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Purpose: To evaluate the outcomes of percutaneous transluminal renal angioplasty with stent implantation (PTRAS) among patients with renal artery stenosis (RAS) who become dialysis-dependent due to acute deterioration of renal function., Materials and Methods: This was a single-center retrospective cohort study of all PTRAS procedures performed from 2003 to 2019 in a referral hospital. A total of 109 procedures were performed in 92 patients. Eleven patients (12%) presented with anuric acute kidney injury (AKI) secondary to high-grade RAS (defined as intraluminal stenosis above 70% per angiography) and underwent PTRAS after starting hemodialysis. Data collected included demographic parameters, medical background, creatinine, blood pressure, indication for intervention, procedure characteristics, adverse events, and long-term data including dialysis treatment and mortality. Among the dialysis-dependent AKI group, outcome measures were defined based on the postprocedural improvement in kidney function and discontinuation of dialysis., Results: Following PTRAS, 8 of 11 patients (73%) demonstrated improved kidney function and were able to discontinue dialysis. The median time on dialysis was 18 days (range, 2-35 days) before PTRAS and 4.5 days (range, 1-24 days) to recovery of kidney function after the time of intervention., Conclusions: Patients with atherosclerotic RAS who develop RAS-related AKI may benefit from PTRAS even after several weeks of anuria and dialysis dependence., (Copyright © 2022 SIR. Published by Elsevier Inc. All rights reserved.)

Preeclampsia is a pregnancy-induced complex of multiple pathological changes. Numerous stresses during pregnancy, including hypoxia, immune activation, inflammatory cytokines, and oxidative stress were reported as contributing factors to the preeclamptic pathology. Seeking common sensors of various stressors in preeclampsia is of new interest and can potentially benefit in disease prevention and treatment. Recent studies have highlighted the role of the Gadd45a protein as a stress sensor in preeclampsia. In response to various pathophysiological stressors, notably hypoxia, oxidative stress, inflammatory cytokines, and AT1-AAs, Gadd45a activates Mkk3-p38 and or JNK signaling. This, in turn, results in immunological and inflammatory changes as well as triggering the production of circulating factors such as sFlt-1, which are believed to account for many of the pathophysiological-related symptoms of preeclampsia. Activation of inflammatory/immune responses in preeclampsia may function in a feedback loop to maintain elevated expression of Gadd45a protein., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Severe congestive heart failure (CHF) patients are prone to hyponatremia. Peritoneal dialysis (PD) is increasingly used for long-term management of refractory CHF patients. The glucose polymer icodextrin was proposed to be a good option for fluid removal in such patients. A small, although statistically significant reduction in serum sodium (∼2mmol/l) consistently observed in multiple trials, is considered as not clinically relevant. Here we reported five refractory CHF patients who demonstrated sodium drop by median of 8meq/l (range 5.4-8.3meq/l) after icodextrin was added to their program. It seems that icodextrin may contribute to clinically relevant hyponatremia if the hyponatremia is compounded by other factors. Patients with extremely severe congestive heart failure are susceptible to this complication., (Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Objectives: Familial Mediterranean fever (FMF) is an autoinflammatory disorder with episodic and persistent inflammation, which is only partially suppressed by continuous colchicine treatment. While chronic inflammation is considered an important cardiovascular risk factor in many inflammatory disorders, its impact in FMF is still disputed. We measured arterial stiffness, a marker of atherosclerotic cardiovascular disease, in a group of FMF patients, in order to evaluate the cardiovascular consequences of inflammation in FMF and the role of colchicine in their development., Methods: Eighty colchicine treated FMF patients, without known traditional cardiovascular risk factors, were randomly enrolled in the study. Demographic, genetic, clinical and laboratory data were retrieved from patient files and examinations. Arterial stiffness was measured using pulse wave velocity (PWV). The recorded values of PWV were compared with those of an age and blood pressure adjusted normal population, using internationally endorsed values., Results: FMF patients displayed normal PWV values, with an even smaller than expected proportion of patients deviating from the 90th percentile of the reference population (5% vs. 10%, p=0.02). The lowest PWV values were recorded in patients receiving the highest dose of colchicine (≥2 mg vs. 0-1 mg, p=0.038), and in patients of North African Jewish origin, whose disease was typically more severe than that of patients of other ethnicities; both observations supporting an ameliorating colchicine effect (p=0.043)., Conclusions: Though subjected to chronic inflammation, colchicine treated FMF patients have normal PWV. Our findings provide direct evidence for a cardiovascular protective role of colchicine in FMF.

