserve Water and Sodium in F344 x Brown Norway Rats

Renal sodium and water concentrating ability declines with age. The potential role of sex differences in the regulation of sodium and water transport proteins with aging has not been determined. To address this, male and female Fisher 344 X Brown Norway (F344BN) rats were studied at 3 months (young) and 30 months (aged) of age, while on a sodium deficient (NaD, Na+ content <0.005%) or a control (C, Na+ content = 0.4%) diet. Urine volume and sodium were increased and urine osmolality was decreased in aged female vs. aged male rats on both diets, but there were no significant differences in these parameters between young males and females. Vasopressin V2 receptor (V2R) and AQP2 protein abundances in the kidney inner medulla were markedly decreased in aged female vs. aged male rats on both diets. The thiazide-sensitive NaCl co-transporter (NCC) abundance in the cortex (CTX) was significantly increased in young females vs. males on C diet. Although the NaD-induced increase of NCC in CTX was blunted with aging, there were no significant differences between males and females on NaD. α-epithelial sodium channel (ENaC) abundance in CTX was similarly increased in females vs. males, but equivalent increases on NaD diet were observed in both young and aged rats. This was true for the 70 kDa band of γ-ENaC as well. In conclusion, relatively down-regulated kidney V2R and AQP2 abundances may contribute to reduced urine concentration in aged female vs. aged male rats. However, the impaired urinary sodium conservation on NaD diet in aged female rats does not appear to be the result of reduced abundances of distal sodium transporters and sodium channel subunits.

Increased abundance of distal sodium transporters in rat kidney during vasopressin escape.

Hyponatremia is associated with inappropriately elevated vasopressin levels. A brisk natriuresis precedes the escape from this antidiuresis. Thus, the hypothesis was that the abundance of one or more of the sodium transporters of the distal tubule (a site for fine tuning of sodium balance) would be altered during vasopressin escape. Semiquantitative immunoblotting was used to examine the regulation of abundance of several sodium transporters/channels of the thick ascending limb through the collecting duct in the rat model. Osmotic minipumps to infuse dDAVP, the V2-selective vasopressin agonist (5 ng/h) for the entire experiment, were implanted in Male Sprague-Dawley rats. After 4 d, rats were divided into a control (dry AIN-76 diet/ad libitum water) or a water-loaded (gelled-agar-AIN-76 diet/ad libitum water) group. Rats were killed after 1, 2, 3, or 7 additional days. The water-loaded rats were hyponatremic (plasma Na+, 98 to 122 mmol/L) and manifested the expected early natriuresis and diuresis of vasopressin escape. Water loading (with dDAVP infusion) resulted in increased whole-kidney abundances of the thiazide-sensitive Na-Cl co-transporter, the alpha-subunit of the epithelial sodium channel (ENaC), and the 70-kD dimer of the gamma-subunit of ENaC. No changes were observed for the ss-subunit of ENaC. Similar protein changes have recently been associated with elevated aldosterone levels in rats. However, plasma aldosterone levels were significantly suppressed in this model. These data suggest that several distal sodium reabsorptive mechanisms are upregulated during vasopressin escape; this may help to attenuate the developing hyponatremia resulting from water loading when vasopressin levels are inappropriately elevated.

Defect in proximal and distal sodium transport in post-obstructive diuresis

Defect in proximal and distal sodium transport in post-obstructive diuresis. Bilateral urinary obstruction was created in rats by tying a ligature around the bladder below the ureteral orifices. Sham-operated rats served as controls. Water intake was limited and food was withheld from both groups during and following obstruction. Release of the obstruction after 30hr was followed by a diuresis lasting 24 to 36hr. One group of rats was kept in balance cages before, during and after obstruction. After release of the obstruction, experimental animals had lower creatinine clearance and higher urine flow, Na, K and solute excretion than did sham-operated controls. Urine flow in the experimental animals exceeded water intake during the postobstructive period. A second group of rats was prepared identically and used for clearance and micropuncture measurements beginning 14hr after release of the obstruction. Plasma concentrations of K, urea and total solutes were elevated. C In was reduced and V, U Na V, U K V, U Osm V and C Osm were elevated. Na and water reabsorption were reduced in the proximal and distal nephron, distal K secretion was increased, and a marked defect in concentrating ability was present. The reduction in proximal Na reabsorption was not associated with a reduced peritubular capillary protein concentration. Tracer microinjection experiments with a third group of rats revealed abnormal permeability to mannitol and inulin. The defect in Na reabsorption is probably due to an increased backflux of Na, reducing net Na reabsorption. GFR is probably not identical to inulin or creatinine clearance in post-obstructive diuresis.

