Na-Cl Cotransporter Research Articles

WCN23-0205 Cyclosporin A-induced activation of the renal NaCl cotransporter depends on the presence of Kir4.1 in the distal convoluted tubule

WCN23-0205 Cyclosporin A-induced activation of the renal NaCl cotransporter depends on the presence of Kir4.1 in the distal convoluted tubule

Activation of Kir4.1/Kir5.1 contributes to the cyclosporin A-induced stimulation of the renal NaCl cotransporter and hyperkalemic hypertension.

Cyclosporin A (CsA) is a widely used immunosuppressive drug that causes hypertension and hyperkalemia. Moreover, CsA-induced stimulation of the thiazide-sensitive NaCl cotransporter (NCC) in the kidney has been shown to be responsible for the development of hyperkalemic hypertension. In this study, we tested whether CsA induces the activation of NCC by stimulating the basolateral Kir4.1/Kir5.1 channel in the distal convoluted tubule (DCT). Electrophysiology, immunoblotting, metabolic cages, and radio-telemetry methods were used to examine the effects of CsA on Kir4.1/Kir5.1 activity in the DCT, NCC function, and blood pressure in wild-type (WT) and kidney-specific Kir4.1 knockout (KS-Kir4.1 KO) mice. The single-channel patch clamp experiment demonstrated that CsA stimulated the basolateral 40 pS K+ channel in the DCT. Whole-cell recording showed that short-term CsA administration (2h) not only increased DCT K+ currents but also shifted the K+ current (IK ) reversal potential to the negative range (hyperpolarization). Furthermore, CsA administration increased phosphorylated NCC (pNCC) levels and inhibited renal Na+ and K+ excretions in WT mice but not in KS-Kir4.1 KO mice, suggesting that Kir4.1 is required to mediate CsA effects on NCC function. Finally, long-term CsA infusion (14 days) increased blood pressure, plasma K+ concentration, and total NCC or pNCC abundance in WT mice, but these effects were blunted in KS-Kir4.1 KO mice. We conclude that CsA stimulates basolateral K+ channel activity in the DCT and that Kir4.1 is essential for CsA-induced NCC activation and hyperkalemic hypertension.

Structure and thiazide inhibition mechanism of the human Na-Cl cotransporter.

The sodium-chloride cotransporter (NCC) is critical for kidney physiology1. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron2,3, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome4. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years5-7. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na-Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.

The serine-threonine protein phosphatases that regulate the thiazide-sensitive NaCl cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

Kidney and blood pressure regulation-latest evidence for molecular mechanisms.

Hypertension is one of the major health problems leading to the development of cardiovascular diseases. Despite a rapid expansion in global hypertension prevalence, molecular mechanisms leading to hypertension are not fully understood largely due to the complexity of pathogenesis involving several factors. Salt intake is recognized as a leading determinant of blood pressure, since reduced dietary salt intake is related to lower morbidity and mortality, and hypertension in relation to cardiovascular events. Compared with salt-resistant populations, salt-sensitive individuals exhibit high sensitivity in blood pressure responses according to changes in salt intake. In this setting, the kidney plays a major role in the maintenance of blood pressure under the hormonal control of the renin-angiotensin-aldosterone system. In the present review, we summarize the current overview on the molecular mechanisms for modulation of blood pressure associated with renal ion channels/transporters including sodium-hydrogen exchanger isoform 3 (NHE3), Na+-K+-2Cl- cotransporter (NKCC2), sodium-chloride cotransporter (NCC), epithelial sodium channel (ENaC) and pendrin expressed in different nephron segments. In particular, recent studies on experimental animal models with deletion of renal ion channels led to the identification of several crucial physiological mechanisms and molecules involved in hypertension. These findings could further provide a potential for novel therapeutic approaches applicable on human patients with hypertension.

NCC regulation by WNK signal cascade.

