Enfermedades renales que cursan con hipomagnesemia. Comentarios acerca de una nueva tubulopatía hipomagnesémica de origen genético / Renal diseases that course with hypomagnesemia. Comments on a new hereditary hypomagnesemic tubulopathy

Las enfermedades renales que cursan con hipomagnesemia son un grupo complejo y variopinto de tubulopatías producidas por mutaciones en genes que codifican proteínas que se expresan en la rama gruesa ascendente del asa de Henle y en el túbulo contorneado distal. En el presente artículo revisamos la descripción inicial, la expresividad clínica y la etiología de cuatro de las primeras causas de tubulopatías hipomagnesémicas que se describieron: las enfermedades de Bartter tipo 3 y Gitelman, la hipomagnesemia con hipocalcemia secundaria autosómica recesiva y la hipomagnesemia familiar con hipercalciuria y nefrocalcinosis. A continuación, se describen los patrones bioquímicos básicos que se observan en las hipomagnesemias tubulares renales y las modalidades de transporte e interacción que concurren entre los transportadores implicados en la reabsorción de magnesio en el túbulo contorneado distal. Finalmente, se comunica la reciente descripción de una nueva tubulopatía hipomagnesémica, la hipomagnesemia con hipocalcemia secundaria tipo 2 causada por una reducción de la actividad del canal TRPM7 (AU)

Renal diseases associated with hypomagnesemia are a complex and diverse group of tubulopathies caused by mutations in genes encoding proteins that are expressed in the thick ascending limb of the loop of Henle and in the distal convoluted tubule. In this paper, we review the initial description, the clinical expressiveness and etiology of four of the first hypomagnesemic tubulopathies described: Type 3 Bartter and Gitelman diseases,Autosomal recessive hypomagnesemia with secondary hypocalcemia and Familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The basic biochemical patterns observed in renal tubular hypomagnesemias and the modalities of transport and interaction that occur between the transporters involved in the reabsorption of magnesium in the distal convoluted tubule are described below. Finally, the recent report of a new renal disease with hypomagnesemia, Type 2 hypomagnesemia with secondary hypocalcemia caused by reduced TRPM7 channel activity is described (AU)

Tamm-Horsfall protein in humane urine: sex-dependent differences in the excretion and N-glycosylation pattern.

Tamm-Horsfall protein (THP) is a highly N-glycosylated protein from epithelial cells of the ascending limb of Henle loop. It is secreted into the urine as part of the innate immune response against uropathogenic pathogens. As women are more likely to suffer from urinary tract infections, biomedical studies were conducted to investigate sex-differences in THP excretion, as well as differences in the THP N-glycosylation pattern. A total of 238 volunteers (92 men, 146 women, 69 with hormonal contraceptives) participated in this study, providing urine samples. Women showed a clear tendency to have higher THP concentration and excretion rates than men (p < 0.16). Regular intake of hormonal contraceptives had no significant influence on urinary THP concentration compared to no regular intake. The individual N-glycosylation pattern of THP in urine samples from randomly selected individuals (10 female, 10 male) was investigated after enzymatic release and MS analysis of the oligosaccharides. Female subjects tended to have an increased proportion of oligomannose type N-glycans and non-fucosylated glycans, whereas men had an increased proportion of fucosylated complex-type glycans. The higher level of oligomannose-type glycans in THP from women might be explained by a self-defence mechanism to overcome the higher infections pressure by the female anatomical properties.

Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle.

The Na+/K+/2Cl- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. cGMP, the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3 ubiquitin ligase families, the cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesized that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, cullin-2, cullin-3, cullin-4A, and cullin-5 were expressed at the protein level, whereas the other members of the cullin family were expressed at the mRNA level, in rat TALs. CRL complex activity is regulated by neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, a process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2 while increasing the expression of cullin-1 by threefold, but this effect was not seen with other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.NEW & NOTEWORTHY The Na+/K+/2Cl- cotransporter (NKCC2) reabsorbs NaCl by the thick ascending limb. Nitric oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs by increasing the second messenger cGMP. The present findings indicate that cGMP increases NKCC2 ubiquitination via a cullin-RING ligase complex and regulates in part surface NKCC2 levels. Identifying the E3 ubiquitin ligases that regulate NKCC2 expression and activity may provide new targets for the development of specific loop diuretics.

Molecular mechanisms underlying paracellular calcium and magnesium reabsorption in the proximal tubule and thick ascending limb.

