Na-Cl Cotransporter Research Articles

Na-Cl Co-transporter (NCC) gene inactivation is associated with improved bone microstructure.

To evaluate the impact of the inactivation of NCC on bone turnover and microarchitecture in Gitelman syndrome patients. A cross-sectional study was conducted in 45 GS patients (25 males and 20 females). Serum procollagen type 1N-terminal propeptide (P1NP), β-carboxy-terminal crosslinked telopeptide of type 1 collagen (β-CTX), and osteocalcin were measured. High-resolution peripheral quantitative computed tomography (HR-pQCT) was conducted to evaluate bone microarchitecture in GS patients and age- and sex-matched healthy controls. Areal bone mineral density (aBMD) was measured by dual-energy X-ray absorptiometry (DXA) simultaneously. GS patients had a relatively lower level of β-CTX. aBMD at several skeletal sites was improved in GS patients. HR-pQCT assessment revealed that GS patients had slightly thinner but significantly more compact trabecular bone (increased trabecular number and decreased thickness), notably decreased cortical porosity, and increased volume BMD (vBMD) at both the radius and tibia compared with controls. The disease severity, represented as the relationship with the minimum level of magnesium during the course and standard base excess, was associated with bone microarchitecture parameters after adjusting for age, sex, and BMI. The decreased vBMD and Tb.BV/TV, and increased Tb.Sp and Ct.Po with aging, were reversed in GS patients to a certain extent. GS patients have superior bone microarchitecture, which suggests that the inactivation of NCC might be beneficial for avoiding osteoporosis.

Inwardly rectifying K+ channels 4.1 and 5.1 (Kir4.1/Kir5.1) in the renal distal nephron.

The inwardly rectifying potassium channel (Kir) 4.1 (encoded by KCNJ10) interacts with Kir5.1 (encoded by KCNJ16) to form a major basolateral K+ channel in the renal distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical collecting duct (CCD). Kir4.1/Kir5.1 heterotetramer plays an important role in regulating Na+ and K+ transport in the DCT, CNT, and CCD. A recent development in the field has firmly established the role of Kir4.1/Kir5.1 heterotetramer of the DCT in the regulation of thiazide-sensitive Na-Cl cotransporter (NCC). Changes in Kir4.1/Kir5.1 activity of the DCT are an essential step for the regulation of NCC expression/activity induced by dietary K+ and Na+ intakes and play a role in modulating NCC by type 2 angiotensin II receptor (AT2R), bradykinin type II receptor (BK2R), and β-adrenergic receptor. Since NCC activity determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), a distal nephron segment from late DCT to CCD, Kir4.1/Kir5.1 activity plays a critical role not only in the regulation of renal Na+ absorption but also in modulating renal K+ excretion and maintaining K+ homeostasis. Thus, Kir4.1/Kir5.1 activity serves as an important component of renal K+ sensing mechanism. The main focus of this review is to provide an overview regarding the role of Kir4.1 and Kir5.1 of the DCT and CCD in the regulation of renal K+ excretion and Na+ absorption.

The European and Japanese eel NaCl cotransporters β exhibit chloride currents and are resistant to thiazide type diuretics.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCCα is located in the kidney and eNCCβ, which is present in the apical membrane of the rectum. Interestingly, the European eNCCβ functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCCβ and jNCCβ are 98% identical. Here, by site-directed mutagenesis, we transformed eNCCβ into jNCCβ. Our data showed that jNCCβ, similar to eNCCβ, is resistant to thiazides. In addition, both NCCβ proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCβ proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCCβ.

Anesthetic Considerations for Cesarean Delivery in a Parturient With Severe Gitelman Syndrome

Gitelman syndrome is an autosomal recessive inherited disorder that impairs the function of thiazide-sensitive sodium-chloride cotransporters in the distal convoluted tubule of the nephron. During labor and delivery, avoidance of sympathetic overactivity, meticulous hemodynamic monitoring, and expedited repletion of potassium and magnesium are required to avoid adverse outcomes. We present a parturient with severe Gitelman syndrome, requiring continuous electrolyte and fluid infusions, who underwent successful cesarean delivery. Potential severe morbidity was avoided with multidisciplinary planning and management.

IL18 signaling causes islet β cell development and insulin secretion via different receptors on acinar and β cells.