Background: Familial hyperkalemia and hypertension (FHHt) is an inherited disorder manifested by hyperkalemia and hypertension. The following four causative genes were identified: WNK1, WNK4, CUL3, and KLHL3. For the first 3 genes, inheritance is autosomal dominant. For KLHL3, inheritance is mostly dominant. A few cases with autosomal recessive disease were described. The mechanism of these 2 modes of inheritance is not clear. In the recessive form, the phenotype of heterozygotes is not well described., Methods: Clinical and genetic investigation of members of 2 families was performed, one with recessive FHHt, and the other, an expansion of a family with Q309R KLHL3 dominant mutation, previously reported by us. Urinary exosomal sodium chloride cotransporter (NCC) was measured., Results: A family with recessive FHHt caused by a new KLHL3 mutation, S553L, is described. This consanguineous Jewish family of Yemenite extraction, included 2 homozygous and 7 heterozygous affected subjects. Increased urinary NCC was found in the affected members of the family with dominant Q309R KLHL3 mutation. In the recessive S553L family, homozygotes appeared to have increased urinary NCC abundance. Surprisingly, heterozygotes seemed to have also increased urinary NCC, though at an apparently lower degree. This was not accompanied by a clinical phenotype., Conclusions: A new recessive mutation in KLHL3 (S553L) was identified in FHHt. Increased urinary NCC was found in affected members (heterozygous) with dominant KLHL3 Q309R, and in affected members (homozygous) of the recessive form. Unexpectedly, in the recessive disease, heterozygotes seemed to have increased urinary NCC as well, apparently not sufficient quantitatively to produce a clinical phenotype., (© 2017 S. Karger AG, Basel.)

Background: Inherited metabolic disorders associated with nephrocalcinosis are rare conditions. The aim of this study was to identify the genetic cause of an Israeli-Arab boy from a consanguineous family with severe nephrocalcinosis and kidney insufficiency., Methods: Clinical and biochemical data of the proband and family members were obtained from both previous and recent medical charts. Genomic DNA was isolated from peripheral blood cells. The coding sequence and splice sites of candidate genes (CYP24A1, CYP27B1, FGF23, KLOTHO, SLC34A3 and SLC34A1) were sequenced directly. Functional studies were performed in Xenopus laevis oocytes and in transfected opossum kidney (OK) cells., Results: Our patient was identified as having nephrocalcinosis in utero, and at the age of 16.5 years, he had kidney insufficiency but no bone disease. Genetic analysis revealed a novel homozygous missense mutation, Arg215Gln, in SLC34A1, which encodes the renal sodium phosphate cotransporter NaPiIIa. Functional studies of the Arg215Gln mutant revealed reduced transport activity in Xenopus laevis oocytes and increased intracellular cytoplasmic accumulation in OK cells., Conclusions: Our findings show that dysfunction of the human NaPiIIa causes severe renal calcification that may eventually lead to reduced kidney function, rather than complications of phosphate loss.

Context: The risk associated with serum uric acid (SUA) levels within the normal range is unknown, especially among lean and apparently healthy adults., Objective: Evaluating whether high-normal SUA levels, 6.8 mg/dL and below, are associated with an increased diabetes risk, compared with low-normal SUA., Design and Setting: This was a cohort study with 10 years of followup involving all clinics of the largest nationally distributed Health Maintenance Organization in Israel., Participants: Participants included 469,947 examinees, 40-70 years old at baseline, who had their SUA measured during 2002. We excluded examinees who had hyperuricemia (SUA > 6.8 mg/dL), impaired fasting glucose, overweight or obesity and chronic cardiovascular or renal disorders. The final cohort was composed of 30 302 participants., Interventions: Participants were followed up to a new diagnosis of diabetes during the study period., Main Outcome Measures: Odds ratio of developing diabetes among participants with high-normal baseline SUA were compared with low-normal (2 ≤ uric acid < 3 and 3 ≤ uric acid < 4 in women and men, respectively)., Results: In a logistic regression model adjusted for age, body mass index, socioeconomic status, smoking, baseline estimated glomerular filtration rate, and baseline glucose, SUA levels of 4-5 mg/dL for women were associated with 61% increased risk for incident diabetes (95% confidence interval, 1.1-2.3). At the highest normal levels for women (SUA, 5-6 mg/dL) the odds ratio was 2.7 (1.8-4.0), whereas men had comparable diabetes risk at values of 6-6.8 mg/dL (hazard ratio, 1.35; 95% confidence interval, 0.9-2.1)., Conclusions: SUA levels within the normal range are associated with an increased risk for new-onset diabetes among healthy lean women when compared with those with low-normal values.

