The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

Uromodulin, the most abundant protein in normal urine, is produced by cells lining the thick ascending limb (TAL) of the loop of Henle. Uromodulin regulates the activity of the potassium channel ROMK in TAL cells. Common variants in KCNJ1, the gene encoding ROMK, are associated with urinary levels of uromodulin in population studies. Here, we investigated the functional link between ROMK and uromodulin in Kcnj1 knock-out mouse models, in primary cultures of mouse TAL (mTAL) cells, and in patients with Bartter syndrome due to KCNJ1 mutations. Both global and kidney-specific Kcnj1 knock-out mice showed reduced urinary levels of uromodulin paralleled by increased levels in the kidney, compared to wild-type controls. Pharmacological inhibition and genetic deletion of ROMK in mTAL cells caused a reduction in apical uromodulin excretion, reflected by cellular accumulation. In contrast, NKCC2 inhibition showed no effect on uromodulin processing. Patients with Bartter syndrome type 2 showed reduced urinary uromodulin levels compared to age and gender matched controls. These results demonstrate that ROMK directly regulates processing and release of uromodulin by TAL cells, independently from NKCC2. They support the functional link between transport activity and uromodulin in the TAL, relevant for blood pressure control and urinary concentrating ability.

Effect of nonsteroidal anti-inflammatory drugs in children with Bartter syndrome.

BACKGROUND: Bartter syndrome (BS) is a salt-wasting tubulopathy with induced expression of cyclooxygenase-2 in the macula densa, leading to increased prostaglandin production and hyperreninemia. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently used in BS; however, there is limited information on the impact of NSAIDs at treatment initiation or the potential utility of plasma renin level to guide therapy in patients with BS. METHODS: We included 19 patients with BS treated with NSAIDs between 1994 and 2016. We assessed serum levels of renin, aldosterone, electrolytes, calcium, phosphorus, vitamin D, and intact parathyroid hormone (iPTH) before and after treatment initiation. We also recorded modifications in sodium and potassium supplements and changes in urine calcium. RESULTS: Median age at diagnosis was 0.9 months [IQR 0-6.9]. Seven patients had BS types 1 or 2, 12 had BS type 3 and two had no mutation identified. There was a trend towards a decrease in sodium chloride supplementation after initiation of NSAIDs. When defining response to treatment based on the normalization of plasma renin level, responders had a greater reduction in their electrolytes supplementation. NSAIDs treatment was associated with a reduction in urine calcium. Before treatment, half of the patients had elevated iPTH, but iPTH normalized following initiation of NSAIDs in all but one patient. CONCLUSIONS: This study confirms that NSAIDs reduce urine wasting of sodium and calcium in patients with BS. Monitoring serum renin levels may be useful to identify the lowest effective dose of NSAIDs that optimizes reduction of urine electrolyte losses.

Gitelman syndrome: consensus and guidance from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Gitelman syndrome (GS) is a rare, salt-losing tubulopathy characterized by hypokalemic metabolic alkalosis with hypomagnesemia and hypocalciuria. The disease is recessively inherited, caused by inactivating mutations in the SLC12A3 gene that encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). GS is usually detected during adolescence or adulthood, either fortuitously or in association with mild or nonspecific symptoms or both. The disease is characterized by high phenotypic variability and a significant reduction in the quality of life, and it may be associated with severe manifestations. GS is usually managed by a liberal salt intake together with oral magnesium and potassium supplements. A general problem in rare diseases is the lack of high quality evidence to inform diagnosis, prognosis, and management. We report here on the current state of knowledge related to the diagnostic evaluation, follow-up, management, and treatment of GS; identify knowledge gaps; and propose a research agenda to substantiate a number of issues related to GS. This expert consensus statement aims to establish an initial framework to enable clinical auditing and thus improve quality control of care.

Unusual case of failure to thrive: Type III Bartter syndrome.

Bartter syndrome Type III is a rare autosomal recessive disorder resulting from an inherited defect in the thick ascending limb of the loop of henle of the nephrons in kidney. The typical clinical manifestations in childhood are failure to thrive and recurrent episodes of vomiting. Typical laboratory findings which help in the diagnosis are hypokalemic metabolic alkalosis, hypomagnesemia and hypercalciuria. We report a case of Type III Bartter syndrome not responding to repeated conventional treatment of failure to thrive.