Diabetic patients show elevated plasma IL18 concentrations. IL18 has two receptors: the IL18 receptor (IL18r) and the Na-Cl co-transporter (NCC). Here, we report that IL18 is expressed on islet α cells, NCC on β cells, and IL18r on acinar cells in human and mouse pancreases. The deficiency of these receptors reduces islet size, β cell proliferation, and insulin secretion but increases β cell apoptosis and exocrine macrophage accumulation after diet-induced glucose intolerance or streptozotocin-induced hyperglycemia. Together with the glucagon-like peptide-1 (GLP1), IL18 uses the NCC and GLP1 receptors on β cells to trigger β cell development and insulin secretion. IL18 also uses the IL18r on acinar cells to block hyperglycemic pancreas macrophage expansion. The β cell-selective depletion of the NCC or acinar-cell-selective IL18r depletion reduces glucose tolerance and insulin sensitivity with impaired β cell proliferation, enhanced β cell apoptosis and macrophage expansion, and inflammation in mouse hyperglycemic pancreas. IL18 uses NCC, GLP1r, and IL18r to maintain islet β cell function and homeostasis.

THE ROLE OF URINARY EXOSOMAL SODIUM CHLORIDE COTRANSPORTER IN SUBTYPING PRIMARY ALDOSTERONISM

Objective: Adrenal venous sampling (AVS) is recognized as the gold standard for subtyping primary aldosteronism (PA), but its invasive nature and technical challenges limit its availability. A recent study reported that sodium chloride cotransporter (NCC) in urinary exosomes is a promising marker for assessing the biological activity of aldosterone and can be treated as a potential biomarker of PA. The current study was conducted to verify the hypothesis that the expression of NCC and its phosphorylated form (pNCC) in urinary exosomes are different in various subtypes and genotypes of PA and can be used to select AVS candidates. Design and method: A total of 27 patients with PA were enrolled in the study.Patients with LI > = 4 and < 4 were classified into high LI (hLI) and low LI (lLI) groups, respectively. Urinary exosomes were isolated from spot urine samples using ultracentrifugation. NCC and pNCC expressions were tested in patients diagnosed with PA who underwent AVS. Sanger sequencing of KCNJ5 was performed on DNA extracted from adrenal adenoma. Results: Compared with the lLI group, the hLI group had higher levels of 24 h urinary aldosterone (28.6 ± 11.8 vs 14.9 ± 4.6, P = 0.0002) and supine PAC (314 ± 115 vs 163 ± 53, P < 0.0001) on admission (Table 1). In addition, the pre-saline infusion test (pre-SIT) supine PAC (after correction of hypokalemia) in the hLI group was higher than that in the lLI group (342 ± 172 vs 224 ± 59, P = 0.0141). pNCC was more abundant in the urinary exosomes in the high lateralization index (hLI, > = 4) group (1.64 folds, P = 0.0080) than in the low LI (lLI, < 4) group (Figure 1). Carriers of the somatic KCNJ5 mutations, compared with non-carriers, had more abundant pNCC expression in urinary exosomes (1.99 folds, P = 0.0399) (Figure 2). Conclusions: The expression of pNCC in urinary exosomes in patients with PA with various subtypes and genotypes was different. They can be used as biomarkers of AVS for PA subtyping.

Acute Intravenous NaCl and Volume Expansion Reduces Sodium-Chloride Cotransporter Abundance and Phosphorylation in Urinary Extracellular Vesicles.

Sodium chloride (NaCl) loading and volume expansion suppress the renin-angiotensin-aldosterone system to reduce renal tubular reabsorption of NaCl and water, but effects on the sodium-chloride cotransporter (NCC) and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. We used urinary extracellular vesicles (uEVs) as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 patients with hypertension who had repeatedly raised aldosterone/renin ratios undergoing infusion of 2 L of 0.9% saline over 4 hours. When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change [FC]=0.70); pendrin (FC=0.84); AQP2 (FC=0.62); and uEV markers, including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52), and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC; FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, whereas the significant decrease in AQP2 persisted (FC=0.62). We conclude that (1) decreases in NCC and pNCC, induced by acute NaCl loading and volume expansion, may be due to diluted post-test urines; (2) the lack of change of NCC and pNCC when corrected for ALIX, despite a fall in plasma aldosterone, may be due to the lack of change in plasma K+; and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion.