Hypertensive emergency (HE) is a life-threatening condition that requires immediate blood pressure (BP) reduction. Although it has been on the decline, the incidence of HE has recently increased in a few countries. The aim of the present retrospective study was to evaluate the incidence, aetiology and 1-year mortality of HE in a large medical centre over a 20-year period (1991-2010). The electronic medical records of all patient files who were hospitalized in the Chaim Sheba Medical Center in Israel from 1991 to 2010 with a primary diagnosis (at admission or discharge) of Malignant Hypertension, Hypertensive Emergency or Accelerated Hypertension were retrieved and analysed. The study interval was divided into four periods of 5 years each. Among 306 files reviewed, only 142 patients had a true HE. Average age at presentation was 63.3±16.5 years. Men were younger than women (59±16 vs 68±16 years; P<0.001). At presentation, most patients (80.3%) had been diagnosed with essential hypertension previously and were undertreated. Average maximum mean arterial pressure (MAP) was higher in men (169±22 mm Hg) than in women (161±17 mm Hg; P=0.026). The rate of HE decreased over the course of the study, from 12.7/100 000 admissions during 1991-1995 to 6.2/100 000 admissions (2006-2010). Similarly, 1-year mortality decreased from 16.7 to 3.6%. The rate of HE has decreased and the prognosis has improved over the last two decades. Appropriate BP control of patients with essential hypertension may further decrease the risk of HE.

There are data describing that cardiovascular risks related to serum uric acid (SUA) levels may begin below the current diagnostic level for hyperuricemia. Values from 5.2 to 6.0 mg/dL were positively associated with higher cardiovascular risk. The risk associated with lower SUA levels has not been fully assessed in healthy adults. The purpose of this study was to evaluate whether normal SUA levels, even below 5-6 mg/dL, might be related to an increased risk of hypertension, compared with low-normal SUA. This cohort study was conducted in an outpatient setting: all clinics of the largest Health Maintenance Organization in Israel, in a national distribution. A total of 118,920 healthy adults (40-70 years old), who had SUA levels screened during 2002, were eligible for the study. They were stratified according to baseline SUA, and were followed for 10 years. The study endpoint was any new diagnosis of hypertension during the study period (until December 31, 2011). During 10 years of follow-up (2002-2011), 28,436 examinees developed hypertension (23.9%). Compared with the pre-defined SUA reference values (2-3 mg/dL), women with SUA within the normal range had a gradual, increased risk of developing new-onset hypertension, starting at values as low as 3-4 mg/dL (adjusted odds ratio, 1.15; 95% confidence interval, 1.01-1.30). Women with SUA 5-6 mg/dL, still accepted as normouricemia, had a 66% increased risk of developing hypertension. Younger women (ages 40-50 years at baseline) in a similar SUA subgroup (5-6 mg/dL) had an even higher risk (odds ratio, 2.25; 95% confidence interval, 1.96-2.60). Similar results were seen among men. The possibility of subtle confounders exists, despite extensive adjustment. SUA within the normal range is associated with new-onset hypertension among healthy adults, compared with once very common low-normal range values. Further study is warranted to determine new cutoffs of hypo-, normo-, and hyperuricemia, which might be far lower than current scales., (Copyright © 2015 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Background: Hypercalcemia is caused by many different conditions and may lead to severe complications. Loss-of-function mutations of CYP24A1, encoding vitamin D-24-hydroxylase, have recently been identified in idiopathic infantile hypercalcemia and in adult kidney stone disease. The aim of this study was to investigate the genetics and clinical features of both infantile and maternal hypercalcemia., Methods: We studied members of four unrelated Israeli families with hypercalcemia, namely, one woman during pregnancy and after delivery and three infants. Clinical and biochemical data were obtained from probands' medical charts. Genomic DNA was isolated from peripheral blood and CYP24A1 was sequenced., Results: Typical symptoms of hypercalcemia associated with the intake of recommended doses of vitamin D developed in the infants and pregnant woman. Four different loss-of-function CYP24A1 mutations were identified, two of which are reported here for the first time (p.Trp134Gly and p.Glu315*). The infants from families 1 and 2, respectively, were found to be compound heterozygotes, and the infant from family 3 and the pregnant woman were found to be homozygous., Conclusions: This is the first report of maternal hypercalcemia caused by a CYP24A1 mutation, showing that not only infants are at risk for this complication. Our findings emphasize the importance of recognition, genetic diagnosis and proper treatment of this recently identified hypercalcemic disorder in this era of widespread vitamin D supplements.