Calcium and magnesium are the most abundant divalent cations in the body. The plasma level is controlled by coordinated interaction between intestinal absorption, reabsorption in the kidney, and, for calcium at least, bone storage and exchange. The kidney adjusts urinary excretion of these ions in response to alterations in their systemic concentration. Free ionized and anion-complexed calcium and magnesium are filtered at the glomerulus. The majority (i.e., >85%) of filtered divalent cations are reabsorbed via paracellular pathways from the proximal tubule and thick ascending limb (TAL) of the loop of Henle. Interestingly, the largest fraction of filtered calcium is reabsorbed from the proximal tubule (65%), while the largest fraction of filtered magnesium is reclaimed from the TAL (60%). The paracellular pathways mediating these fluxes are composed of tight junctional pores formed by claudins. In the proximal tubule, claudin-2 and claudin-12 confer calcium permeability, while the exact identity of the magnesium pore remains to be determined. Claudin-16 and claudin-19 contribute to the calcium and magnesium permeable pathway in the TAL. In this review, we discuss the data supporting these conclusions and speculate as to why there is greater fractional calcium reabsorption from the proximal tubule and greater fractional magnesium reabsorption from the TAL.

Olfactomedin 4 as a novel loop of Henle-specific acute kidney injury biomarker.

Acute kidney injury (AKI) is associated with morbidity and mortality. Urinary biomarkers may disentangle its clinical heterogeneity. Olfactomedin 4 (OLFM4) is a secreted glycoprotein expressed in stressed neutrophils and epithelial cells. In septic mice, OLFM4 expression localized to the kidney's loop of Henle (LOH) and was detectable in the urine. We hypothesized that urine OLFM4 (uOLFM4) will be increased in patients with AKI and sepsis. Urine from critically ill pediatric patients was obtained from a prospective study based on AKI and sepsis status. uOLFM4 was quantified with a Luminex immunoassay. AKI was defined by KDIGO severe criteria. Sepsis status was extracted from the medical record based on admission diagnosis. Immunofluorescence on pediatric kidney biopsies was performed with NKCC2, uromodulin and OLFM4 specific antibodies. Eight patients had no sepsis, no AKI; 7 had no sepsis but did have AKI; 10 had sepsis, no AKI; 11 had sepsis and AKI. Patients with AKI had increased uOLFM4 compared to no/stage 1 AKI (p = 0.044). Those with sepsis had increased uOLFM4 compared to no sepsis (p = 0.026). uOLFM4 and NGAL were correlated (r2 0.59, 95% CI 0.304-0.773, p = 0.002), but some patients had high uOLFM4 and low NGAL, and vice versa. Immunofluorescence on kidney biopsies demonstrated OLFM4 colocalization with NKCC2 and uromodulin, suggesting expression in the thick ascending LOH (TALH). We conclude that AKI and sepsis are associated with increased uOLFM4. uOLFM4 and NGAL correlated in many patients, but was poor in others, suggesting these markers may differentiate AKI subgroups. Given OLFM4 colocalization to human TALH, we propose OLFM4 may be a LOH-specific AKI biomarker.

Unrecognized role of claudin-10b in basolateral membrane infoldings of the thick ascending limb.

Claudin-10b is an important component of the tight junction in the thick ascending limb (TAL) of Henle's loop and allows paracellular sodium transport. In immunofluorescence stainings, claudin-10b-positive cells exhibited extensive extra staining of basolateral, column-like structures. The precise localization and function have so far remained elusive. In isolated cortical TAL segments from C57BL/6J mice, kidney-specific claudin-10 knockout mice (cKO), and respective litter mates (WT), we investigated the localization and protein expression and function by fluorescence microscopy and electrophysiological measurements. Ultrastructural analysis of TAL in kidney sections was performed by electron microscopy. Claudin-10b colocalized with the basolateral Na+ -K+ ATPase and the Cl- channel subunit barttin, but the lack of claudin-10b did not influence the localization or abundance of these proteins. However, the accessibility of the basolateral infolded extracellular space to ouabain or fluorescein was increased by basolateral Ca2+ removal and in the absence of claudin-10b. Ultrastructural analysis by electron microscopy revealed a widening of basolateral membrane infoldings in cKO in comparison to WT. We hypothesize that claudin-10b shapes neighboring membrane invaginations by trans interaction to stabilize and facilitate high-flux salt transport in a water-tight epithelium.

Anatomophysiology of the Henle's Loop: Emphasis on the Thick Ascending Limb.

