Vasorin as an actor of bone turnover?

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/ß-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.

State of knowledge on ammonia handling by the kidney.

The disposal of ammonia, the main proton buffer in the urine, is important for acid-base homeostasis. Renal ammonia excretion is the predominant contributor to renal net acid excretion, both under basal condition and in response to acidosis. New insights into the mechanisms of renal ammonia production and transport have been gained in the past decades. Ammonia is the only urinary solute known to be produced in the kidney and selectively transported through the different parts of the nephron. Both molecular forms of total ammonia, NH3 and NH4+, are transported by specific proteins. Proximal tubular ammoniagenesis and the activity of these transport processes determine the eventual fate of total ammonia produced and excreted by the kidney. In this review, we summarized the state of the art of ammonia handling by the kidney and highlighted the newest processes described in the last decade.

Low exposition to lithium prevents nephrogenic diabetes insipidus but not microcystic dilations of the collecting ducts in long-term rat model.

Lithium induces nephrogenic diabetes insipidus (NDI) and microcystic chronic kidney disease (CKD). As previous clinical studies suggest that NDI is dose-dependent and CKD is time-dependent, we investigated the effect of low exposition to lithium in a long-term experimental rat model. Rats were fed with a normal diet (control group), with the addition of lithium (Li+ group), or with lithium and amiloride (Li+/Ami group) for 6 months, allowing obtaining low plasma lithium concentrations (0.25 ± 0.06 and 0.43 ± 0.16 mmol/L, respectively). Exposition to low concentrations of plasma lithium levels prevented NDI but not microcystic dilations of kidney tubules, which were identified as collecting ducts (CDs) on immunofluorescent staining. Both hypertrophy, characterized by an increase in the ratio of nuclei per tubular area, and microcystic dilations were observed. The ratio between principal cells and intercalated cells was higher in microcystic than in hypertrophied tubules. There was no correlation between AQP2 messenger RNA levels and cellular remodeling of the CD. Additional amiloride treatment in the Li+/Ami group did not allow consistent morphometric and cellular composition changes compared to the Li+ group. Low exposition to lithium prevented overt NDI but not microcystic dilations of the CD, with differential cellular composition in hypertrophied and microcystic CDs, suggesting different underlying cellular mechanisms.

Claudin-10 Expression and the Gene Expression Pattern of Thick Ascending Limb Cells.

Many genomic, anatomical and functional differences exist between the medullary (MTAL) and the cortical thick ascending limb of the loop of Henle (CTAL), including a higher expression of claudin-10 (CLDN10) in the MTAL than in the CTAL. Therefore, we assessed to what extent the Cldn10 gene expression is a determinant of differential gene expression between MTAL and CTAL. RNAs extracted from CTAL and MTAL microdissected from wild type (WT) and Cldn10 knock out mice (cKO) were analyzed by RNAseq. Differential and enrichment analyses (GSEA) were performed with interactive R Shiny software. Between WT and cKO MTAL, 637 genes were differentially expressed, whereas only 76 were differentially expressed between WT and cKO CTAL. Gene expression patterns and GSEA analyses in all replicates showed that WT MTAL did not cluster with the other replicates; no hierarchical clustering could be found between WT CTAL, cKO CTAL and cKO MTAL. Compared to WT replicates, cKO replicates were enriched in Cldn16, Cldn19, Pth1r, (parathyroid hormone receptor type 1), Casr (calcium sensing receptor) and Vdr (Vitamin D Receptor) mRNA in both the cortex and medulla. Cldn10 is associated with gene expression patterns, including genes specifically involved in divalent cations reabsorption in the TAL.

Global guidance for the recognition, diagnosis, and management of tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome caused by mesenchymal tumors that secrete fibroblast growth factor 23 (FGF23). Patients present with progressive bone pain, muscle weakness, and fragility fractures. TIO is characterized by hypophosphatemia, excess renal phosphate excretion, and low/inappropriately normal 1,25-dihydroxyvitamin D (1,25(OH)2 D) levels. Rarity and enigmatic clinical presentation of TIO contribute to limited awareness among the medical community. Accordingly, appropriate diagnostic tests may not be requested, leading to delayed diagnosis and poorer patient outcomes. We have developed a global guidance document to improve the knowledge of TIO in the medical community, enabling the recognition of patients with TIO and appropriate referral. We provide recommendations aiding diagnosis, referral, and treatment, helping promote a global standard of patient management. We reviewed the literature and conducted a three-round Delphi survey of TIO experts. Statements were drafted based on published evidence and expert opinions (≥70% consensus required for final recommendations). Serum phosphate should be measured in patients presenting with chronic muscle pain or weakness, fragility fractures, or bone pain. Physical examination should establish features of myopathy and identify masses that could be causative tumors. Priority laboratory evaluations should include urine/serum phosphate and creatinine to assess renal tubular reabsorption of phosphate and TmP/GFR, alkaline phosphatase, parathyroid hormone, 25-hydroxyvitamin D, 1,25(OH)2 D, and FGF23. Patients with the clinical/biochemical suspicion of TIO should be referred to a specialist for diagnosis confirmation, and functional imaging should be used to localize causative tumor(s). Recommended treatment is tumor resection or, with unresectable/unidentifiable tumors, phosphate salts plus active vitamin D, or burosumab.