Gitelman syndrome with normocalciuria – a case report

BackgroundGitelman Syndrome (GS) is a hereditary tubulopathy associated with a biallelic inactivating mutations of the SLC12A3 gene encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT). The typical clinical manifestation is a hypokalemic metabolic alkalosis with significant hypomagnesemia, and low urinary calcium excretion. Hypocalciuria is widely believed to be a hallmark of GS that distinguishes it from Barter’s syndrome, presenting as hypercalciuria. The pathomechanism of hypocalciuria in GS is not fully elucidated. Up to date, a clinical course of GS with normocalciuria has been reported only in men, while women have a milder course of the disease with typical hypocalciuria, which is believed as the result of sex hormone. Additionally, there is a growing evidence that calcium channels of the distal nephron could be regulated by a variety of hormones, including aldosterone (Aldo).Case presentationWe present the case of a 28-year-old Caucasian woman with asymptomatic, chronic hypokalemia, hypomagnesemia, hypochloremic alkalosis and normal urinary calcium excretion. A high renin levels with normal concentration of Aldo in serum have also been found. The values of blood pressure were low. Based on genetic studies, two heterozygous mutations in the trans position were confirmed: c.2186G>T (p.Gly729Val) and c.1247G>C (p.Cys416Ser) in the SLC12A3 gene, which ultimately confirmed the diagnosis of GS.ConclusionsWe report here the first case of genetically confirmed GS manifested as normocalciuria in a Caucasian woman. Thus, our result does not confirm a role of sex hormones on the level of calciuria. Based on the results of normal Aldo concentration despite high renin level in our patient, we hypothesized that Aldo may be connecting with the level of urinary calcium excretion in patients with the GS.

Genetic and Biological Effects of SLC12A3, a Sodium-Chloride Cotransporter, in Gitelman Syndrome and Diabetic Kidney Disease.

The SLC12A3 (Solute carrier family 12 member 3) gene encodes a sodium-chloride cotransporter and mediates Na+ and Cl− reabsorption in the distal convoluted tubule of kidneys. An experimental study has previously showed that with knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage, suggesting that SLC12A3 may have genetic effects in renal disorders. Many clinical reports have demonstrated that the function-loss mutations in the SLC12A3 gene, mainly including Thr60Met, Asp486Asn, Gly741Arg, Leu859Pro, Arg861Cys, Arg913Gln, Arg928Cys and Cys994Tyr, play the pathogenic effects in Gitelman syndrome. This kidney disease is inherited as an autosomal recessive trait. In addition, several population genetic association studies have indicated that the single nucleotide variant Arg913Gln in the SLC12A3 gene is associated with diabetic kidney disease in type 2 diabetes subjects. In this review, we first summarized bioinformatics of the SLC12A3 gene and its genetic variation. We then described the different genetic and biological effects of SLC12A3 in Gitelman syndrome and diabetic kidney disease. We also discussed about further genetic and biological analyses of SLC12A3 as pharmacokinetic targets of diuretics.

Interaction between CD81 and NCC is associated with LPS induced preeclampsia

CD81, a member of the tetraspain superfamily, is an important component in the pathogenesis of pregnancy. In order to determine whether there is a connection between CD81 and renal sodium transporters, we studied the interaction of CD81 and Na+‐Cl‐cotransporter (NCC) in vivo and in vitro. A rat model of pregnancy hypertension was built by injecting a small amount of lipopolysaccharide (LPS, 0.5μg/kg in 2ml saline) into the tail vein of pregnant rats on GD 5. On GD 18, BPs (SBP :99.9±1.4 vs 116.9±1.8, DBP :85.9±2.0 vs 94.4±1.8,mmHg, n=7/group) and urine protein (1185±35.0 vs 3550±158.0, μg/day, n=4/group) were higher in LPS ‐rats relative to vehicle‐rats while renal protein abundance of CD81 (186.7±20, % of vehiclel, n=4/group, same as below) and NCC (278.0±53) was increased. CD81 was colocalized and co‐immunoparticipated with NCC, not with NHE3, in rat kidney. The immunofluorescent staining of NCC and CD81 was located in the sub‐cellular membrane and cytoplasm. In order to further explore the relationship between renal CD81 and NCC, we treated mouse kidney distal tubule cells with LPS for 24h to construct an in vitro experimental model. The protein abundances of CD81, and NCC in mDCT cells were increased in the cell groups treated with LPS at concentration of 10, 20, 30 g/mL while LDH and CCK‐8 were not altered. The cd81‐siRNA (32nM) for 48 hrs totally deleted CD81 protein. It also decrease NCC protein(70.9±4.8) expression without changing NCC mRNA(124.9±14) and reversed the increase of NCC(126.0±5.2,86.9±8.8) induced by LPS(10μg/mL) for the last 24 hrs conforming the interaction of CD81 with NCC in vitro. Thus, the interaction between CD81 and NCC are determined in vivo and its role in preeclampsia remains to be studied in future.