With-no-lysine (K) (WNK) kinases have been identified as the causal genes for pseudohypoaldosteronism type II (PHAII), a rare hereditary hypertension condition characterized by hyperkalemia, hyperchloremic metabolic acidosis, and thiazide-hypersensitivity. We thought that clarifying the link between WNK and NaCl cotransporter (NCC) would bring us new mechanism(s) of NCC regulation. For the first time, we were able to produce a knock-in mouse model of PHAII and anti-phosphorylated NCC antibodies against the putative NCC phosphorylation sites and discover that constitutive activation of NCC and increased phosphorylation of NCC are the primary pathogenesis of the disease in vivo. We have since demonstrated that this regulatory mechanism is mediated by the kinases oxidative stress-response protein 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK) (WNK-OSR1/SPAK-NCC signaling cascade) and that the signaling is not only important in the pathological condition of PHAII but also plays a crucial physiological role in the regulation of NCC.

Control of sodium and potassium homeostasis by renal distal convoluted tubules.

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

PS-BPB10-4: CREATION AND CHARACTERIZATION OF THE UBIQUITIN LIGASE KLHL3-S433A KNOCK-IN MICE

Background and aim: Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure and electrolyte homeostasis. Mutation and inactivation of KLHL3 are known to cause hypertension and hyperkalemia mainly due to the impaired with-no-lysine (WNK) degradation and increased Na-Cl cotransporter (NCC) activity in the kidney. Previously, we have reported that angiotensin II (Shibata et al. PNAS 2014), potassium deficiency, and impaired glucose tolerance (Ishizawa et al. BBRC 2016 and JASN2019) can inactivate KLHL3 by altering phosphorylation at S433 in the Kelch-domain. We and others have also shown that decreased KLHL3 expression is also involved in the pathology of hypertension and hyperkalemia. The current study was designed to demonstrate the physiological importance of S433 in KLHL3 function in vivo in mice. Method: Using CRISPR/Cas9, S433 of KLHL3 was substituted to alanine in C57BL/6 mice (KLHL3-S433A Knock in mice: KI). Creation of KI was confirmed by DNA sequencing and RFLP analysis. Blood pressure was evaluated by telemetry, and blood gas was analyzed by iSTAT. Levels of KLHL3, WNK1/4, and NCC in the kidney were determined by Western blot analysis. Gene expression of KLHL3, WNK1 and WNK4 was evaluated by real time RT-PCR. Results: On a normal salt diet, blood pressure levels were similar between WT and KI; however, KI mice showed significant increase in response to a high salt diet compared with WT on the same diet. KI mice showed a significant increase in plasma potassium levels and a significant decrease in bicarbonate levels already at baseline, confirming that S433 in KLHL3 is also critical in regulating potassium and acid/base homeostasis. Urinalysis indicated that the increase in plasma potassium levels is mainly attributable to the decreased potassium excretion. Of interest, we found that total KLHL3 abundance was drastically reduced by Western blot analysis, which was accompanied by the increase in KS-WNK1 and WNK4 and an increase in NCC both. Real time RT-PCR showed that gene expression of Klhl3, as well as those of Wnk1 and Wnk4, was unchanged, indicating that S433A substitution promotes degradation of KLHL3, leading to hyperkalemia and increased salt sensitivity. Conclusion: The study confirmed the critical importance of S433 in KLHL3 in controlling the ubiquitin ligase function and electrolyte homeostasis in vivo. It also indicates that the modification of KLHL3-S433 influences the protein abundance without affecting gene transcription.

Pharmacology of Compounds Targeting Cation-Chloride Cotransporter Physiology.

Transporters of the solute carrier family 12 (SLC12) carry inorganic cations such as Na+ and/or K+ alongside Cl across the plasma membrane of cells. These tightly coupled, electroneutral, transporters are expressed in almost all tissues/organs in the body where they fulfil many critical functions. The family includes two key transporters participating in salt reabsorption in the kidney: the Na-K-2Cl cotransporter-2 (NKCC2), expressed in the loop of Henle, and the Na-Cl cotransporter (NCC), expressed in the distal convoluted tubule. NCC and NKCC2 are the targets of thiazides and "loop" diuretics, respectively, drugs that are widely used in clinical medicine to treat hypertension and edema. Bumetanide, in addition to its effect as a loop diuretic, has recently received increasing attention as a possible therapeutic agent for neurodevelopmental disorders. This chapter also describes how over the past two decades, the pharmacology of Na+ independent transporters has expanded significantly to provide novel tools for research. This work has indeed led to the identification of compounds that are 100-fold to 1000-fold more potent than furosemide, the first described inhibitor of K-Cl cotransport, and identified compounds that possibly directly stimulate the function of the K-Cl cotransporter. Finally, the recent cryo-electron microscopy revolution has begun providing answers as to where and how pharmacological agents bind to and affect the function of the transporters.