Possible role for rare TRPM7 variants in patients with hypomagnesaemia with secondary hypocalcaemia.

BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.

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.

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA.

BACKGROUND: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. METHODS: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. RESULTS: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. CONCLUSION: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.

The importance of kidney calcium handling in the homeostasis of extracellular fluid calcium.

Extracellular fluid calcium concentration must be maintained within a narrow range in order to sustain many biological functions, encompassing muscle contraction, blood coagulation, and bone and tooth mineralization. Blood calcium value is critically dependent on the ability of the renal tubule to reabsorb the adequate amount of filtered calcium. Tubular calcium reabsorption is carried out by various and complex mechanisms in 3 distinct segments: the proximal tubule, the cortical thick ascending limb of the loop of Henle, and the late distal convoluted/connecting tubule. In addition, calcium reabsorption is tightly controlled by many endocrine, paracrine, and autocrine factors, as well as by non-hormonal factors, in order to adapt the tubular handling of calcium to the metabolic requirements. The present review summarizes the current knowledge of the mechanisms and factors involved in calcium handling by the kidney and, ultimately, in extracellular calcium homeostasis. The review also highlights some of our gaps in understanding that need to be addressed in the future.

Parathyroid hormone and phosphate homeostasis in patients with Bartter and Gitelman syndrome: an international cross-sectional study.

BACKGROUND: Small cohort studies have reported high parathyroid hormone (PTH) levels in patients with Bartter syndrome and lower serum phosphate levels have anecdotally been reported in patients with Gitelman syndrome. In this cross-sectional study, we assessed PTH and phosphate homeostasis in a large cohort of patients with salt-losing tubulopathies. METHODS: Clinical and laboratory data of 589 patients with Bartter and Gitelman syndrome were provided by members of the European Rare Kidney Diseases Reference Network (ERKNet) and the European Society for Paediatric Nephrology (ESPN). RESULTS: A total of 285 patients with Bartter syndrome and 304 patients with Gitelman syndrome were included for analysis. Patients with Bartter syndrome type I and II had the highest median PTH level (7.5 pmol/L) and 56% had hyperparathyroidism (PTH >7.0 pmol/L). Serum calcium was slightly lower in Bartter syndrome type I and II patients with hyperparathyroidism (2.42 versus 2.49 mmol/L; P = .038) compared to those with normal PTH levels and correlated inversely with PTH (rs -0.253; P = .009). Serum phosphate and urinary phosphate excretion did not correlate with PTH. Overall, 22% of patients had low serum phosphate levels (phosphate-standard deviation score < -2), with the highest prevalence in patients with Bartter syndrome type III (32%). Serum phosphate correlated with tubular maximum reabsorption of phosphate/glomerular filtration rate (TmP/GFR) (rs 0.699; P < .001), suggesting renal phosphate wasting. CONCLUSIONS: Hyperparathyroidism is frequent in patients with Bartter syndrome type I and II. Low serum phosphate is observed in a significant number of patients with Bartter and Gitelman syndrome and appears associated with renal phosphate wasting.

mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness.

BACKGROUND: The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. METHODS: A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. RESULTS: We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. CONCLUSIONS: Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

Differential localization patterns of claudin 10, 16, and 19 in human, mouse, and rat renal tubular epithelia.

Functional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated.

Monitoring acid base status in CKD patients: can urinary citrate help?

Metabolic acidosis is an early and deleterious complication of chronic kidney disease. Because it is frequently eubicarbonatemic, diagnosis may be difficult. In this issue, Gianella et al. suggest that lower urinary citrate excretion, considered as an homeostatic response to metabolic acidosis, may be helpful for early diagnosis and monitoring of alkali treatment. This study should be an incentive for further assessment of the tubular handling of urinary citrate in CKD patients and determination of the performance of urinary citrate for the diagnosis of eubicarbonatemic metabolic acidosis and monitoring of alkali therapy.