DCT‐specific COP9 Signalosome Deletion Activates the WNK4‐NCC Pathway and Mimics Familial Hyperkalemic Hypertension

With‐no‐lysine kinase 4 (WNK4) activates the NaCl cotransporter (NCC) which is the main pathway for sodium reabsorption in the distal convoluted tubule (DCT) of the kidney. The cullin‐RING ubiquitin ligase cullin 3 (CUL3) regulates WNK4 abundance via interaction through the substrate adaptor KLHL3. The monogenic disease familial hyperkalemic hypertension (FHHt) is caused by mutations in this pathway. Mutations in KLHL3 and WNK4 mainly disrupt formation of this complex, whereas, the CUL3 mutations impair binding to the COP9 signalosome (CSN), a deneddylase and upstream regulator of cullin‐RING ligases. Since FHHt is mostly a disease that affects the DCT, we proposed that DCT‐specific knockdown of the main CSN subunit, JAB1, would phenocopy FHHt. We deleted Jab1 in the DCT using the tamoxifen inducible NCC‐Cre‐ERT2 mouse model (DCT‐Jab1‐/‐). After five days of tamoxifen administration, proximal tubule, thick ascending limb, DCT, and connecting tubule/collecting duct segments were individually dissected and isolated to analyze protein abundance of each specific segment via Western blot. JAB1 and CUL3 protein abundance were substantially reduced in the DCT of DCT‐Jab1‐/‐ mice, whereas, WNK4 and phosphorylated NCC (pNCC) abundance were markedly elevated. Western blot analysis of cortical kidney tissue showed lower KLHL3 abundance and significantly higher abundance of phosphorylated SPAK (pSPAK), the intermediary kinase between WNK4 and NCC. Immunofluorescent staining of WNK4 and pSPAK showed increased expression and puncta formation in DCT1 and DCT2 tubules of DCT‐Jab1‐/‐ mice. This was not observed in other tubule segments that express WNK4 or pSPAK, such as the thick ascending limb and connecting tubule/collecting duct. Although there was obvious activation of the WNK4‐SPAK‐NCC pathway DCT‐Jab1‐/‐ mice, plasma analysis showed no significant differences in potassium, chloride or TCO2. However, challenging the DCT‐Jab1‐/‐ mice with a high potassium diet for seven days exaggerated the differences in pSPAK and pNCC protein abundance and led to a significant increase in plasma potassium. The results indicate that DCT‐specific disruption of JAB1 recapitulates many aspects of FHHt, including decreased KLHL3 and CUL3 abundance which leads to activation of the WNK4‐NCC pathway and a tendency toward hyperkalemia. These results implicate defective CSN binding as a key component of CUL3‐mediated familial hyperkalemic hypertension.

Uncovering Distal Convoluted Tubule (DCT) Heterogeneity with Enriched DCT Single‐Nucleus RNA‐Sequencing

The mammalian kidneys play a critical role in regulation of electrolyte balance and blood pressure. At least 14 kidney tubule segments are described, of which the distal convoluted tubule (DCT) is the shortest; yet this short segment plays a crucial role in a variety of homeostatic processes, including sodium, potassium, calcium and magnesium handling. The DCT is morphologically and functionally heterogeneous and comprises DCT1 and DCT2. A “shotgun” single‐cell RNA sequencing (scRNAseq) approach transcriptionally profiles all cells from a piece of kidney tissue, which may allow abundant cell types to overwhelm minority cell types, such as DCT, thereby masking their complex heterogeneity. Recently, Chen et al. used the targeted scRNAseq analysis and identified 6 populations of DCT1 cells; yet the DCT2 was not well represented and therefore poorly resolved.To understand the heterogeneity within the DCT2 population, we used “Isolation of Nuclei TAgged in specific Cell Types” (INTACT), which fluorescently labels nuclei from genetically targetable cells. Combined with the NCC (sodium chloride cotransporter)‐driven inducible Cre recombinase, DCT nuclei can be enriched using Fluorescence‐Activated Nuclei Sorting (FANS). Female NCC‐Cre‐INTACT mice were treated with Tamoxifen to induce expression of the GFP‐tagged SUN1, a nuclear membrane protein. Mice were provided either normal (NK) or potassium deficient diet (KD) for 4 days and kidneys were harvested. Nuclei from one kidney cortex were isolated and 10,000 GFP‐positive nuclei from each mouse were input into 10X Genomics Chromium Controller and the libraries were sequenced using NovaSeq. The resulting dataset was analyzed using Seurat to determine the number of populations and their identities using the UMAP algorithm for analysis and visualization.After data quality control, we detected 24,054 genes across 70,233 cells. Unbiased clustering and UMAP visualization revealed 12 clusters, with most cells from the DCT (78%) indicating the success of the enrichment process. Among those, 70% of the cells were from DCT1 and 30% from DCT2. There was clear separation of DCT1 from DCT2, with DCT1 enriched in Trpm7, Egf, Umod and Erbb4, and DCT2 in Slc8a1, Calb1, S100g, Trpv5, and Scnn1g. After further analysis of the DCT2 population, we identified two specialized populations, DCT2‐α and DCT2‐β. The DCT2‐α is enriched in NCC, magnesium and calcium‐related genes (Slc12a3, Wnk1, Slc8a1, Calb1, Trpm6, etc.) whereas DCT2‐β is enriched in mineralocorticoid receptors and epithelial sodium channel‐related genes (Nr3c2, Klk1, Scnn1b, Scnn1g, etc.), indicating that DCT2‐α is more similar to DCT1 and DCT2‐β to collecting duct. Four‐day KD treatment decreased the Klk1, Scnn1b and Scnn1g expression levels in DCT2‐β, indicating that some DCT2 cells may reprogram to retain potassium in response to the short‐term potassium deficiency.Our results suggest that DCT2 cells fall into specialized populations that likely serve different functions in electrolyte handling. The DCT2 is plastic and can transform from potassium excretion to potassium sparing in response to dietary potassium restriction to maintain electrolyte balance.