The loop of Henle plays a variety of important physiological roles through the concerted actions of ion transport systems in both its apical and basolateral membranes. It is involved most notably in extracellular fluid volume and blood pressure regulation as well as Ca2+ , Mg2+ , and acid-base homeostasis because of its ability to reclaim a large fraction of the ultrafiltered solute load. This nephron segment is also involved in urinary concentration by energizing several of the steps that are required to generate a gradient of increasing osmolality from cortex to medulla. Another important role of the loop of Henle is to sustain a process known as tubuloglomerular feedback through the presence of specialized renal tubular cells that lie next to the juxtaglomerular arterioles. This article aims at describing these physiological roles and at discussing a number of the molecular mechanisms involved. It will also report on novel findings and uncertainties regarding the realization of certain processes and on the pathophysiological consequences of perturbed salt handling by the thick ascending limb of the loop of Henle. Since its discovery 150 years ago, the loop of Henle has remained in the spotlight and is now generating further interest because of its role in the renal-sparing effect of SGLT2 inhibitors. © 2022 American Physiological Society. Compr Physiol 12:1-21, 2022.

Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Bartter and Gitelman Syndromes: A Primer for Clinicians.

Gitelman and Bartter syndromes are rare inherited diseases that belong to the category of renal tubulopathies. The genes associated with these pathologies encode electrolyte transport proteins located in the nephron, particularly in the Distal Convoluted Tubule and Ascending Loop of Henle. Therefore, both syndromes are characterized by alterations in the secretion and reabsorption processes that occur in these regions. Patients suffer from deficiencies in the concentration of electrolytes in the blood and urine, which leads to different systemic consequences related to these salt-wasting processes. The main clinical features of both syndromes are hypokalemia, hypochloremia, metabolic alkalosis, hyperreninemia and hyperaldosteronism. Despite having a different molecular etiology, Gitelman and Bartter syndromes share a relevant number of clinical symptoms, and they have similar therapeutic approaches. The main basis of their treatment consists of electrolytes supplements accompanied by dietary changes. Specifically for Bartter syndrome, the use of non-steroidal anti-inflammatory drugs is also strongly supported. This review aims to address the latest diagnostic challenges and therapeutic approaches, as well as relevant recent research on the biology of the proteins involved in disease. Finally, we highlight several objectives to continue advancing in the characterization of both etiologies.

Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs.

Dahl salt-sensitive (SS) rat kidneys produce less nitric oxide (NO) than those of salt-resistant (SR) rats. Thick ascending limb (TAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in TALs from SS rats primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal-salt (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused TALs and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35 ± 6 arbitrary units (AU)/min in TALs from SR rats but only 11 ± 2 AU/min in TALs from SS (P < 0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (P < 0.020). The NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) decreased flow-induced superoxide by 36 ± 8% in TALs from SS rats (P < 0.02) but had no effect in TALs from SR rats. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR rats: 44 ± 10 vs. SS: 9 ± 2 AU/min, P < 0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (P < 0.012). l-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in TALs from SR rats but not in TALs from SS rats (P < 0.003). We conclude that 1) on NS, flow-induced NO is diminished in TALs from SS rats mainly due to NOS3 uncoupling such that it produces superoxide and 2) on HS, the difference is enhanced due to failure of TALs from SS rats to increase NOS3 expression.NEW & NOTEWORTHY The Dahl rat has been used extensively to study the causes and effects of salt-sensitive hypertension. Our study suggests that more complex processes other than simple scavenging of nitric oxide (NO) by superoxide lead to less NO production in thick ascending limbs of the Dahl salt-sensitive rat. The predominant mechanism involved depends on dietary salt. Impaired flow-induced NO production in thick ascending limbs most likely contributes to the Na+ retention associated with salt-sensitive hypertension.

Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle.

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl- (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

[Role of distal nephron in the control of extracellular volume in physiology and in nephrotic syndrome]. / Rôle du néphron distal dans le contrôle du volume extracellulaire en condition physiologique et dans le syndrome néphrotique.

The kidney plays a major role to maintain the constancy of the "milieu intérieur" by adjusting the urinary excretion of water and solutes to the requirement of the body balance. This function is coordinated with elimination of waste products generated among others by the catabolism of proteins and nucleic acids. To cope with these two major functions, the human kidneys generate each day about 180 L of ultrafiltrate from plasma and reabsorbs the vast majority of filtered water and solutes to excrete daily about one-two liter(s) of urine containing concentrations of sodium, potassium and chloride ranging from 20 to 200 mM. The final adjustment of urine composition is finely tuned along the aldosterone-sensitive distal nephron (ASDN) which includes the distal convoluted tubule and the collecting system (connecting tubule and collecting duct). Sodium reabsorption is predominant along the distal tubule if potassium must be spared, or along the collecting system when large amounts of potassium must be secreted. Nephrotic syndrome is characterized by heavy proteinuria consecutive to a glomerular injury, associated with renal sodium and water retention taking initially place along ASDN and leading to edema.