Urinary exosomes in the diagnosis of Gitelman and Bartter syndromes.

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary salt-losing tubulopathies (SLTs) resulting from defects of renal proteins involved in electrolyte reabsorption, as for sodium-chloride cotransporter (NCC) and furosemide-sensitive sodium-potassium-chloride cotransporter (NKCC2) cotransporters, affected in GS and BS Type 1 patients, respectively. Currently, definitive diagnosis is obtained through expensive and time-consuming genetic testing. Urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, represent an ideal source of markers for renal dysfunction and injury, because UE molecular composition stands for the cell of origin. On these assumptions, the aim of this work is to evaluate the relevance of UE for the diagnosis of SLTs. METHODS: UE were purified from second morning urines collected from 32 patients with genetically proven SLTs (GS, BS1, BS2 and BS3 patients), 4 with unclassified SLTs and 22 control subjects (age and sex matched). The levels of NCC and NKCC2 were evaluated in UE by SDS-PAGE/western blotting with specific antibodies. RESULTS: Due to their location on the luminal side of tubular cells, NCC and NKCC2 are well represented in UE proteome. The NCC signal is significantly decreased/absent in UE of Gitelman patients compared with control subjects (Mann-Whitney t-test, P < 0.001) and, similarly, the NKCC2 in those of Bartter type 1 (P < 0.001). The difference in the levels of the two proteins allows recognition of Gitelman and Bartter type 1 patients from controls and, combined with clinical data, from other Bartter patients. Moreover, the receiver operating characteristic curve analysis using UE NCC densitometric values showed a good discriminating power of the test comparing GS patients versus controls and BS patients (area under the curve value = 0.92; sensitivity 84.2% and specificity 88.6%). CONCLUSIONS: UE phenotyping may be useful in the diagnosis of GS and BS, thus providing an alternative/complementary, urine-based diagnostic tool for SLT patient recognition and a diagnostic guidance in complex cases.

Large-scale proteomics and phosphoproteomics of urinary exosomes.

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Novel mutations of the chloride channel Kb gene in two Japanese patients clinically diagnosed as Bartter syndrome with hypocalciuria.

Hypokalemic metabolic tubulopathy, such as in Bartter syndrome and Gitelman syndrome, is caused by the dysfunction of renal electrolyte transporters. Despite advances in molecular genetics with regard to hypokalemic metabolic tubulopathy, recent reports have suggested that the phenotype-genotype correlation is still confusing, especially in classic Bartter and Gitelman syndromes. We report here two Japanese patients who suffered from clinically diagnosed classic Bartter syndrome but who presented hypocalciuria. Hypocalciuria is generally believed to be a pathognomonic finding of NCCT malfunction. To better understand the genotype-phenotype correlation in these two cases, we screened four renal electrolyte transporter genes [Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), Cl channel Kb (ClC-Kb), and Na-Cl cotransporter (NCCT)] by the PCR direct sequencing method. We identified three ClC-Kb allelic variants, including two new mutations (L27R and W610X in patient 1 and a G to C substitution of a 3' splice site of intron 2 and W610X in patient 2). We did not find any mutations in the other three genes. Our present data suggest that some ClC-Kb mutations may affect calcium handling in renal tubular cells.

Angiotensin II reduces calcium uptake into bone.

Children with neonatal Bartter syndrome (NBS) have hypercalciuria, nephrocalcinosis, and osteopenia. A complex of basic-fibroblast growth factor (b-FGF) and a naturally occurring glycosaminoglycan has been identified in the serum and urine of NBS patients. This complex increases bone resorption in a bone disc bioassay system. Angiotensin II (AT II), which is increased in Bartter syndrome, increases the synthesis of b-FGF by cultured endothelial cells. Addition of 10(-8) M AT II to the bioassay, a concentration reported in Bartter syndrome patients, significantly decreased calcium uptake into bone discs [E/C 0.60 (0.04), P < 0.001 compared with buffer, normal E/C >0.90]. Adding b-FGF monoclonal antibody at 10 microg/ml [E/C 0.90 (0.06), P=NS] or indomethacin [E/C 1.00 (0.03), P=NS] to 10(-8 )M AT II neutralized this effect. In separate experiments, newborn rats were given intraperitoneal injections of AT II. Bone discs from these animals were used in the bioassay system and calcium uptake was markedly reduced compared with discs from rats injected with phosphate-buffered saline [AT II 6.6 x 10(-9), E/C 0.10 (0.04), P<0.001, AT II 3.3 x 10(-8), E/C 0.10 (0.05), P<0.001]. AT II decreases calcium uptake in the bone disc bioassay system. This effect can be abrogated by antibody to b-FGF or prostaglandin synthetase inhibition. These results support the hypothesis that in children with NBS, elevated levels of AT II stimulate local skeletal b-FGF synthesis, with a resultant increase in bone resorption via a prostaglandin-dependent pathway.