Immunohistochemical localization of receptor protein tyrosine phosphatase γ in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule

Most normal physiological processes require that both intracellular (i) and extracellular (o) pH be maintained within a tight range. pHo is contingent on arterial (a) pH, which is determined by the ratio of the arterial carbon dioxide to the arterial bicarbonate concentration, [CO2]a/[HCO3−]a. Pulmonary ventilation controls [CO2]a. The kidney controls [HCO3−]a by adjusting HCO3− transport rates (JHCO3) across the basal membranes of particular nephron epithelia into the blood to maintain normal pHa, or compensate for an acid‐base insult. In an effort to identify the sensor proteins for pHa, [CO2]a, or [HCO3−]a, our laboratory previously reported that, in isolated proximal tubules (PTs) from receptor protein tyrosine phosphatase γ (RPTPγ) knockout (KO) mice, JHCO3 is insensitive to changes in the basolateral [CO2] or [HCO3−]. Furthermore, RPTPγ‐KO mice have a deficiency in defending against a whole‐body metabolic acidosis. These data implicate RPTPγ as a major [CO2] or [HCO3−] sensor. RPTPγ possesses an extracellular catalytically inactive carbonic‐anhydrase‐like domain (CALD) and fibronectin type III (FNIII) domain, a single transmembrane helix, and intracellular domains D1 and D2. Dimerization of RPTPγ presumably allows the inhibitory D2 of one monomer to inhibit the otherwise catalytically active D1 of the other monomer. Thus, the dimerization/monomerization state controls downstream signaling. We hypothesize that the binding of CO2 or HCO3− to the CALD alters the CALD conformation, thereby transmitting signals that control the dimerization status of RPTPγ and thereby regulate downstream effects on JHCO3. We hypothesize that RPTPγ is localized in nephron segments important for regulating HCO3− transport/reabsorption, in the basal membranes that face the blood. To determine the location of RPTPγ in wild‐type mouse kidney cryosections, we use a chicken IgY anti‐RPTPγ antibody (Ab) raised against an epitope within the FNIII domain, and co‐stain with rabbit polyclonal Abs against the Na/HCO3 cotransporter (NBCe1‐A) as a PT marker, the Na/K/Cl cotransporter 2 (NKCC2) as a thick ascending limb (TAL) marker, and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. Imaging sections by confocal microscopy, we observe in 3 WT mice that RPTPγ localizes primarily to the PT basal membrane. However, 54% of PTs (NBCe1‐A positive) also exhibit apical RPTPγ staining of comparable intensity to the RPTPγ staining observed in the basal membrane. We also observe both apical and basal RPTPγ staining in Bowman’s capsules. We stained additional sections from the same animals with a rabbit polyclonal primary Ab that targets the RPTPγ CALD domain. In these sections, co‐stained with the anti‐FNIII Ab, we similarly observe both apical and basal staining in PTs. We observe RPTPγ staining in a minority of TALs, and at much lower intensity than observed in the PT. We observe minimal to no RPTPγ staining in the DCT. We validated the specificity of the RPTPγ Abs on RPTPγ‐KO kidney sections, which exhibit low background levels of fluorescence for both the anti‐FNIII and anti‐CALD Abs. In conclusion, these data confirm RPTPγ expression in PT basal membranes, but also describe the first examples of apical RPTPγ expression in renal epithelia.