PL-4: INSIGHTS INTO MOLECULAR MECHANISMS OF HYPERTENSION FROM HUMAN GENETICS

Identification and investigation of rare patients with extreme forms of high and low blood pressure has led to the identification of specific genes whose mutation results in these outlier phenotypes. These rare mutations converge on genes that mediate or regulate the renal reabsorption of Na-Cl. Mutations that increase or decrease renal salt reabsorption respectively increase or decrease blood pressure. Mutations that cause hypertension include gain of function mutations in subunits of the epithelial Na+ channel (ENaC) and in any of eight genes that cause constitutive activity of ENaC by its stimulation via its major regulator, the mineralocorticoid receptor, the target of aldosterone signaling; these increase salt reabsorption in the distal nephron. Conversely, mutations that cause hypotension include loss of function mutations in ENaC subunits, but also mutations the thiazide-sensitive Na-Cl cotransporter (NCC) or a K+ channel required for cotransporter function that act in the distal convoluted tubule (DCT), or mutations in the Na-K-2Cl cotransporter as well as K+ and Cl- channels expressed in the thick ascending limb of Henle that are required for normal Na-Cl reabsorption in this nephron segment. ENaC, the Na-Cl cotransporter and the Na-K-2Cl cotransporter, as well as the mineralocorticoid receptor are all commonly used targets for treatment of hypertension and/or diuresis. In addition to these genes, genetic studies also identified a previously unrecognized pathway that regulates the balance between renal salt reabsorption and K+ secretion. Mutations in the kinases WNK1 or WNK4, or genes that mediate their degradation via ubiquitylation, CUL3 and KLHL3 cause hypertension with hyperkalemia. Activity of WNK1 and WNK4 stimulates activation of NCC promoting Na-Cl reabsorption in the DCT, reducing Na-Cl- delivery distally, thereby limiting ENaC activity and limiting K+ secretion in the distal nephron. Conversely, hyperkalemia inhibits WNK activity, promoting distal Na-Cl delivery and maximizing K+ secretion. Angiotensin II signaling promotes WNK activity, increasing Na-Cl reabsorption in the DCT and inhibiting K+ secretion, while hyperkalemia appears to inhibit WNK activity, providing distal Na+ delivery that is used to drive K+ secretion. The importance of this pathway is that it can explain the mechanism by which increased dietary K+ lowers blood pressure. Both reduced dietary salt intake and increased dietary K+ can reduce blood pressure in controlled settings, and recent large population studies have shown that increased dietary K+ and reduced Na-Cl intake not only lower blood pressure but reduce the incidence of cardiovascular disease and death.

Dietary potassium intake and blood pressure: possible beneficial effect of Paleolithic diet.

Potassium is one of the most important elements of human organism. Its main distribution in the intracellular space makes it an essential component of intracellular volume regulation on the other hand extracellular/ intracellular potassium ratio determines the resting membrane potential. This later capability of potassium makes essential the necessity of maintaining extracellular potassium levels in a narrow range limit between 3.5 and 5.5 mEq/L in order to avoid dangerous dysfunction of excitable cells such as myocardium, neuronal cells and muscle cells. Because of asymmetric potassium distribution between intracellular and extracellular space there is a continuous need to excrete the excess of potassium ingested by food in a diurnal basis. The main site of potassium excretion is the distal nephron and it is coupled directly with the amount of sodium and solute delivered to this segment of the nephron. Although the very early segment of distal convoluted tubule has no ability to excrete potassium it is capable to regulate the amount of sodium chloride delivered downstream the nephron and by this way is implicated indirectly in potassium excretion. Newer data suggest that this segment of the nephron responds to increased extracellular levels of potassium by reducing sodium chloride cotransporter activity and so increases sodium delivery to distal nephron for exchange with potassium and leads to kaliuresis as well as natriuresis. It is believed that this mechanism is responsible for the beneficial effect of increased potassium intake in ameliorating hypertension even in cases of increased salt intake. In this review article, under the light of recent discoveries, we try to elucidate the complex underlying mechanisms of increased potassium intake and blood pressure regulation as well as the possible beneficial effect of Paleolithic diet upon hypertension.