TITLE: Rôle du néphron distal dans le contrôle du volume extracellulaire en condition physiologique et dans le syndrome néphrotique. ABSTRACT: Les reins jouent un rôle majeur dans le maintien de la composition du milieu intérieur. Cette fonction est coordonnée avec l'élimination des déchets du métabolisme, impliquant la production par les reins d'environ 180 litres de filtrat par jour et la réabsorption de la grande majorité de l'eau et des solutés filtrés. L'ajustement final de la composition de l'urine est réalisé dans le segment distal sensible à l'aldostérone (ASDN), qui inclut le tube contourné distal et le système collecteur. La réabsorption de sodium prédomine dans le tube distal si le potassium doit être épargné, ou dans le système collecteur si le potassium doit être sécrété. Le syndrome néphrotique est caractérisé par une protéinurie massive causée par des lésions glomérulaires, associée à une rétention hydrosodée prenant place dans l'ASDN et induisant chez le patient des œdèmes parfois volumineux.

Thick ascending limb claudins are altered to increase calciuria and magnesiuria in metabolic acidosis.

Urinary calcium and magnesium wasting is a characteristic feature of metabolic acidosis, and this study focused on the role of the thick ascending limb of Henle's loop in metabolic acidosis-induced hypercalciuria and hypermagnesiuria because thick ascending limb is an important site of paracellular calcium and magnesium reabsorption. Male Sprague-Dawley rats were used to determine the effects of acid loading (by adding NH4Cl, 7.2 mmol/220 g body wt/day to food slurry for 7 days) on renal expression of claudins and then to evaluate whether the results were reversed by antagonizing calcium-sensing receptor (using NPS-2143). At the end of each animal experiment, the kidneys were harvested for immunoblotting, immunofluorescence microscopy, and quantitative PCR (qPCR) analysis of claudins and the calcium-sensing receptor. As expected, NH4Cl loading lowered urinary pH and increased excretion of urinary calcium and magnesium. In NH4Cl-loaded rats, renal protein and mRNA expression of claudin-16, and claudin-19, were decreased compared with controls. However, claudin-14 protein and mRNA increased in NH4Cl-loaded rats. Consistently, the calcium-sensing receptor protein and mRNA were up-regulated in NH4Cl-loaded rats. All these changes were reversed by NPS-2143 coadministration and were confirmed using immunofluorescence microscopy. Hypercalciuria and hypermagnesiuria in NH4Cl-loaded rats were significantly ameliorated by NPS-2143 coadministration as well. We conclude that in metabolic acidosis, claudin-16 and claudin-19 in the thick ascending limb are down-regulated to produce hypercalciuria and hypermagnesiuria via the calcium-sensing receptor.NEW & NOTEWORTHY This study found that the thick ascending limb of Henle's loop is involved in the mechanisms of hypercalciuria and hypermagnesiuria in metabolic acidosis. Specifically, expression of claudin-16/19 and claudin-14 was altered via up-regulation of calcium-sensing receptor in NH4Cl-induced metabolic acidosis. Our novel findings contribute to understanding the regulatory role of paracellular tight junction proteins in the thick ascending limb.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Molecular characteristics and physiological roles of Na+ -K+ -Cl- cotransporter 2.

Na+ -K+ -Cl- cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na+ , K+ , and Cl- ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+ , Cl- , Ca2+ , Mg2+ , and HCO3- loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na+ -K+ -Cl- cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses.

Lipopolysaccharide directly inhibits bicarbonate absorption by the renal outer medullary collecting duct.

Acidosis is associated with E. coli induced pyelonephritis but whether bacterial cell wall constituents inhibit HCO3 transport in the outer medullary collecting duct from the inner stripe (OMCDi) is not known. We examined the effect of lipopolysaccharide (LPS), on HCO3 absorption in isolated perfused rabbit OMCDi. LPS caused a ~ 40% decrease in HCO3 absorption, providing a mechanism for E. coli pyelonephritis-induced acidosis. Monophosphoryl lipid A (MPLA), a detoxified TLR4 agonist, and Wortmannin, a phosphoinositide 3-kinase inhibitor, prevented the LPS-mediated decrease, demonstrating the role of TLR4-PI3-kinase signaling and providing proof-of-concept for therapeutic interventions aimed at ameliorating OMCDi dysfunction and pyelonephritis-induced acidosis.

Metabolic acidosis and hyperkalemia differentially regulate cation HCN3 channel in the rat nephron.