Effects of Potassium Supplementation and Kir7.1 Knockout on Renal Function During the Progression of Salt‐Sensitive Hypertension

The inward‐rectifying potassium channel (Kir) family has been shown to play an important role in the kidney’s ability to maintain electrolyte homeostasis as well as blood pressure. Kir7.1 (encoded by the Kcnj13 gene) is expressed in the kidney, though its role in renal function is rudimentary. To examine the role of Kir7.1 in renal electrolyte handling and the development of salt‐induced hypertension, we generated a knockout of Kcnj13 on the Dahl salt‐sensitive (SS) rat background. Rats with the knockout of Kcnj13 were embryonically lethal; therefore, to study the Kir7.1 channel, we used heterozygous (SSKcnj13+/‐) rats. Immunohistochemistry staining for Kir7.1 revealed basolateral staining in the cortical sections of the kidneys in wild‐type littermates (WT), while SSKcnj13+/‐ rats showed little to no staining. In addition, immunofluorescence analysis confirmed that Kir7.1 was localized on the basolateral side in tubules and revealed that Kir7.1 co‐localized with aquaporin 2 and sodium‐chloride cotransporter (NCC). This data indicates that Kir7.1 is expressed in the cortical collecting duct (CCD) and the distal convoluted tubule (DCT). SSKcnj13+/‐ rats were further used to measure blood pressure and electrolyte handling changes during the development of salt‐induced hypertension. DSI telemeters were implanted in animals at 8 weeks of age; after a week to recover from surgery, animals were fed a high salt 4% NaCl diet (HS) for three weeks. Both WT and SSKcnj13+/‐rats become hypertensive after three weeks of HS; no difference between mean arterial pressures (153.1 ± 5.7 vs. 159.2 ± 6.9 mmHg for WT and SSKcnj13+/‐rats, respectively) was identified. Serum electrolytes were measured and also showed no difference in the amount of potassium (3.4 ± 0.1 vs. 3.5 ± 0.1 mmol/L) or sodium (142 ± 0.8 vs. 142 ± 0.4 mmol/L). Furthermore, urinary electrolyte excretion was measured and revealed no differences between WT and SSKcnj13+/‐ animals for potassium (14.0 ± 0.6 vs. 15.6 ± 1.9 K+/Cre) or sodium excretion (91 ± 11 vs. 117 ± 16 Na+/Cre). Potassium supplementation has previously been shown to augment renal Kir7.1 expression. Thus, we repeated the previous protocol but instead using a high salt and high potassium 4% NaCl and 2% KCl (HSHK) diet. After three weeks on this diet, animals exhibited no difference in blood pressure (154.2 ± 9.1 vs 145.6 ± 4.1 mmHg) and no differences in plasma potassium (4.0 ± 0.1 vs 4.0 ± 0.1 mmol/L) or sodium (139 ± 1 vs 139 ± 1 mmol/L). Urinary electrolyte excretion showed no differences in potassium excretion (60.9 ± 3.3 vs 54.8 ± 1.1 K+/Cre), but SSKcnj13+/‐ animals did have a significant decrease in sodium excretion (130 ± 8 vs 103 ± 3 Na+/Cre). Altogether, we have confirmed the presence of Kir7.1 in the CCD and DCT, found that heterozygous knockout of Kir7.1 does not affect the development of blood pressure on a HS or HSHK diet. However, we do see a reduction in sodium excretion in SSKcnj13+/‐rats on the HSHK diet, suggesting that Kir7.1’s role may be more pronounced under conditions of higher potassium.

Loop‐Diuretics Increase Pendrin through alpha‐ketoglutarate receptor, Oxgr1

IntroductionIt is well appreciated that loop diuretic resistance is governed by pendrin and activation of the thiazide‐sensitive sodium chloride cotransporter, NCC. Recently, our lab discovered pendrin increases as the natriuretic response wanes, coincident with the activation of a‐ketoglutarate (AKG) synthesis, secretion of a‐KG into the pro‐urine, and increased gene expression of the AKG GPCR, Oxgr1. Here we test the hypothesis that activation of Oxgr1 is necessary for the development of loop diuretic resistance.Methods10 week old male OXGR1 KO and WT (C57B6J) mice were challenged with furosemide (100 mg/kg) for 4 days. Urine was collected over 24 hours, daily, and urinary electrolytes were measured. Mice were euthanized at the end of the experiment and plasma electrolytes and aldosterone levels were measured using standard methods. Cortical protein and RNA abundances were measured using western blot and qRT‐PCR, respectively.ResultsWT and OXGR1 KO displayed distinguishing phenotypes only after furosemide administration. The loop‐diuretic challenge increased pendrin protein in WT mice but not in the OXGR1 KO mice despite identical increases in the chief pendrin stimulators, aldosterone and metabolic alkalosis. Pendrin transcript increased to the same extent in both genotypes, indicating OXGR1 augments pendrin via a post‐translational mechanism. Even with compromised upregulation of pendrin, OXGR1 KO mice exhibited the same diuretic response as WT mice. Upregulation of NCC was not compromised in the OXGR1 mice.ConclusionOXGR1 is required for post‐translational upregulation of pendrin protein post diuretic challenge, consistent with activation of a aKG‐OXGR1‐pendrin signaling axis. Upregulation of NCC and residual pendrin activity may be sufficient to brake the loop‐diuretic response.