Hipokalemija - rani marker autozomno recesivne tubulopatije (Gitelmanov sindrom) - prikaz slučaja

Hypokalemia is the most common feature of Gitelman syndrome, which is a rare, inherited, autosomal recessive kidney disease associated with tubule disease. In addition to hypokalemia, it is also characterized by hypomagnesemia, metabolic alkalosis, hyperrenemic hyperaldosteronism, normal or lower blood pressure, while the presence of arterial hypertension does not exclude the diagnosis. It affects men and women equally, with a prevalence of 1 to 10 cases per 40,000 inhabitants. The most common cause are mutations in the SLC12A3 gene, which encodes the thiazidesensitive sodium chloride cotransporter (NCCT) in the renal distal tubules, and the TRPM6 (cation channel subfamily 6 protein claudin 16) gene, which controls distal tubular magnesium transport. The aim of the paper is to present an adult patient with pronounced hypokalemia as part of Gitelman's syndrome. Case report: We present a 21-year-old man with severe hypokalemia as part of Gitelman's syndrome. The disease manifested itself in non-specific complaints, and laboratory findings showed hypokalemia of 2.0 mmol/L, which was the reason for urgent hospitalization. Further examinations of the patient verified the following: hypomagnesemia, hypocalciuria, metabolic alkalosis, preserved kidney function and arterial hypotension. Other potential causes of hypokalemia were excluded by differential diagnosis. He was treated with potassium and magnesium replacement therapy, after which the symptoms of hypokalemia disappeared, and the electrolyte values were closer to the reference values. The diagnosis of Gitelman's syndrome was made based on clinical and laboratory findings. A geneticist was also consulted. Hypokalemia as part of Gitelman's syndrome is rarely encountered in clinical practice, and it is rarely thought of. Severe forms of hypokalemia should arouse suspicion of its existence and lead to a final diagnosis, for which rich clinical experience and teamwork are necessary. The patients with symptoms should be treated symptomatically, and those without symptoms should be monitored 1-2 times a year.

The Expressions of Sodium Chloride Cotransporter (NCC mRNAs) in the Kidney Hypertensive Rats

The Expressions of Sodium Chloride Cotransporter (NCC mRNAs) in the Kidney Hypertensive Rats

S-51-1: SYMPATHETIC REGULATION OF SALT SENSITIVITY

This presentation will cover recent advances relating the role of the sympathetic nervous system in the regulation of the salt sensitivity of blood pressure. Recent studies from our laboratory have elucidated the previously unknown role of PVN specific Galphai2 signal transduction in the prevention of the development of the salt sensitivity of blood pressure. We will discuss these findings and their potential translational relevance. Highlighting the role(s) of the renal sympathetic nerves in the modulation of sodium reabsorption and long-term blood pressure regulation we will highlight recent advances in the role(s) of the afferent renal nerves in the salt sensitivity of blood pressure and how increased renal sympathetic outflow influences the expression and activity of the renal sodium chloride cotransporter.