Increasing magnitude of electrical potential along the renal distal tubule.

Increasing magnitude of electrical potential along the renal distal tubule.

Influence of postglomerular hematocrit and protein concentration on rat nephron fluid transfer.

Influence of postglomerular hematocrit and protein concentration on rat nephron fluid transfer.

Micropuncture study of effects of urea infusion in tubular reabsorption in the rat.

ARTICLESMicropuncture study of effects of urea infusion in tubular reabsorption in the ratML Kauker, WE Lassiter, and CW GottschalkML Kauker, WE Lassiter, and CW GottschalkPublished Online:01 Jul 1970https://doi.org/10.1152/ajplegacy.1970.219.1.45MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByMild Chronic Hyponatremia in the Ambulatory SettingClinical Journal of the American Society of Nephrology, Vol. 10, No. 12Diuretics: a review10 July 2012 | Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, Vol. 49, No. 5Effects of chronic, urea-induced osmotic diuresis on kidney weight and function in ratsDiabetologia, Vol. 37, No. 3Renal Handling of Urea1 January 2011Renal tubular transport of 3H-digoxin in saline diuresis in rats.Circulation Research, Vol. 38, No. 3Factors influencing sodium and water excretion in uremic manKidney International, Vol. 7, No. 2Defect in proximal and distal sodium transport in post-obstructive diuresisKidney International, Vol. 2, No. 6Alterations in renal tubular sodium and water reabsorption in chronic renal disease in manKidney International, Vol. 2, No. 3Glomerular filtration changes during renal artery infusion of various hypertonic solutions in the dogPfl�gers Archiv European Journal of Physiology, Vol. 337, No. 1FUNCTIONAL ASPECTS OF COMPENSATORY RENAL GROWTHPharmaceutical Sciences—1970: Literature Review of PharmaceuticsJournal of Pharmaceutical Sciences, Vol. 60, No. 7The relation between the reabsorption of urea and of water in the distal tubule of the rat kidneyPfl�gers Archiv European Journal of Physiology, Vol. 329, No. 2 More from this issue > Volume 219Issue 1July 1970Pages 45-50 Copyright & PermissionsCopyright © 1970 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1970.219.1.45PubMed5424857History Published online 1 July 1970 Published in print 1 July 1970 Metrics

Oxygen cost of sodium reabsorption in proximal and distal parts of the nephron.

The relationship between changes in tubular sodium reabsorption and changes in renal oxygen consumption (Na/O2) was determined in dogs made anemic, as the arteriovenous difference in oxygen saturation was more than doubled by antecedent bleeding. Tubular sodium reabsorption was reduced by 45 ± 3%, and oxygen consumption by 36 ± 4% after blocking distal sodium transport with ethacrynic acid and chlorothiazide. On the basis of these data it could be calculated that 34 ± 5 Eq Na+ were transported in the distal nephron per/mol O2 consumed. Similar Na/O2 ratios were obtained by reducing proximal sodium reabsorption by lowering glomerular filtration rate after blocking distal sodium reabsorption. During infusion of chlorothiazide and ethacrynic acid, oxygen consumption was not further decreased by reduction of net proximal sodium reabsorption with mannitol infusion. This observation supports the hypothesis that energy-dependent sodium reabsorption in the proximal tubules is not reduced during mannitol diuresis....

Effects of hypotonic saline loading in hydrated dog: evidence for a saline-induced limit on distal tubular sodium transport.

We performed studies on dogs under hydrated conditions, utilizing the rate of free water formation (C(H2O)) as an index of the rate of distal tubular sodium transport. Since C(H2O) could be progressively increased with no evidence of a maximal rate during loading with hypotonic (2.5%) mannitol, it was concluded that there is no limit on distal tubular sodium transport during mannitol loading. In contrast, during hypotonic (0.45%) saline loading C(H2O) rose initially, but as urine flow (V) exceeded 25% of the filtered load C(H2O) attained maximal levels (up to 20% of the filtered load) and remained stable as V increased to 50% of the filtered load. It was concluded that saline loading progressively inhibits proximal sodium reabsorption. Initially, the distal tubule absorbes a large fraction of the proximal rejectate and sodium excretion rises slightly. Eventually, an alteration in distal sodium transport appears which culminates in a maximal rate or transport limit. This distal transport limit provoked by saline loading could not be characterized by a classical Tm as seen with glucose and does not seem to be consequent to high rates of flow through the distal tubule. Regardless of the precise nature of this limit, the major increment in sodium excretion develops during saline loading only after saline alters the capacity of the distal tubule to transport sodium.