Sodium Status Modulates the Effects of Aldosterone on Renal Transporter Expression and Tubular Remodeling

Aldosterone (aldo) is a steroid hormone that has a key role in extracellular volume and K+ homeostasis. While aldo levels are expected to be suppressed with high Na+intake, 5‐10% of hypertensives have chronic hyperaldosteronism regardless of Na+ intake. This combination of high sodium and high aldo can worsen end organ damage, especially in the kidney. We examined the effects of elevated aldo in the absence or presence of a high salt diet (HSD) on the expression of Na+ transporters in the distal nephron and on distal nephron remodeling to understand if these changes may play a role in the altered pathology. Male C57Bl/6 mice received either a control (0.4% NaCl) or HSD (4 weeks, 8% NaCl), +/‐ aldo via minipump for the final 2 weeks of treatment (240 μg/kg/day). Transporter expression levels and localization were analyzed by Western blot, qPCR and immunofluorescent staining. Expression of the α subunit of the epithelial sodium channel (ENaC) was increased with aldosterone regardless of Na+ intake, while subunits increased at both a transcript and protein level in mice receiving aldo and HSD. Both total levels of sodium chloride cotransporter (NCC) and active pNCC significantly increased with aldo alone, in association with a fall in blood [K+]. However, the addition of HSD markedly attenuated this increase in NCC and pNCC, despite more severe hypokalemia. This change was not associated with a difference in the expression of its upstream kinase SPAK. NKCC2 expression was not significantly altered in any of the conditions. We imaged whole tubules in thick kidney slices (300μm) to assess whether remodeling of the distal tubule could account for NCC expression differences. Sections were incubated with antibodies against pNCC and AQP2 and cleared with ethyl cinnamate to allow for imaging and analysis of whole distal tubules. Length of the distal tubule was analyzed as the pNCC positive portion. While aldo has previously been shown to promote hypertrophy of the distal tubule, we found a significant shortening of tubules in animals treated chronically with 2 weeks of aldo (Ctl 434m ± 63μm versus aldo 375m ± 88m, P<0.05, n=38 and 30 respectively). However, the addition of HSD with aldo caused a significant increase in distal tubule length (486m ±68μm, P<0.05, n=23). HSD by itself did not alter distal tubule length (428m ± 55m, n=17). Our results suggest that aldo‐mediated transcriptional regulation of ENaC subunits in the kidney changes according to dietary Na+ status. The attenuation of increased NCC and pNCC expression in kidneys of mice receiving HSD with aldo, despite profound hypokalemia and an increase in DCT length, suggest that factors other than blood [K+] have important roles in regulating NCC expression. Finally, aldo‐dependent DCT remodeling is dependent on dietary Na+.

Targeting Calcineurin Inhibitor-Induced Arterial Hypertension in Liver Transplanted Children Using Hydrochlorothiazide.

Arterial hypertension (AH) is the most common toxic effect of calcineurin inhibitor (CNI)-based immunosuppression in children after liver transplantation (LT). Activation of the renal sodium chloride cotransporter (NCC) by CNIs has been described as a major cause of CNI-induced AH. Thiazides, for example, hydrochlorothiazide (HCTZ), can selectively block the NCC and may ameliorate CNI-induced AH after pediatric LT. From 2005 thru 2015 we conducted a retrospective, single-center analysis of blood pressure in 2 pediatric cohorts (each n = 33) with or without HCTZ in their first year after LT. All patients received CNI-based immunosuppression. According to AAP guidelines, AH was defined as stage 1 and stage 2. Cohort 1 received an HCTZ-containing regimen to target the CNI-induced effect on the NCC, leading to AH. Cohort 2 received standard antihypertensive therapy without HCTZ. In children who have undergone LT and been treated with CNI, AH overall was observed less frequently in cohort 1 vs cohort 2 (31% vs 44%; ns). Moreover, severe AH (stage 2) was significantly lower in cohort 1 vs 2 (1% vs 18%; p < 0.001). Multivariate analysis revealed HCTZ as the only significant factor with a protective effect on occurrence of severe stage 2 AH. While monitoring safety and tolerability, mild asymptomatic hypokalemia was the only adverse effect observed more frequently in cohort 1 vs 2 (27% vs 3%; p = 0.013). Targeting NCC by HCTZ significantly improved control of severe CNI-induced AH and was well tolerated in children who underwent LT. This effect may reduce the risk of long-term end-organ damage and improve quality of life.

