Hypomagnesemia, Hypocalcemia, and Tubulointerstitial Nephropathy Caused by Claudin-16 Autoantibodies.

BACKGROUND: Chronic hypomagnesemia is commonly due to diarrhea, alcoholism, and drugs. More rarely, it is caused by genetic defects in the effectors of renal magnesium reabsorption. METHODS: In an adult patient with acquired severe hypomagnesemia, hypocalcemia, tubulointerstitial nephropathy, and rapidly progressing kidney injury, similarities between the patient's presentation and features of genetic disorders of renal magnesium transport prompted us to investigate whether the patient had an acquired autoimmune cause of renal magnesium wasting. To determine if the patient's condition might be explained by autoantibodies directed against claudin-16 or claudin-19, transmembrane paracellular proteins involved in renal magnesium absorption, we conducted experiments with claudin knockout mice and transfected mouse kidney cells expressing human claudin-16 or claudin-19. We also examined effects on renal magnesium handling in rats given intravenous injections of IgG purified from sera from the patient or controls. RESULTS: Experiments with the knockout mice and in vitro transfected cells demonstrated that hypomagnesemia in the patient was causally linked to autoantibodies directed against claudin-16, which controls paracellular magnesium reabsorption in the thick ascending limb of Henle's loop. Intravenous injection of IgG purified from the patient's serum induced a marked urinary waste of magnesium in rats. Immunosuppressive treatment combining plasma exchange and rituximab was associated with improvement in the patient's GFR, but hypomagnesemia persisted. The patient was subsequently diagnosed with a renal carcinoma that expressed a high level of claudin-16 mRNA. CONCLUSIONS: Pathogenic claudin-16 autoantibodies represent a novel autoimmune cause of specific renal tubular transport disturbances and tubulointerstitial nephropathy. Screening for autoantibodies targeting claudin-16, and potentially other magnesium transporters or channels in the kidney, may be warranted in patients with acquired unexplained hypomagnesemia.

Dysfunction of the circadian clock in the kidney tubule leads to enhanced kidney gluconeogenesis and exacerbated hyperglycemia in diabetes.

The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes.

Diversity of functional alterations of the ClC-5 exchanger in the region of the proton glutamate in patients with Dent disease 1.

Mutations in the CLCN5 gene encoding the 2Cl- /1H+ exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low-molecular-weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells, where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the "proton glutamate" that serves as a crucial H+ -binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for the second group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations might alter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.

Chemically based transmissible ER stress protocols are unsuitable to study cell-to-cell UPR transmission.

Renal epithelial cells regulate the destructive activity of macrophages and participate in the progression of kidney diseases. Critically, the Unfolded Protein Response (UPR), which is activated in renal epithelial cells in the course of kidney injury, is required for the optimal differentiation and activation of macrophages. Given that macrophages are key regulators of renal inflammation and fibrosis, we suppose that the identification of mediators that are released by renal epithelial cells under Endoplasmic Reticulum (ER) stress and transmitted to macrophages is a critical issue to address. Signals leading to a paracrine transmission of ER stress (TERS) from a donor cell to a recipient cells could be of paramount importance to understand how ER-stressed cells shape the immune microenvironment. Critically, the vast majority of studies that have examined TERS used thaspigargin as an inducer of ER stress in donor cells in cellular models. By using multiple sources of ER stress, we evaluated if human renal epithelial cells undergoing ER stress can transmit the UPR to human monocyte-derived macrophages and if such TERS can modulate the inflammatory profiles of these cells. Our results indicate that carry-over of thapsigargin is a confounding factor in chemically based TERS protocols classically used to induce ER Stress in donor cells. Hence, such protocols are not suitable to study the TERS phenomenon and to identify its mediators. In addition, the absence of TERS transmission in more physiological models of ER stress indicates that cell-to-cell UPR transmission is not a universal feature in cultured cells.

Analysis of CLCNKB mutations at dimer-interface, calcium-binding site, and pore reveals a variety of functional alterations in ClC-Kb channel leading to Bartter syndrome.

Pathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC-Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC-Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH-sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH-sensitivity, p.A254V abolishes the calcium-sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties.

Defective bicarbonate reabsorption in Kir4.2 potassium channel deficient mice impairs acid-base balance and ammonia excretion.