The kidney controls body fluids, electrolyte and acid-base balance. Previously, we demonstrated that hyperpolarization-activated and cyclic nucleotide-gated (HCN) cation channels participate in ammonium excretion in the rat kidney. Since acid-base balance is closely linked to potassium metabolism, in the present work we aim to determine the effect of chronic metabolic acidosis (CMA) and hyperkalemia (HK) on protein abundance and localization of HCN3 in the rat kidney. CMA increased HCN3 protein level only in the outer medulla (2.74 ± 0.31) according to immunoblot analysis. However, immunofluorescence assays showed that HCN3 augmented in cortical proximal tubules (1.45 ± 0.11) and medullary thick ascending limb of Henle's loop (4.48 ± 0.45) from the inner stripe of outer medulla. HCN3 was detected in brush border membranes (BBM) and mitochondria of the proximal tubule by immunogold electron and confocal microscopy in control conditions. Acidosis did not alter HCN3 levels in BBM and mitochondria but augmented them in lysosomes. HCN3 was also immuno-detected in mitoautophagosomes. In the distal nephron, HCN3 was expressed in principal and intercalated cells from cortical to medullary collecting ducts. CMA did not change HCN3 abundance in these nephron segments. In contrast, HK doubled HCN3 level in cortical collecting ducts and favored its basolateral localization in principal cells from the inner medullary collecting ducts. These findings further support HCN channels contribution to renal acid-base and potassium balance.

Colectomía derecha robótica con escisión completa del mesocolon y linfadenectomía D3 mediante abordaje suprapúbico «bottom-to-up»: aspectos técnicos clave / Robotic right colectomy with Complete Mesocolic Excision (CME) and D3 lymphadenectomy with a suprapubic bottom-to-up approach: technical key points

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Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka.

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 µM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

Multiple acid-base and electrolyte disturbances upregulate NBCn1, NBCn2, IRBIT and L-IRBIT in the mTAL.

KEY POINTS: The roles of the Na+ /HCO3- cotransporters NBCn1 and NBCn2 as well as their activators IRBIT and L-IRBIT in the regulation of the mTAL transport of NH4+ , HCO3- , and NaCl are investigated. Dietary challenges of NH4 Cl, NaHCO3 or NaCl all increase the abundance of NBCn1 and NBCn2 in the outer medulla. The three challenges generally produce parallel increases in the abundance of IRBIT and L-IRBIT in the outer medulla. Both IRBIT and L-IRBIT powerfully stimulate the activities of the mTAL isoforms of NBCn1 and NBCn2 as expressed in Xenopus oocytes. Our findings support the hypothesis that NBCn1, NBCn2, IRBIT and L-IRBIT appropriately promote NH4+ shunting but oppose HCO3- and NaCl reabsorption in the mTAL, and thus are at the nexus of the regulation pathways for multiple renal transport processes. ABSTRACT: The medullary thick ascending limb (mTAL) plays a key role in urinary acid and NaCl excretion. NBCn1 and NBCn2 are present in the basolateral mTAL, where NBCn1 promotes NH4+ shunting. IRBIT and L-IRBIT (the IRBITs) are two powerful activators of certain acid-base transporters. Here we use western blotting and immunofluorescence to examine the effects of multiple acid-base and electrolyte disturbances on expression of NBCn1, NBCn2 and the IRBITs in rat kidney. We also use electrophysiology to examine the functional effects of IRBITs on NBCn1 and NBCn2 in Xenopus oocytes. NH4 Cl-induced metabolic acidosis (MAc) substantially increases protein expression of NBCn1 and NBCn2 in the outer medulla (OM) of rat kidney. Surprisingly, NaHCO3 -induced metabolic alkalosis (MAlk) and high-salt diet (HSD) also increase expression of NBCn1 and NBCn2 (effect of NaHCO3  > HSD). Moreover, all three challenges generally increase OM expression of the IRBITs. In Xenopus oocytes, the IRBITs substantially increase the activities of NBCn1 and NBCn2. We propose that upregulation of basolateral NBCn1 and NBCn2 plus the IRBITs in the mTAL: (1) promotes NH4+ shunting by increasing basolateral HCO3- uptake to neutralize apical NH4+ uptake during MAc; (2) inhibits HCO3- reabsorption during MAlk by opposing HCO3- efflux via the basolateral anion exchanger AE2; and (3) inhibits NaCl reabsorption by mediating (with AE2) net NaCl backflux into the mTAL cell during HSD. Thus, NBCn1, NBCn2 and the IRBITs are at the nexus of the regulatory pathways for multiple renal transport processes.