Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

Identification of an Electrogenic 2Cl-/H+ Exchanger, ClC5, as a Chloride-Secreting Transporter Candidate in Kidney Cyst Epithelium in Tuberous Sclerosis.

Kidney cyst expansion in tuberous sclerosis complex (TSC) or polycystic kidney disease (PKD) requires active secretion of chloride (Cl-) into the cyst lumen. In PKD, Cl- secretion is primarily mediated via the cystic fibrosis transmembrane conductance regulator (CFTR) in principal cells. Kidney cystogenesis in TSC is predominantly composed of type A intercalated cells, which do not exhibit noticeable expression of CFTR. The identity of the Cl--secreting molecule(s) in TSC cyst epithelia remains speculative. RNA-sequencing analysis results were used to examine the expression of FOXi1, the chief regulator of acid base transporters in intercalated cells, along with localization of Cl- channel 5 (ClC5), in various models of TSC. Results from Tsc2+/- mice showed that the expansion of kidney cysts corresponded to the induction of Foxi1 and correlated with the appearance of ClC5 and H+-ATPase on the apical membrane of cyst epithelia. In various mouse models of TSC, Foxi1 was robustly induced in the kidney, and ClC5 and H+-ATPase were expressed on the apical membrane of cyst epithelia. Expression of ClC5 was also detected on the apical membrane of cyst epithelia in humans with TSC but was absent in humans with autosomal dominant PKD or in a mouse model of PKD. These results indicate that ClC5 is expressed on the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in TSC.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

Pathogenesis of Hypertension in Metabolic Syndrome: The Role of Fructose and Salt.

Metabolic syndrome is manifested by visceral obesity, hypertension, glucose intolerance, hyperinsulinism, and dyslipidemia. According to the CDC, metabolic syndrome in the US has increased drastically since the 1960s leading to chronic diseases and rising healthcare costs. Hypertension is a key component of metabolic syndrome and is associated with an increase in morbidity and mortality due to stroke, cardiovascular ailments, and kidney disease. The pathogenesis of hypertension in metabolic syndrome, however, remains poorly understood. Metabolic syndrome results primarily from increased caloric intake and decreased physical activity. Epidemiologic studies show that an enhanced consumption of sugars, in the form of fructose and sucrose, correlates with the amplified prevalence of metabolic syndrome. Diets with a high fat content, in conjunction with elevated fructose and salt intake, accelerate the development of metabolic syndrome. This review article discusses the latest literature in the pathogenesis of hypertension in metabolic syndrome, with a specific emphasis on the role of fructose and its stimulatory effect on salt absorption in the small intestine and kidney tubules.

Metabolic Alkalosis Pathogenesis, Diagnosis, and Treatment: Core Curriculum 2022.

Metabolic alkalosis is a widespread acid-base disturbance, especially in hospitalized patients. It is characterized by the primary elevation of serum bicarbonate and arterial pH, along with a compensatory increase in Pco2 consequent to adaptive hypoventilation. The pathogenesis of metabolic alkalosis involves either a loss of fixed acid or a net accumulation of bicarbonate within the extracellular fluid. The loss of acid may be via the gastrointestinal tract or the kidney, whereas the sources of excess alkali may be via oral or parenteral alkali intake. Severe metabolic alkalosis in critically ill patients-arterial blood pH of 7.55 or higher-is associated with significantly increased mortality rate. The kidney is equipped with sophisticated mechanisms to avert the generation or the persistence (maintenance) of metabolic alkalosis by enhancing bicarbonate excretion. These mechanisms include increased filtration as well as decreased absorption and enhanced secretion of bicarbonate by specialized transporters in specific nephron segments. Factors that interfere with these mechanisms will impair the ability of the kidney to eliminate excess bicarbonate, therefore promoting the generation or impairing the correction of metabolic alkalosis. These factors include volume contraction, low glomerular filtration rate, potassium deficiency, hypochloremia, aldosterone excess, and elevated arterial carbon dioxide. Major clinical states are associated with metabolic alkalosis, including vomiting, aldosterone or cortisol excess, licorice ingestion, chloruretic diuretics, excess calcium alkali ingestion, and genetic diseases such as Bartter syndrome, Gitelman syndrome, and cystic fibrosis. In this installment in the AJKD Core Curriculum in Nephrology, we will review the pathogenesis of metabolic alkalosis; appraise the precipitating events; and discuss clinical presentations, diagnoses, and treatments of metabolic alkalosis.