The kidneys excrete the daily acid load mainly by generating and excreting ammonia but the underlying molecular mechanisms are not fully understood. Here we evaluated the role of the inwardly rectifying potassium channel subunit Kir4.2 (Kcnj15 gene product) in this process. In mice, Kir4.2 was present exclusively at the basolateral membrane of proximal tubular cells and disruption of Kcnj15 caused a hyperchloremic metabolic acidosis associated with a reduced threshold for bicarbonate in the absence of a generalized proximal tubule dysfunction. Urinary ammonium excretion rates in Kcnj15- deleted mice were inappropriate to acidosis under basal and acid-loading conditions, and not related to a failure to acidify urine or a reduced expression of ammonia transporters in the collecting duct. In contrast, the expression of key proteins involved in ammonia metabolism and secretion by proximal cells, namely the glutamine transporter SNAT3, the phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase enzymes, and the sodium-proton exchanger NHE-3 was inappropriate in Kcnj15-deleted mice. Additionally, Kcnj15 deletion depolarized the proximal cell membrane by decreasing the barium-sensitive component of the potassium conductance and caused an intracellular alkalinization. Thus, the Kir4.2 potassium channel subunit is a newly recognized regulator of proximal ammonia metabolism. The kidney consequences of its loss of function in mice support the proposal for KCNJ15 as a molecular basis for human isolated proximal renal tubular acidosis.

A novel CLCN5 pathogenic mutation supports Dent disease with normal endosomal acidification.

Dent disease is an X-linked recessive renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. Inactivating mutations of CLCN5, the gene encoding the 2Cl- /H+ exchanger ClC-5, have been reported in patients with Dent disease 1. In vivo studies in mice harboring an artificial mutation in the "gating glutamate" of ClC-5 (c.632A > C, p.Glu211Ala) and mathematical modeling suggest that endosomal chloride concentration could be an important parameter in endocytosis, rather than acidification as earlier hypothesized. Here, we described a novel pathogenic mutation affecting the "gating glutamate" of ClC-5 (c.632A>G, p.Glu211Gly) and investigated its molecular consequences. In HEK293T cells, the p.Glu211Gly ClC-5 mutant displayed unaltered N-glycosylation and normal plasma membrane and early endosomes localizations. In Xenopus laevis oocytes and HEK293T cells, we found that contrasting with wild-type ClC-5, the mutation abolished the outward rectification, the sensitivity to extracellular H+ and converted ClC-5 into a Cl- channel. Investigation of endosomal acidification in HEK293T cells using the pH-sensitive pHluorin2 probe showed that the luminal pH of cells expressing a wild-type or p.Glu211Gly ClC-5 was not significantly different. Our study further confirms that impaired acidification of endosomes is not the only parameter leading to defective endocytosis in Dent disease 1.

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock.

Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. To decipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysis performed on control mice and mice with an inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that approximately 30% of RNAs, approximately 20% of proteins, and approximately 20% of metabolites are rhythmic in the kidneys of control mice. Several key metabolic pathways, including NAD+ biosynthesis, fatty acid transport, carnitine shuttle, and ß-oxidation, displayed impairments in kidneys of cKOt mice, resulting in perturbed mitochondrial activity. Carnitine reabsorption from primary urine was one of the most affected processes with an approximately 50% reduction in plasma carnitine levels and a parallel systemic decrease in tissue carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.

Renal tubular peroxisomes are dispensable for normal kidney function.

Peroxisomes are specialized cellular organelles involved in a variety of metabolic processes. In humans, mutations leading to complete loss of peroxisomes cause multiorgan failure (Zellweger's spectrum disorders, ZSD), including renal impairment. However, the (patho)physiological role of peroxisomes in the kidney remains unknown. We addressed the role of peroxisomes in renal function in mice with conditional ablation of peroxisomal biogenesis in the renal tubule (cKO mice). Functional analyses did not reveal any overt kidney phenotype in cKO mice. However, infant male cKO mice had lower body and kidney weights, and adult male cKO mice exhibited substantial reductions in kidney weight and kidney weight/body weight ratio. Stereological analysis showed an increase in mitochondria density in proximal tubule cells of cKO mice. Integrated transcriptome and metabolome analyses revealed profound reprogramming of a number of metabolic pathways, including metabolism of glutathione and biosynthesis/biotransformation of several major classes of lipids. Although this analysis suggested compensated oxidative stress, challenge with high-fat feeding did not induce significant renal impairments in cKO mice. We demonstrate that renal tubular peroxisomes are dispensable for normal renal function. Our data also suggest that renal impairments in patients with ZSD are of extrarenal origin.

Cell stress response impairs de novo NAD+ biosynthesis in the kidney.

The biosynthetic routes leading to de novo nicotinamide adenine dinucleotide (NAD+) production are involved in acute kidney injury (AKI), with a critical role for quinolinate phosphoribosyl transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. The molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD), are unknown. We demonstrate that a high urinary quinolinate-to-tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiac surgery and is predictive of progression to CKD in kidney transplant recipients. We also provide evidence that the endoplasmic reticulum (ER) stress response may impair de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This finding may lead to identification of noninvasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.

Impaired fatty acid metabolism perpetuates lipotoxicity along the transition to chronic kidney injury.