S-27-6: ADAPTIVE IMMUNITY REGULATES RENAL SALT-RETENTION AND BLOOD PRESSURE

Hypertension is a major public health problem, which contributes to a plethora of other cardiovascular diseases. A long-time postulate is that a defect in the kidney plays a key role in the development of hypertension. Years of intense research have confirmed that distal nephron plays important role in excessive salt retention, which contributes to the pathogenesis of hypertension. Yet the precise identity of this kidney defect and the downstream signaling mechanisms that impair sodium handling remain unclear. In the past decade, T cells, in particular CD8+ T cells (CD8Ts), have garnered attention as an important contributor to hypertension. T cells infiltrate into the kidneys of hypertensive animals, possibly driven by elevated perfusion pressure. Subsequently elevation of blood pressure and salt content stimulate T cell-activation and cytokine production. We recently reported a new mechanism by which renal infiltrated CD8Ts contribute to salt retention and hypertension: CD8Ts upregulate the sodium-chloride cotransporter (NCC) in the renal distal tubules, resulting in excessive salt retention via direct interactions between CD8Ts and renal distal convoluted tubule cells (DCTs) (Image in enclosed figure). This finding raises the exciting prospect that kidney-accumulation of CD8Ts and their interactions with DCTs represent one kidney defect in the pathogenesis of salt-sensitive hypertension. Although blocking NCC using thiazide temporarily lowered blood pressure in hypertensive animals, it failed to diminish T cell-homing to the kidneys, and hypertension returned after withdrawal of thiazide. Thus, in order to design effective therapeutics for salt-sensitive hypertension which interrupt the initial stimuli that trigger renal sodium retention, it is critical for us to define mechanisms of T cell-homing to the kidney and identify the key molecules mediating the CD8T-DCT interaction. Here, we further identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFNγ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFNγ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in two different mouse models. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

S-40-5: ROLE OF INTERFERON GAMMA IN T CELL MEDIATED SALT-RETENTION IN THE KIDNEY AND PROGRESSION OF HYPERTENSION

Objective: In accordance with previous studies implicating the immune system in the development of hypertension, we found that CD8+ T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), to upregulate sodium-chloride co-transporter (NCC) activity, leading to excessive salt retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that gamma interferon (IFNg), released by activated CD8Ts, primes the tubular cell to promote interactions between activated CD8Ts and DCTs during the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Design &amp; Method: In co-culture of mouse CD8Ts and DCTs, recombinant mIFNg, IFNg-neutralizing antibody, and specific siRNAs against the IFNg-receptor or PDL1 were used to determine the role of IFNg-PDL1 pathway in the CD8T-DCT interaction. To validate these studies, we employed IFNg knockout (KO) mice, mice with systemically blocked PDL1 using anti-PDL1 antibody or with renal tubule-specific knockdown of PDL1 (mediated via nanoparticles) in the DOCA-salt model and the CD8T adoptive transfer hypertension models. Blood pressure was monitored using radio-biotelemetry. Endpoint analysis involved flow cytometry, RT-qPCR, and western blot, and immunohistochemistry. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice produce more IFNg and exhibit augmented ability to interact with DCTs compared to those from sham normotensive mice. IFNg stimulation to mDCTs increased expression of CD8-specific antigen presenting molecule MHC-I and CD8T-co-signaling molecule PDL1, which enhanced the interaction between CD8Ts and DCTs, consequently upregulating NCC expression in DCTs, leading to enhanced salt retention. These effects on MHC-I, PDL1, or NCC expression were abolished by neutralizing IFNg in co-culture of DCTs and activated CD8Ts, and knockdown of PDL1 in DCTs blunted NCC expression and sodium retention. In-vivo results verified the in-vitro studies. We found that in both IFNg-KO mice and WT mice receiving PDL1-antagonizing antibodies, DOCA-salt treatment failed to elevate their blood pressure and reduced T cell infiltration and NCC expression within their kidneys compared to WT mice receiving the same treatment (anti-PDL1 results shown in Figure 1). Similar results were observed in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1. Conclusion: In salt-sensitive hypertension, activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNg-PDL1 pathway mediated mechanism. Blocking the IFNg-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo, ameliorating hypertension.

Novel SLC12A3 gene mutations and clinical characteristics in two pedigrees with Gitelman syndrome.