Background: Familial hyperkalemia and hypertension (FHHt) is a rare genetic disorder manifested by hyperkalemia and early hypertension. Hypercalciuria is another accompanying feature. Mutations in WNK4 and WNK1 were found initially, and recently additional mutations were found in two genes, KLHL3 and CUL3, which are components of the Ubiquitin system. It was not reported whether these latter mutations are accompanied by hypercalciuria., Methods: We compared urinary calcium excretion (UCa) in affected subjects with FHHt and KLHL3 mutations, and in their unaffected family members, and in affected subjects with FHHt and WNK4 Q565E mutation., Results: Two new families with FHHt including a total number of 23 subjects, 10 of them affected, in whom previously described mutations in KLHL3 (Q309R and R528H) were identified. Presenting features were short stature in the first family, and transient tachypnea of the newborn (TTN) in the second. Affected subjects had hypercalciuria. UCa levels in affected subjects in the two families were significantly higher than in unaffected subjects (0.608 ± 0.196 vs. 0.236 ± 0.053 mmol Ca per mmol creatinine, respectively (p < 0.0001)). Hypercalciuria in FHHt with KLHL3 mutations is less severe than that observed in FHHt with the Q565E WNK4 mutation (0.608 ± 0.196 (n = 10) mmol Ca per mmol creatinine versus 0.860 ± 0.295 (n = 29), respectively (p = 0.0168))., Conclusions: FHHt caused by KLHL3 mutations is accompanied by hypercalciuria as well as hyperkalemia and hypertension. The similar phenomena observed for FHHt caused by WNK4 mutations fits the other evidence that WNK4 mutations are activating, and the aberrant mechanism of calcium handling by the kidney in FHHt., (© 2015 S. Karger AG, Basel.)

Proinflammatory cytokines play a pathogenic role in congestive heart failure. In this study, the effect of peritoneal dialysis treatment on inflammatory cytokines levels in refractory congestive heart failure patients was investigated. During the treatment, the patients reached a well-tolerated edema-free state and demonstrated significant improvement in NYHA functional class. Brain natriuretic peptide decreased significantly after 3 months of treatment and remained stable at 6 months. C-reactive protein, a plasma marker of inflammation, decreased significantly following the treatment. Circulating inflammatory cytokines TNF-α and IL-6 decreased significantly after 3 months of peritoneal dialysis treatment and remained low at 6 months. The reduction in circulating inflammatory cytokines levels may be partly responsible for the efficacy of peritoneal dialysis for refractory congestive heart failure.

Non-tuberculous mycobacteria are a rare but serious cause of peritoneal dialysis-related peritonitis. There are no clear guidelines for treating non-tuberculous mycobacteria peritoneal dialysis-associated infections. It has been recommended that at least two antibiotics be given for a prolonged period and peritoneal catheter should be removed. This paper describes the clinical course and treatment of a patient with M. chelonae peritoneal dialysis-related peritonitis and reviews the previously published cases.

Background: Uromodulin (Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals. Despite 60 years of research, its physiological role remains rather elusive. Familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease Type 2 are autosomal dominant tubulointerstitial nephropathies characterized by gouty arthritis and progressive renal insufficiency, caused by uromodulin (UMOD) mutations. The aim of this study was to compare the cellular effects of mutant and wild-type UMOD., Methods: Wild-type UMOD cDNA was cloned from human kidney cDNA into pcDNA3 expression vector. A mutant UMOD construct, containing the previously reported mutation, V273, was created by in vitro mutagenesis. Transient and stable transfection studies were performed in human embryonic kidney cells and mouse distal convoluted tubular cells, respectively. Expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Oligosaccharide cleavage by glycosidases was performed to characterize different forms of UMOD. Nuclear translocation of P65 and degradation of IκBα and IRAK1 in response to interleukin (IL)-1β were used to evaluate the effects of wild-type and mutant UMOD on the IL-1R-NFκB pathway., Results: The mutant protein was shown to be retained in the endoplasmic reticulum and was not excreted to the cell medium, as opposed to the wild-type protein. NFκB activation in cells expressing mutant UMOD was similar to that of untransfected cells. In contrast, cells over-expressing wild-type UMOD showed markedly reduced NFκB activation., Conclusion: Our findings suggest that UMOD may have a physiologic function related to its inhibitory effect on the NFκB pathway.