Pathogenetic role of cyclooxygenase-2 in hyperprostaglandin E syndrome/antenatal Bartter syndrome: therapeutic use of the cyclooxygenase-2 inhibitor nimesulide.

Patients with hyperprostaglandin E syndrome/antenatal Bartter syndrome typically have renal salt wasting, hypercalciuria with nephrocalcinosis, and secondary hyperaldosteronism. Antenatally, these patients have fetal polyuria, leading to polyhydramnios and premature birth. Hyperprostaglandin E syndrome/antenatal Bartter syndrome is accompanied by a pathologically elevated synthesis of prostaglandin E(2), thought to be responsible for aggravation of clinical symptoms such as salt and water loss, vomiting, diarrhea, and failure to thrive. In this study administration of the cyclooxygenase-2 (COX-2) specific inhibitor nimesulide to patients with hyperprostaglandin E syndrome/antenatal Bartter syndrome blocked renal prostaglandin E(2) formation and relieved the key parameters hyperprostaglandinuria, secondary hyperaldosteronism, and hypercalciuria. Partial suppression of serum thromboxane B(2) synthesis resulting from platelet COX-1 activity and complete inhibition of urinary 6-keto-prostaglandin F(1alpha), reflecting endothelial COX-2 activity, indicate preferential inhibition of COX-2 by nimesulide. Amelioration of the clinical symptoms by use of nimesulide indicates that COX-2 may play an important pathogenetic role in hyperprostaglandin E syndrome/antenatal Bartter syndrome. Moreover, on the basis of our data we postulate that COX-2-derived prostaglandin E(2) is an important mediator for stimulation of the renin-angiotensin-aldosterone system in the kidney.

Bartter syndrome and focal segmental glomerulosclerosis: a possible link between two diseases.

We describe a patient with signs and symptoms of classic Bartter syndrome. The patient tested negative for all known genetic abnormalities associated with this tubular disorder. Proteinuria was found within 1 year after the diagnosis of Bartter syndrome. A renal biopsy performed 6 months later, when her kidney function was normal, revealed focal segmental glomerulosclerosis (FSGS). We propose a link between stimulation of the renin-angiotensin system and sclerotic changes in the glomerulus. This lesion may explain previous reports of kidney failure in patients with Bartter syndrome.

Adrenomedullin and nitrite levels in children with Bartter syndrome.

Children with Bartter syndrome have lower than normal vascular reactivity with normotension in spite of biochemical and hormonal abnormalities which are typical of hypertension. Nitric oxide (NO) is a potent endogenous vasodilator, and plays an important role in the control of vascular tone. Adrenomedullin (AM) is a novel hypotensive peptide originally isolated from human pheochromocytoma. The possible role of NO and AM in maintaining this reduced vascular reactivity was examined by studying plasma and urinary nitrite, a stable metabolite of NO, and AM levels in ten children with Bartter syndrome, ten healthy controls, and five children with hypokalemia of causes other than Bartter syndrome (pseudo-Bartter). Urinary excretion of nitrite (mumol/mg urinary creatinine) was 8.9 +/- 1.2 in children with Bartter syndrome, 4.7 +/- 0.9 in healthy controls, and 2.9 +/- 0.8 in pseudo-Bartter (P < 0.05). Plasma nitrite levels (mumol/l) were 101.9 +/- 23.4, 59.9 +/- 14.7, and 65.0 +/- 29.7, respectively (P < 0.05), in the three groups. Urinary excretion of AM (pmol/mg urinary creatinine) was 187 +/- 40, 65 +/- 10, and 160 +/- 50, respectively (P < 0.05), in the three groups. Plasma AM levels were 47.4 +/- 1.8, 39.9 +/- 5.9, and 42.4 +/- 3.9, respectively (P > 0.05), in the three groups. The same parameters were repeated in the two groups of controls and in the Bartter patients in the 6th month of therapy. Urinary nitrite and AM levels were still higher in the Bartter patients than in the other groups. We conclude that in Bartter syndrome the increased NO production may be responsible for the reduced vascular response of the disease. Initially, increased levels of AM in Bartter syndrome and pseudo-Bartter may be a compensatory response to acute hypokalemia; however, continuation of a high level of urinary excretion of AM in children with Bartter syndrome may suggest also the possible role of AM in the reduced vascular response of the disease.