Gitelman syndrome (GS) is an autosomal recessive tubulopathy resulting from inactivating mutations in the SLC12A3 gene that encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). To date, more than 500 mutations have been identified in the SLC12A3 gene. In this study, we identified two new mutations in the SLC12A3 gene in two Chinese GS pedigrees. The clinical characteristics and laboratory examination of two suspected GS patients in our hospital were analyzed. In addition, two pedigrees including 11 members and 2 patients underwent SLC12A3 gene analysis. Both patients were middle-aged women with characteristics of hypokalemic metabolic alkalosis, hypomagnesemia, low level of urinary calcium and the elevated levels of renin-angiotensin-aldosterone system. So, they were clinically diagnosed as GS. Patient 2 also had type 2 diabetes and Graves'disease. Both patients were found to carry two mutations of SLC12A3 gene by Sanger direct sequencing, which were all compound heterozygous mutations. We identified three mutations in these two Chinese GS pedigrees, one of which was c.179C>T (Thr60Met). The novel c.2159G>T (p. Gly720Val) and c.2675T>C (p. Leu892Pro) mutations were strongly predicted to be pathogenic using four network programs-Polyphen-2, SIFT, Mutation Taster and LRT. We identified two novel SLC12A3 genetic variant [c.2159G>T (p.Gly720Val) and c.2675T>C (p.Leu892Pro)] in two Chinese GS pedigrees. The discovery of new mutations has enriched the spectrum of SLC12A3 genotypes.

Molecular Mechanisms of Na-Cl Cotransporter in Relation to Hypertension in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a common clinical disease with an increasing incidence, affecting 10 to 15% of the world's population. Hypertension is the most common and modifiable risk factor for preventing adverse cardiovascular outcomes in patients with CKD. A survey from developed countries shows that 47% of hypertensive patients over the age of 20 have uncontrolled blood pressure (BP), and the control rate is even lower in developing countries. CKD is both a common cause of uncontrolled hypertension and a risk factor for altered sequelae. In particular, studies have demonstrated that abnormal blood-pressure patterns in CKD patients, such as non-dipping-blood-pressure patterns, are associated with a significantly increased risk of cardiovascular (CV) disease. The distal convoluted tubule (DCT) is a region of the kidney, and although only 5-10% of the sodium (Na+) filtered by the glomerulus is reabsorbed by DCT, most studies agree that Na-Cl cotransporter (NCC) in human, rabbit, mouse, and rat kidneys is the most important route of sodium reabsorption across the DCT for maintaining the homeostasis of sodium. The regulation of NCC involves a large and complex network structure, including certain physiological factors, kinases, scaffold proteins, transporter phosphorylation, and other aspects. This regulation network includes various levels. Naturally, cross-talk between the components of this system must occur in order to relay the important signals to the transporter to play its role. Knowledge of the mechanisms regulating NCC activation is critical for understanding and treating hypertension and CKD. Previous studies from our laboratory have investigated the mechanisms through which NCC is activated in several different models. In the following sections, we review the literature on the mechanisms of NCC in relation to hypertension in CKD.

Differential IL18 signaling via IL18 receptor and Na-Cl co-transporter discriminating thermogenesis and glucose metabolism regulation.

White adipose tissue (WAT) plays a role in storing energy, while brown adipose tissue (BAT) is instrumental in the re-distribution of stored energy when dietary sources are unavailable. Interleukin-18 (IL18) is a cytokine playing a role in T-cell polarization, but also for regulating energy homeostasis via the dimeric IL18 receptor (IL18r) and Na-Cl co-transporter (NCC) on adipocytes. Here we show that IL18 signaling in metabolism is regulated at the level of receptor utilization, with preferential role for NCC in brown adipose tissue (BAT) and dominantly via IL18r in WAT. In Il18r-/-Ncc-/- mice, high-fat diet (HFD) causes more prominent body weight gain and insulin resistance than in wild-type mice. The WAT insulin resistance phenotype of the double-knockout mice is recapitulated in HFD-fed Il18r-/- mice, whereas decreased thermogenesis in BAT upon HFD is dependent on NCC deletion. BAT-selective depletion of either NCC or IL18 reduces thermogenesis and increases BAT and WAT inflammation. IL18r deletion in WAT reduces insulin signaling and increases WAT inflammation. In summary, our study contributes to the mechanistic understanding of IL18 regulation of energy metabolism and shows clearly discernible roles for its two receptors in brown and white adipose tissues.