Renal complications in patients with chronic hypoparathyroidism on conventional therapy: a systematic literature review : Renal disease in chronic hypoparathyroidism.

A systematic literature review was performed to summarize the frequency and nature of renal complications in patients with chronic hypoparathyroidism managed with conventional therapy. Methodology was consistent with the recommendations outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Peer-reviewed journal articles with specified medical subject heading terms were identified using the PubMed, EMBASE, and Cochrane databases. Data were extracted from eligible articles based on prespecified parameters for clinical outcomes of renal calcifications and disease. Because of the heterogeneity of the data, a meta-analysis could not be conducted. From 1200 potentially relevant articles, data were extracted from 13 manuscripts that reported data for ≥1 of the 19 predefined renal outcomes for ≥10 adult patients (n = 11 manuscripts) or pediatric patients (n = 2 manuscripts). The collective data provide evidence that adult and pediatric patients with chronic hypoparathyroidism and treated with conventional therapy (oral calcium and active vitamin D) had an increased risk of renal complications. The reported rate of nephrolithiasis was up to 36%, with the lowest rates in studies reporting shorter duration of disease. The rate of nephrocalcinosis was up to 38%. Some studies reported a combined nephrolithiasis/nephrocalcinosis outcome of 19% to 31%. Data for renal disease that encompassed a range of renal insufficiency to chronic kidney disease were reported in 10 articles; the reported rates ranged from 2.5% to 41%. In patients who receive long-term treatment with oral calcium and active vitamin D, chronic hypoparathyroidism may be associated with an increased risk of renal complications compared with the general population.

The PARADIGHM (physicians advancing disease knowledge in hypoparathyroidism) registry for patients with chronic hypoparathyroidism: study protocol and interim baseline patient characteristics.

BACKGROUND: The PARADIGHM registry of adult and pediatric patients with chronic hypoparathyroidism evaluates the long-term safety and effectiveness of treatment with recombinant human parathyroid hormone, rhPTH(1-84), and describes the clinical disease course under conditions of routine clinical practice. In this first report, we detail the registry protocol and describe the baseline characteristics of two adult patient cohorts from an interim database analysis. One cohort after study entry were prescribed rhPTH(1-84), and the other cohort received conventional therapy of calcium and active vitamin D. METHODS: An observational study of patients with chronic hypoparathyroidism in North America and Europe, collecting data for ≥10 years per patient. Main outcome measures were baseline patient demographics, clinical characteristics, medications, and disease outcome variables of symptoms, biochemical parameters, and health assessments. Baseline is the enrollment assessment for all variables except biochemical measurements in patients treated with rhPTH(1-84); those measurements were the most recent value before the first rhPTH(1-84) dose. Exclusion criteria applied to the analysis of specified outcomes included pediatric patients, patients who initiated rhPTH(1-84) prior to enrollment, and those who received rhPTH(1-34). Clinically implausible biochemical outlier data were excluded. RESULTS: As of 30 June 2019, data of 737 patients were analyzed from 64 centers; 587 (80%) were women, mean ± SD age 49.1±16.45 years. At enrollment, symptoms reported for patients later prescribed rhPTH(1-84) (n=60) and those who received conventional therapy (n=571), respectively, included fatigue (51.7%, 40.1%), paresthesia (51.7%, 29.6%), muscle twitching (48.3%, 21.9%), and muscle cramping (41.7%, 33.8%). Mean serum total calcium, serum phosphate, creatinine, and estimated glomerular filtration rate were similar between cohorts. Health-related quality of life (HRQoL) 36-item Short Form Health Survey questionnaire scores for those later prescribed rhPTH(1-84) were generally lower than those for patients in the conventional therapy cohort. CONCLUSIONS: At enrollment, based on symptoms and HRQoL, a greater percentage of patients subsequently prescribed rhPTH(1-84) appeared to have an increased burden of disease than those who received conventional therapy despite having normal biochemistry measurements. PARADIGHM will provide valuable real-world insights on the clinical course of hypoparathyroidism in patients treated with rhPTH(1-84) or conventional therapy in routine clinical practice. TRIAL REGISTRATION: EUPAS16927, NCT01922440.

Performance of ion chromatography to measure picomole amounts of magnesium in nanolitre samples.