Background: Loss-of-function mutations of vitamin D-24 hydroxylase have recently been recognized as a cause of hypercalcaemia and nephrocalcinosis/nephrolithiasis in infants and adults. True prevalence and natural history of this condition are still to be defined., Methods: We describe two adult patients with homozygous mutations and six related heterozygous carriers. Mineral and hormonal data in these patients were compared with that in 27 patients with stage 2-3 chronic kidney disease and 39 healthy adult kidney donors., Results: Probands had recurrent nephrolithiasis, chronic hypercalcaemia with depressed parathyroid hormone (PTH) and increased 1,25(OH)(2)D levels; carriers had nephrolithiasis (two of six), hypercalciuria (two of six) and high or normal-high 1,25(OH)(2)D (four of four). Corticosteroids did not reduce plasma and urine calcium levels, but ketoconazole did, indicating that 1,25(OH)(2)D production is not maximally depressed despite coexisting hypercalcaemia, high 1,25(OH)(2)D and depressed PTH, and that 1,25(OH)(2)D degradation through vitamin D-24 hydroxylase is a regulator of plasma 1,25(OH)(2)D levels. Both probands had vascular calcifications and high bone mineral content. One developed stage 3b renal failure: in this patient 1,25(OH)(2)D decreased within normal limits as glomerular filtration rate (GFR) fell and PTH rose to high-normal values, yet hypercalcaemia persisted and the ratio of 1,25(OH)(2)D to GFR remained higher than normal for any degree of GFR., Conclusions: This natural model indicates that vitamin D-24 hydroxylase is a key physiologic regulator of calcitriol and plasma calcium levels, and that balanced reduction of 1,25(OH)(2)D and GFR is instrumental for the maintenance of physiologic calcium levels and balance in chronic kidney diseases.

Purpose: Hypercalciuria is the most common cause of kidney stone disease and genetic factors have an important role in nearly half of these cases. Recently loss-of-function mutations of CYP24A1, the gene encoding vitamin D 24-hydroxylase, were identified in idiopathic infantile hypercalcemia. We describe the clinical and molecular basis of severe long-standing kidney stone disease in adults caused by CYP24A1 mutations., Materials and Methods: Three subjects from 2 Israeli families with nephrolithiasis and nephrocalcinosis were clinically characterized. Genomic DNA was isolated from peripheral blood and sequencing of CYP24A1 was performed., Results: All subjects presented with severe kidney stone disease, the cause of which was not discovered for decades despite extensive evaluation. They all had hypercalciuria, nephrocalcinosis and intermittent hypercalcemia, and chronic kidney insufficiency developed in the oldest subject. All patients had a typical pattern of test results, including normal-high serum calcium, low parathyroid hormone levels, high vitamin D 25-(OH)D3 and 1,25-(OH)2D3, and low 24,25-(OH)2D3. Overall 3 CYP24A1 loss-of-function mutations were identified, including a homozygous deletion (delE143) in consanguinous family 1, and compound heterozygous mutations L409S and the novel W268-stop in family 2., Conclusions: Loss-of-function mutations of CYP24A1 gene, encoding for 1,25-dihydroxyvitamin D3 24-hydroxylase, cause severe hypercalciuric nephrolithiasis and nephrocalcinosis. The mutations may present in adults and may lead to chronic renal insufficiency. Our results support a recessive mode of inheritance. CYP24A1 mutations should be considered in the differential diagnosis of hypercalciuric nephrolithiasis, especially as many adults are now prescribed supplemental oral vitamin D., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Accumulating evidence suggests that placental stresses during pregnancy can play an important role in the pathogenesis of preeclampsia. A common signal pathway that senses and converts placental stresses into intracellular stress response may be contributing to this pathology. Based on our previous findings, we extended our investigation to establish that Gadd45a stress signaling regulates sFlt-1 levels, particularly in placenta, when exposed to various preeclampsia-associated stresses including AT-1 receptor agonist (Angiotensin II), hypoxia, and inflammatory cytokines. Using a placental explant model, we found that Gadd45a was induced in response to all the preeclampsia stresses stated above. Although stress induced Gadd45a was associated with the activation of its downstream effectors phospho-p38 and phospho-JNK, the subsequent regulation of sFlt-1 levels occurred through either one of these effectors, but not both. These observations indicate that Gadd45a signaling may work as a hub connecting placental stresses and the pathogenesis of preeclampsia. It also provides evidence to justify testing the role of Gadd45 in the etiology of preeclampsia using in vivo mouse (i.e., Gadd45a null mice) models., (Copyright © 2012 Wiley Periodicals, Inc.)