Kidney Injury in COVID-19: Epidemiology, Molecular Mechanisms and Potential Therapeutic Targets.

As of December 2021, SARS-CoV-2 had caused over 250 million infections and 5 million deaths worldwide. Furthermore, despite the development of highly effective vaccines, novel variants of SARS-CoV-2 continue to sustain the pandemic, and the search for effective therapies for COVID-19 remains as urgent as ever. Though the primary manifestation of COVID-19 is pneumonia, the disease can affect multiple organs, including the kidneys, with acute kidney injury (AKI) being among the most common extrapulmonary manifestations of severe COVID-19. In this article, we start by reflecting on the epidemiology of kidney disease in COVID-19, which overwhelmingly demonstrates that AKI is common in COVID-19 and is strongly associated with poor outcomes. We also present emerging data showing that COVID-19 may result in long-term renal impairment and delve into the ongoing debate about whether AKI in COVID-19 is mediated by direct viral injury. Next, we focus on the molecular pathogenesis of SARS-CoV-2 infection by both reviewing previously published data and presenting some novel data on the mechanisms of cellular viral entry. Finally, we relate these molecular mechanisms to a series of therapies currently under investigation and propose additional novel therapeutic targets for COVID-19.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

New Insights into the Critical Importance of Intratubular Na+/H+ Exchanger 3 and Its Potential Therapeutic Implications in Hypertension.

PURPOSE OF REVIEW: The sodium (Na+) and hydrogen (H+) exchanger 3 (NHE3), known as solute carrier family 9 member 3 (SLC9A3), mediates active transcellular Na+ and bicarbonate reabsorption in the small intestine of the gut and proximal tubules of the kidney. The purpose of this article is to review and discuss recent findings on the critical roles of intestinal and proximal tubule NHE3 in maintaining basal blood pressure (BP) homeostasis and their potential therapeutic implications in the development of angiotensin II (Ang II)-dependent hypertension. RECENT FINDINGS: Recently, our and other laboratories have generated or used novel genetically modified mouse models with whole-body, kidney-specific, or proximal tubule-specific deletion of NHE3 to determine the critical roles and underlying mechanisms of NHE3 in maintaining basal BP homeostasis and the development of Ang II-induced hypertension at the whole-body, kidney, or proximal tubule levels. The new findings demonstrate that NHE3 contributes to about 10 to 15 mmHg to basal blood pressure levels, and that deletion of NHE3 at the whole-kidney or proximal tubule level, or pharmacological inhibition of NHE3 at the kidney level with an orally absorbable NHE3 inhibitor AVE-0657, attenuates ~ 50% of Ang II-induced hypertension in mice. The results support the proof-of-concept hypothesis that NHE3 plays critical roles in physiologically maintaining normal BP and in the development of Ang II-dependent hypertension. Our results also strongly suggest that NHE3 in the proximal tubules of the kidney may be therapeutically targeted to treat poorly controlled hypertension in humans.

SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium.

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl- between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN-), a property that does not correspond with any of the Cl- channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN- permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl- channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN- conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN--selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

A role for tubular Na+/H+ exchanger NHE3 in the natriuretic effect of the SGLT2 inhibitor empagliflozin.

Inhibitors of proximal tubular Na+-glucose cotransporter 2 (SGLT2) are natriuretic, and they lower blood pressure. There are reports that the activities of SGLT2 and Na+-H+ exchanger 3 (NHE3) are coordinated. If so, then part of the natriuretic response to an SGLT2 inhibitor is mediated by suppressing NHE3. To examine this further, we compared the effects of an SGLT2 inhibitor, empagliflozin, on urine composition and systolic blood pressure (SBP) in nondiabetic mice with tubule-specific NHE3 knockdown (NHE3-ko) and wild-type (WT) littermates. A single dose of empagliflozin, titrated to cause minimal glucosuria, increased urinary excretion of Na+ and bicarbonate and raised urine pH in WT mice but not in NHE3-ko mice. Chronic empagliflozin treatment tended to lower SBP despite higher renal renin mRNA expression and lowered the ratio of SBP to renin mRNA, indicating volume loss. This effect of empagliflozin depended on tubular NHE3. In diabetic Akita mice, chronic empagliflozin enhanced phosphorylation of NHE3 (S552/S605), changes previously linked to lesser NHE3-mediated reabsorption. Chronic empagliflozin also increased expression of genes involved with renal gluconeogenesis, bicarbonate regeneration, and ammonium formation. While this could reflect compensatory responses to acidification of proximal tubular cells resulting from reduced NHE3 activity, these effects were at least in part independent of tubular NHE3 and potentially indicated metabolic adaptations to urinary glucose loss. Moreover, empagliflozin increased luminal α-ketoglutarate, which may serve to stimulate compensatory distal NaCl reabsorption, while cogenerated and excreted ammonium balances urine losses of this "potential bicarbonate." The data implicate NHE3 as a determinant of the natriuretic effect of empagliflozin.