Energy metabolism failure in proximal tubule cells (PTCs) is a hallmark of chronic kidney injury. We combined transcriptomic, metabolomic, and lipidomic approaches in experimental models and patient cohorts to investigate the molecular basis of the progression to chronic kidney allograft injury initiated by ischemia/reperfusion injury (IRI). The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was substantially enriched with long chain fatty acids (FAs). We identified a renal FA-related gene signature with low levels of carnitine palmitoyltransferase 2 (Cpt2) and acyl-CoA synthetase medium chain family member 5 (Acsm5) and high levels of acyl-CoA synthetase long chain family member 4 and 5 (Acsl4 and Acsl5) associated with IRI, transition to chronic injury, and established chronic kidney disease in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-Acsl4+Acsl5+Acsm5- PTCs failing to recover from IRI as identified by single-nucleus RNA-Seq. In vitro experiments indicated that ER stress contributed to CPT2 repression, which, in turn, promoted lipids' accumulation, drove profibrogenic epithelial phenotypic changes, and activated the unfolded protein response. ER stress through CPT2 inhibition and lipid accumulation engaged an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule, sustaining the progression to chronic kidney allograft injury.

Renal Function Decline With Small Interfering RNA Silencing Aminolevulinic Acid Synthase 1 (ALAS1).

INTRODUCTION: Givosiran is an RNA interference therapeutic designed to block the synthesis of the aminolevulinic acid (ALA) synthase 1 (ALAS1) enzyme in patients with acute intermittent porphyria (AIP). Givosiran may have adverse effects on the kidney. METHODS: We performed a descriptive case series of renal function parameters of all the patients who received givosiran in France. Twenty patients receiving givosiran between March 2018 and July 2020 in France were analyzed: 7 patients in the ENVISION trial and 13 patients treated in collaboration with the Centre de Référence Maladies Rares Prophyries. RESULTS: A transient decrease in renal function was observed in all but 2 patients (90%) within the 3 months following givosiran initiation. None of the patients developed acute kidney injury or disease. Patients of the ENVISION cohort were followed for at least 30 months: 2 patients did not experience estimated glomerular filtration rate (eGFR) loss, 3 patients experienced a modest decline in renal function (-3.4 ml/min per 1.73 m2 per year in average), and 2 patients had a clearly abnormal eGFR loss (-5.8 ml/min per 1.73 m2 per year in average). None of the patients had biochemical signs of active tubular or glomerular injury. One patient's kidney was biopsied without finding any signs of an active kidney disease and with normal ALAS1 tubular expression. CONCLUSIONS: Givosiran is associated with a transient moderate increase in serum creatinine (sCr) without sign of kidney injury. A long-term deleterious impact of ALAS1 inhibition on renal function is not excluded. Because AIP promotes chronic kidney disease, it is difficult to separate the long-term effects of givosiran from the natural progression of the renal disease.

The cellular prion protein is a stress protein secreted by renal tubular cells and a urinary marker of kidney injury.

Endoplasmic Reticulum (ER) stress underlies the pathogenesis of numerous kidney diseases. A better care of patients with kidney disease involves the identification and validation of ER stress biomarkers in the early stages of kidney disease. For the first time to our knowledge, we demonstrate that the prion protein PrPC is secreted in a conventional manner by ER-stressed renal epithelial cell under the control of the transcription factor x-box binding protein 1 (XBP1) and can serve as a sensitive urinary biomarker for detecting tubular ER stress. Urinary PrPC elevation occurs in patients with chronic kidney disease. In addition, in patients undergoing cardiac surgery, detectable urine levels of PrPC significantly increase after cardiopulmonary bypass, a condition associated with activation of the IRE1-XBP1 pathway in the kidney. In conclusion, our study has identified PrPC as a novel urinary ER stress biomarker with potential utility in early diagnosis of ongoing acute or chronic kidney injury.

In silico model of the human ClC-Kb chloride channel: pore mapping, biostructural pathology and drug screening.

The human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target.

Rhodnius prolixus smells repellents: Behavioural evidence and test of present and potential compounds inducing repellency in Chagas disease vectors.

Insect repellents are known since many decades ago and constitute a major tool for personal protection against the biting of mosquitoes. Despite their wide use, the understanding of why and how repellents repel is relatively recent. In particular, the question about to what extent insects other than mosquitoes are repulsed by repellents remains open. We developed a series of bioassays aimed to test the performance of well established as well as potential repellent molecules on the Chagas disease vector Rhodnius prolixus. Besides testing their ability to prevent biting, we tested the way in which they act, i.e., by obstructing the detection of attractive odours or by themselves. By using three different experimental protocols (host-biting, open-loop orientation to odours and heat-triggered proboscis extension response) we show that DEET repels bugs both in the presence and in the absence of host-associated odours but only at the highest quantities tested. Piperidine was effective with or without a host and icaridine only repelled in the absence of a living host. Three other molecules recently proposed as potential repellents due to their affinity to the Ir40a(+) receptor (which is also activated by DEET) did not evoke significant repellency. Our work provides novel experimental tools and sheds light on the mechanism behind repellency in haematophagous bugs.