Preeclampsia is a pregnancy-induced complex of multiple pathological changes. Numerous stresses during pregnancy, including hypoxia, immune activation, inflammatory cytokines, and oxidative stress were reported as contributing factors to the preeclamptic pathology. Seeking common sensors of various stressors in preeclampsia is of new interest and can potentially benefit in disease prevention and treatment. Recent studies have highlighted the role of the Gadd45a protein as a stress sensor in preeclampsia. In response to various pathophysiological stressors, notably hypoxia, inflammatory cytokines, and AT1-AAs, Gadd45a activates Mkk3-p38 and or JNK signaling. This, in turn, results in immunological and inflammatory changes as well as triggering the production of circulating factors such as sFlt-1, which are believed to account for many of the pathophysiological-related symptoms of preeclampsia. Activation of inflammatory/immune responses in preeclampsia may function in a feedback loop to maintain elevated expression of Gadd45a protein.

Background: Peritoneal dialysis (PD) is increasingly used for long-term management of refractory congestive heart failure (CHF). In this study, we investigated the outcome of patients with refractory CHF treated with PD, aiming to identify potential prognostic factors for long term-survival., Methods: This was a prospective observational study over a period of 42 months which included 37 refractory CHF patients., Results: Median survival on PD was 14 months (1-41 months). Long survivors had serum sodium >132 mEq/l (p < 0.001), serum albumin >3.2 g/dl (p < 0.001) and hospitalization rate <2 days per month a year before starting the treatment (p = 0.008). Patients in the lowest survival quartile had lower serum albumin (2.8 vs. 3.5 g/dl in longer survivors, p = 0.003) and serum sodium (126 vs. 137 mEq/l, p < 0.0001), higher serum leukocyte count (7,500 vs. 6,800/μl in long survivors, p = 0.033), higher glomerular filtration rate (39.4 vs. 29.9 ml/min/1.73 m(2), p = 0.035), had more hospitalization before starting the treatment (3.4 vs. 1.9 days per month, p = 0.003) and lower estimated left ventricular mass index (113 vs. 137 g/m(2), p = 0.035). Long-term survivors demonstrated significant improvement in the New York Heart Association functional class by a median of one class, reduced hospitalization rate by 55% and decrease in dependence on intravenous diuretics and vasoactive medications (73% drop in CHF day care visits during the first year of treatment)., Conclusions: Survival of patients with refractory CHF treated with PD is highly variable. Serum sodium, serum albumin and hospitalization rate are important prognostic factors for long-term survival. Long survivors demonstrated improved functional status, reduced hospitalization and mortality rates., (Copyright © 2013 S. Karger AG, Basel.)

Background: Organic anion transporters (OATs) are located on either the basolateral or the apical membrane of the proximal tubule cell and mediate the absorption and secretion of various drugs and endogenous metabolites. It has been shown that cellular damage in acute kidney injury (AKI) involves three forms of injury: sublethal damage resulting in loss of cell polarity, cell death through apoptosis and necrosis. We hypothesize that cellular mistargeting of OAT proteins in AKI will change the profile of OAT proteins in urine., Methods: Thirty AKI patients were included in the study. AKI was defined by clinical course, daily urine output, response to fluid repletion, urinary sediment, fractional excretion of sodium (FeNa) and urine osmolality. Urinary OAT1, OAT3 and OAT4 protein abundance was measured from semiquantitative immunoblots of urine membrane fraction samples (exosome) collected from patients with AKI and from control subjects., Results: Although all patients studied reached a similar severity of renal failure measured by serum creatinine, some of them recovered from AKI with supportive care only, while others required renal replacement therapy (RRT). OAT1 and OAT3, which are normally localized in the basolateral membrane of the proximal tubule cell, were detected at low levels in urine from control subjects and were increased significantly in all patients with AKI. OAT4 protein, which is normally localized in the luminal membrane of proximal tubule cells, was present in abundance in urine of control subjects. Interestingly, in patients with AKI who eventually recovered, urinary OAT4 was found to be significantly lower than in controls, while in patients who needed RRT, it was higher than in controls., Conclusions: We have shown that OATs are mistargeted in AKI. The urinary OAT protein profile can help us to learn about the pathophysiology of the disease and might be a marker of AKI severity. AKI patients with early reversible proximal tubular damage will have high urine OAT1 and OAT3 and low OAT4, while patients with severe AKI will have high urine OAT1, OAT3 and OAT4.