Composition of the sugar moiety of Tamm-Horsfall protein in patients with urinary diseases.

The sugar moiety of Tamm-Horsfall protein (THP) is altered by pathological conditions. The aim of this study was to investigate the composition of THP glycans in urinary diseases. THP was isolated from the urine of patients with urinary tract infection (group A), glomerulonephritis or interstitial nephritis (group B), and Bartter's syndrome (BS) (group C). Monosaccharides, N-glycan profile, THP reactivity with specific lectins and some other proteins were analyzed. THP of patients from groups A, B, and C showed lower amounts of N-acetylgalactosamine (P<0.05, P<0.005, and P<0.05, respectively) than controls; this was reflected in lower reactivity with Phaseolus vulgaris lectin (P<0.005, P<0.05, and P<0.005). Reduced amounts of N-acetylglucosamine were noticed in groups A (P<0. 05) and B (P<0.05). In group A lower amounts of galactose and alpha2, 6-linked sialic acid, as determined by reactivity with Datura stramonium lectin (P<0.005) and Sambucus nigra lectin (P<0.005), were observed. In patients with BS there was a shift from tetrasialylated glycans towards less-sialylated chains. We found also that THP of all patients binds more strongly to IgG(1) (P<0.005, for all patient groups). Our results indicate that the urinary diseases examined affect the THP sugar moiety and the binding of THP to IgG(1).

Biochemical examination of mother's urine is useful for prenatal diagnosis of Bartter syndrome.

Bartter syndrome is characterized by renal potassium and chloride loss, hypokalaemia, hypochloraemic metabolic alkalosis and increased plasma renin activity along with elevated angiotensin II and hyperaldosteronism. For diagnosis we conducted biochemical examinations of both amniotic fluid and the mother's urine. Except for potassium, amniotic fluid electrolytes in a mother with a fetus with Bartter syndrome were high. Urinary chloride, sodium and calcium were very low. Thus, the latter parameters may allow prediction of fetal Bartter syndrome during the prenatal period.

Conjunctive effects of fibroblast growth factor and glycosaminoglycan on bone metabolism in neonatal bartter syndrome.

The calciotropic activity of urine from a subject with neonatal Bartter syndrome (NBS) has been partially purified using ion-exchange and gel chromatographic techniques. A bioassay using bone disks from rat calvaria was used to estimate calciotropic activity, which in the urine of the subject with NBS appears to be due to basic fibroblast growth factor (bFGF) bound to a glycosaminoglycan susceptible to heparitinase digestion. The calciotropic activity is eluted from DEAE-Sephacel and Sepharose CL-6B in a narrow band in association with metachromatic material and is destroyed by heparitinase and blocked by an antibody to bFGF. After treatment of purified preparations with heparitinase, a component that is inactive alone but develops calciotropic activity in association with heparin can be isolated by affinity chromatography on heparin-Sepharose columns. This component is recovered from the column at NaCl concentrations expected to elute bFGF and is inactivated by antibodies to bFGF. No calciotropic activity can be shown in glycosaminoglycan-containing fractions from urine from a normal boy or a normal man, but such fractions exhibit calciotropic activity if bFGF is added to the assay system. When bFGF is added to urine from either normal subject followed by ion-exchange chromatography on DEAE-Sephacel, calciotropic activity is eluted at NaCl concentrations closely similar to those found to elute calciotropic activity from the urine of the NBS subject. It appears that the abnormal findings in NBS urine are due to excess bFGF, although they could be due to some abnormality of the glycosaminoglycan component.