Ablation of polyamine catabolic enzymes provokes Purkinje cell damage, neuroinflammation, and severe ataxia.

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

Acute Kidney Injury in SARS-CoV-2 Infection: Direct Effect of Virus on Kidney Proximal Tubule Cells.

Coronaviruses (CoVs), including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and the novel coronavirus disease-2 (SARS-CoV-2) are a group of enveloped RNA viruses that cause a severe respiratory infection which is associated with a high mortality [...].

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells.

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Effect of renal tubule-specific knockdown of the Na+/H+ exchanger NHE3 in Akita diabetic mice.

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

High concentrations of urinary calcium counteract vasopressin action via the activation of the calcium-sensing receptor (CaSR) that is expressed in the luminal membrane of collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). Pendrin/NaCl cotransporter double-knockout (dKO) mice display significant calcium wasting and develop severe volume depletion, despite increased circulating vasopressin levels. We hypothesized that the CaSR-mediated impairment of AQP2 expression/trafficking underlies vasopressin resistance in dKO mice. Compared with wild-type mice, in renal inner medulla, dKO mice had reduced total AQP2 sensitive to proteasome inhibitors, higher levels of AQP2-pS261, ubiquitinated AQP2, and p38-MAPK, an enzyme that is activated by CaSR signaling and known to phosphorylate AQP2 at Ser261. CaSR inhibition with the calcilytic NPS2143 reversed these effects, which indicates that CaSR mediates the up-regulation of AQP2-pS261, ubiquitination, and degradation. Of note, dKO mice demonstrated significantly higher AQP2-targeting miRNA-137 that was reduced upon CaSR inhibition, supporting a critical role for CaSR in the down-regulation of AQP2 expression. Our data indicate that CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the p38-MAPK/AQP2-pS261/ubiquitination/proteasomal axis. These effects may contribute to the reduced renal concentrating ability that has been observed in dKO mice and underscore a physiologic mechanism of the CaSR-dependent regulation of AQP2 abundance via a novel microRNA pathway.-Ranieri, M., Zahedi, K., Tamma, G., Centrone, M., Di Mise, A., Soleimani, M., Valenti, G. CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

Polyamine Catabolism in Acute Kidney Injury.

Acute kidney injury (AKI) refers to an abrupt decrease in kidney function. It affects approximately 7% of all hospitalized patients and almost 35% of intensive care patients. Mortality from acute kidney injury remains high, particularly in critically ill patients, where it can be more than 50%. The primary causes of AKI include ischemia/reperfusion (I/R), sepsis, or nephrotoxicity; however, AKI patients may present with a complicated etiology where many of the aforementioned conditions co-exist. Multiple bio-markers associated with renal damage, as well as metabolic and signal transduction pathways that are involved in the mediation of renal dysfunction have been identified as a result of the examination of models, patient samples, and clinical data of AKI of disparate etiologies. These discoveries have enhanced our ability to diagnose AKIs and to begin to elucidate the mechanisms involved in their pathogenesis. Studies in our laboratory revealed that the expression and activity of spermine/spermidine N1-acetyltransferase (SAT1), the rate-limiting enzyme in polyamine back conversion, were enhanced in kidneys of rats after I/R injury. Additional studies revealed that the expression of spermine oxidase (SMOX), another critical enzyme in polyamine catabolism, is also elevated in the kidney and other organs subjected to I/R, septic, toxic, and traumatic injuries. The maladaptive role of polyamine catabolism in the mediation of AKI and other injuries has been clearly demonstrated. This review will examine the biochemical and mechanistic basis of tissue damage brought about by enhanced polyamine degradation and discuss the potential of therapeutic interventions that target polyamine catabolic enzymes or their byproducts for the treatment of AKI.

Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.

BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.

Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis.

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