KEY POINTS: An UHPLC method to measure picomole amounts of magnesium has been developed. The method is sensitive, specific, accurate and reproducible. The method is suitable for quantifying magnesium transport across intact epithelia. ABSTRACT: Magnesium is involved in many biological processes. Extracellular magnesium homeostasis mainly depends on the renal handling of magnesium, the study of which requires measurement of low concentrations of magnesium in renal tubular fluid. We developed an ultra-high-performance liquid chromatography method to measure millimolar concentrations of magnesium in nanolitre samples. Within-assay and between-assay coefficients of variation were lower than 5% and 6.6%, respectively. Measurement of magnesium concentration was linear (r2  = 0.9998) over the range 0-4 mmol/l. Absolute bias ranged from -0.03 to 0.05 mmol/l. The lower limit of quantification was 0.2 mmol/l. Recovery was 97.5-100.3%. No significant interference with calcium, another divalent cation present in the same samples, was detected. The method was successfully applied to quantify transepithelial magnesium transport by medullary and cortical thick ascending limbs during ex vivo microperfusion experiments. In conclusion, ultra-high-performance liquid chromatography is suitable for measurement of picomole amounts of magnesium in renal tubular fluid. The method allows detailed studies of transepithelial magnesium transport across native epithelium.

Medullary and cortical thick ascending limb: similarities and differences.

The thick ascending limb of the loop of Henle (TAL) is the first segment of the distal nephron, extending through the whole outer medulla and cortex, two regions with different composition of the peritubular environment. The TAL plays a critical role in the control of NaCl, water, acid, and divalent cation homeostasis, as illustrated by the consequences of the various monogenic diseases that affect the TAL. It delivers tubular fluid to the distal convoluted tubule and thereby affects the function of the downstream tubular segments. The TAL is commonly considered as a whole. However, many structural and functional differences exist between its medullary and cortical parts. The present review summarizes the available data regarding the similarities and differences between the medullary and cortical parts of the TAL. Both subsegments reabsorb NaCl and have high Na+-K+-ATPase activity and negligible water permeability; however, they express distinct isoforms of the Na+-K+-2Cl- cotransporter at the apical membrane. Ammonia and bicarbonate are mostly reabsorbed in the medullary TAL, whereas Ca2+ and Mg2+ are mostly reabsorbed in the cortical TAL. The peptidic hormone receptors controlling transport in the TAL are not homogeneously expressed along the cortical and medullary TAL. Besides this axial heterogeneity, structural and functional differences are also apparent between species, which underscores the link between properties and role of the TAL under various environments.

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.

Performance of creatinine-based equations for estimating glomerular filtration rate changes over time.

BACKGROUND: Glomerular filtration rate (GFR) is commonly used to monitor chronic kidney disease (CKD) progression, but its validity for evaluating kidney function changes over time has not been comprehensively evaluated. We assessed the performance of creatinine-based equations for estimating GFR slope according to patient characteristics and specific CKD diagnosis. METHODS: In the NephroTest cohort study, we measured GFR 5324 times by chromium 51-labeled ethylenediamine tetraacetic acid renal clearance in 1955 adult patients with CKD Stages 1-4 referred to nephrologists (Stages 1-2, 19%) and simultaneously estimated GFR with both the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and Modification of Diet in Renal Disease (MDRD) equations for isotope dilution mass spectrometry traceable creatinine; absolute and relative GFR slopes were calculated using a linear mixed model. RESULTS: Over a median follow-up of 3.4 [interquartile range (IQR) 2.0-5.6] years, the decline in mean absolute and relative measured GFR (mGFR) and CKD-EPI and MDRD estimated GFR (eGFR) was 1.6 ± 1.2, 1.5 ± 1.4 and 1.3 ± 1.3 mL/min/1.73 m2/year and 5.9 ± 5.3, 5.3 ± 5.3 and 4.8 ± 5.2%/year, respectively; 52% and 55% of the patients had MDRD and CKD-EPI eGFR slopes within 30% of mGFR slopes. Both equations tended to overestimate the GFR slope in the youngest patients and underestimate it in the oldest, thus producing inverse associations between age and mGFR versus eGFR slope. Other patient characteristics and specific CKD diagnoses had little effect on the performance of the equations in estimating associations. CONCLUSIONS: This study shows little bias, but poor precision in GFR slope estimation for both MDRD and CKD-EPI equations. Importantly, bias strongly varied with age, possibly due to variations in muscle mass over time, with implications for clinical care and research.