Phenotype resembling Gitelman's syndrome in mice lacking the apical Na+-Cl- cotransporter of the distal convoluted tubule.

Mutations in the gene encoding the thiazide-sensitive Na+-Cl- cotransporter (NCC) of the distal convoluted tubule cause Gitelman's syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman's syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc-/-) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure. Ncc-/- mice retain Na+ as well as wild-type mice when fed a Na+-depleted diet; however, after 2 weeks of Na+ depletion the mean arterial blood pressure of Ncc-/- mice was significantly lower than that of wild-type mice. In addition, Ncc-/- mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg2+ and Ca2+ are altered, as observed in Gitelman's syndrome.

Humoral factor in children with neonatal Bartter syndrome reduces bone calcium uptake in vitro.

The neonatal Bartter syndrome (NBS) is associated with a complex disorder of mineral metabolism in children, including hypercalciuria, nephrocalcinosis, and diminished bone mineral density. Although cyclooxygenase inhibition usually brings about improvement in these findings, there is a variable component which is resistant to such therapy in many children. The factor mediating this disorder has not been identified. Blood and urine from 12 children with NBS were examined. When compared with samples from normal children and adults, all (NBS) sera reduced bone calcium uptake in a bone disc bioassay. This effect persisted in the presence of parathyroid hormone (PTH) antibody and PTH receptor blockade, indicating that neither PTH nor PTH related peptide was responsible. It was eliminated by indomethacin, suggesting that prostanoid generation was essential. Protamine was also inhibitory, as was the addition of ecteola, an anion binder. Activity could be recovered from ecteola by elution with hypertonic buffer. Urine samples from children with NBS had the same calcitropic effect. The agent was removed by ecteola and recovered by hypertonic elution. Activity was eliminated by protamine and by heparinase, but not by trypsin digestion. Size exclusion centrifugation showed that the activity was associated with a material between 10 and 30 kilodaltons. Finally, urine ecteola eluates from NBS patients raised serum concentrations of calcium after intraperitoneal injection in rats. These data suggest that children with NBS have a calcitropic substance in their serum and urine which is not found in normal individuals. The substance is heparin like, and mediates its effects through prostanoid production. These studies provide additional evidence against a direct renal cause of the urinary calcium disturbance characteristic of the disorder.

Studies on the pathogenesis of hypokalemia in Gitelman's syndrome: role of bicarbonaturia and hypomagnesemia.

OBJECTIVE: Hypokalemia and renal potassium (K) wasting are hallmarks of the group of disorders called Bartter's syndrome. The presence of hypomagnesemia and a low rate of excretion of calcium are currently used to characterize a subgroup of these patients as having Gitelman's syndrome (GS) in which the molecular lesion is a defect in the thiazide-sensitive NaCl cotransporter in the distal convoluted tubule. This study was undertaken to examine whether bicarbonaturia or hypomagnesemia exacerbates the kaliuresis in patients with GS. METHODS: Six patients with most of the diagnostic features of GS were examined. To examine the role of bicarbonaturia, the transtubular K concentration gradient (TTKG) was assessed before and after an oral load of NH4Cl which caused the urine pH to be < 6. To evaluate the role of hypomagnesemia, the TTKG was examined after an infusion of enough magnesium (Mg) to achieve normal levels of Mg in plasma for close to 24 h. RESULTS: The TTKG remained very high even when the pH of the urine was < 6.0. An infusion of Mg caused the TTKG to approach expected values for hypokalemia in 4 of 6 patients. The infusion of Mg was extended in 1 patient who had a sustained high TTKG for 24 h; the TTKG remained elevated for 96 h despite normal plasma Mg levels. CONCLUSIONS: Bicarbonaturia does not play a critical role in maintaining the very high TTKG in these patients. The K wasting in 4 of 6 of these patients could largely be attributed to hypomagnesemia and/or Mg depletion. The plasma aldosterone level tended to be higher in patients who did not respond to the infusion of Mg. Therefore, these patients may not represent a homogeneous group with regard to the pathophysiology of their renal K wasting.