The E3 ubiquitin‐protein ligase Nedd4‐2 regulates the sodium chloride cotransporter (NCC) but is not required for a potassium‐induced reduction of NCC expression

Study objectiveThe activity of the sodium chloride cotransporter (NCC) in the kidney distal convoluted is important for kidney sodium (Na+) and potassium (K+) handling. NCC abundance is reduced by high plasms K+ levels. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4‐2 (Nedd4‐2) is involved in regulation of NCC, but its precise role is unclear. The aim of this study was to examine the functional role of Nedd4‐2 on NCC regulation and whether Nedd4‐2 is involved in the high K+ effects on NCC.HypothesisNedd4‐2 regulates NCC membrane abundance by influencing NCC trafficking or degradation and plays an important role in the high K+ induced reduction in NCC.MethodsIn vitro studies were performed using tetracycline inducible MDCKI cell lines stably expressing human NCC combined with Nedd4‐2 knockdown using shRNA. The cell lines were characterized using immunoprecipitation coupled to biotin‐based membrane abundance assays. The effects of normal or high extracellular K+ on NCC in ex vivo isolated mouse kidney tubules from wildtype or Nedd4‐2 knockout (KO) mice were assessed.ResultsIn MDCK cells the levels of NCC and phosphorylated NCC (pNCC) were increased with Nedd4‐2 deletion, and NCC levels on the membrane were elevated. No significant changes were seen for the SPS1‐related proline/alanine‐rich serine‐threonine kinase (SPAK) and phosphorylated SPAK (pSPAK) after Nedd4‐2 knockdown. Deletion of Nedd4‐2 had no effect on the endocytosis rate of NCC, but NCC half‐life was increased. In tubules high K+ reduced total and pNCC regardless of genotype. The levels of the inwardly rectifying potassium channel subunits Kir4.1 and Kir5.1 were increased in Nedd4‐2 KO tubules. High K+ treatment decreased the level of Kir4.1, but not Kir5.1, only when Nedd4‐2 was eliminated. pSPAK was decreased with high K+ treatment regardless of the genotype.ConclusionNedd4‐2 is involved in ubiquitylation of NCC, regulation of NCC membrane abundance and degradation, but it does not play role in the K+ induced inhibition of NCC.

The role of mineralocorticoid receptor in the potassium switch: effect on NCC regulation+

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

Resemblance in Physiological Systems during Phylogeny and Ontogeny

Although it is not certain whether some characteristics of the mammalian fetus/embryo may repeat the adult stage of ancestral vertebrates, comparison of these two time‐dependent processes should provide new insight into the molecular and functional evolution and its significance. Here we discuss two important biological models: 1) Renin‐angiotensin system (RAS) and 2) Urine concentration mechanism.Renin is a hormone and substrate‐dependent enzyme secreted mostly by the juxtaglomerular (JG) cells of the afferent arteriole located at the vascular pole of the glomerulus. Renin acts on renin substrates and forms angiotensin (Ang) I which is subsequently converted to Ang II by Ang converting enzyme (ACE). Renin containing cells evolved early both in phylogeny and ontogeny, whereas, the macula densa (tubule component), and extracellular mesangium appeared at later stages.Renin containing cells are distributed more widely intrarenally and/or extrarenally in early stages during phylogeny and ontogeny. Granulated cells and renin expression become progressively restricted to JG areas with phylogenic and ontogenic advancement. Kidney renin activities tend to be higher in primitive fishes than in more advanced, while the relative level of extrarenal renin expression to that of the kidney is higher in embryos than in more mature chicken and mice. Extra renal renin is noted in primitive vertebrates. Also, in both the mammalian fetus and nonmammalian vertebrates, the vasopressor and vascular action of Ang II evolved early. A single application of ACE inhibitors decreases the basal level of blood pressure and increase plasma renin activity in conscious adult teleost fish. Ang II stimulates, whereas ACE inhibitors inhibit, arteriolar branching in zebrafish. Likewise, the RAS is important during early ontogeny of mammals in vascular growth and development.Distal tubules of freshwater trout show secondary active NaCl cotransport mechanism without accompaniment of water and act as the so‐called “diluting segment.” Teleost fish cannot concentrate urine because they lack structural and functional countercurrent mechanisms. Only birds and mammals can form urine clearly hyperosmotic to plasma and conserve body water. The structure and function of adult quail kidneys have similarities to those of developing rodents. Both have a loop of Henle, consisting of descending limb and thick ascending limb, but they lack the thin ascending limb. Countercurrent urine concentration of adult quail and developing rodents heavily depends on NaCl recycling without participation of urea transport. Moreover, in both adult quail and developing rodent kidneys, aquaporin 2 gene expression and protein are present in the apical membrane of the collecting tubules, but the water conservation effect of neurohypophysial hormones is smaller than that of adult rodent kidneys. Hence, comparing these two time‐dependent processes may provide new insights into the interpretation of existing findings and provide perspective for future investigations.