Background: Elevated serum uric acid (UA) is associated with gout, hypertension, cardiovascular and renal disease. Hereditary renal hypouricemia type 1 (RHUC1) is caused by mutations in the renal tubular UA transporter URAT1 and can be complicated by nephrolithiasis and exercise-induced acute renal failure (EIARF). We have recently shown that loss-of-function homozygous mutations of another UA transporter, GLUT9, cause a severe type of hereditary renal hypouricemia with similar complications (RHUC2)., Methods: Two unrelated families with renal hypouricemia were clinically characterized. DNA was extracted and SLC22A12 and SLC2A9 coding for URAT1 and GLUT9, respectively, were sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of the GLUT9 mutations found. A molecular modeling study was undertaken to structurally characterize and probe the effects of these mutations., Results: Two novel homozygous GLUT9 missense mutations were identified: R171C and T125M. Mean serum UA level of the four homozygous subjects was 0.15 ± 0.06 mg/dL and fractional excretion of UA was 89-150%. None of the affected subjects had nephrolithiasis, EIARF or any other complications. Transport assays revealed that both mutant proteins had a dramatically reduced ability to transport UA. Modeling showed that both R171C and T125M mutations are located within the inner channel that transports UA between the cytoplasmic and extracellular regions., Conclusions: This is the second report of renal hypouricemia caused by homozygous GLUT9 mutations. Our findings confirm the pivotal role of GLUT9 in UA transport and highlight the similarities and differences between RHUC1 and RHUC2.

Background: Cyclosporine is used for treatment of transplanted patients and for immune-mediated diseases. Cyclosporine is known to cause a combination of metabolic side effects including hypertension, hyperkalemia, hypercalciuria and hypomagnesemia. These side effects except for hypomagnesemia are the cardinal features of familial hyperkalemia and hypertension (FHHt), also called pseudohypoaldosteronism type II (PHA II). FHHt is caused by mutations in the kinases WNK1 and WNK4 resulting in an increase in renal Na-Cl cotransporter (NCC) apical distribution and function. Therefore, we studied whether cyclosporine's metabolic side effects are mediated by WNK4 and NCC., Design: Sprague-Dawley (SD) rats were treated by cyclosporine 25 mg kg(-1) subcutaneously for 14 days. Blood pressure, blood chemistry values and kidney WNK4 protein were determined. In addition, mDCT cells were exposed to cyclosporine, and their WNK4 mRNA and protein content, and their NCC protein content and phosphorylation were determined., Results: The rats developed an FHHt-like syndrome including hypertension, hyperkalemia and salt-sensitive hypercalciuria. A significant increase in their kidney WNK4 protein content (0·13 ± 0·01 vs. 0·67 ± 0·16 WNK4/GAPDH in controls, P = 0·0183) was found. In mDCT cells, cyclosporine caused a rise in WNK4 mRNA levels and also a threefold rise in WNK4 protein content. This rise was followed by a rise in NCC protein content and pSer71 phosphorylation., Conclusions: These observations may explain in part the mechanism of cyclosporine-induced hypertension, hyperkalemia and hypercalciuria., (© 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Hereditary renal hypouricemia may be complicated by nephrolithiasis or exercise-induced acute renal failure. Most patients described so far are of Japanese origin and carry the truncating mutation W258X in the uric acid transporter URAT1 encoded by SLC22A12. Recently, we described severe renal hypouricemia in Israeli patients with uric acid transporter GLUT9 (SLC2A9) loss-of-function mutations. Renal hypouricemia in Iraqi Jews has been previously reported, but its molecular basis has not been ascertained., Methods: Three Jewish Israeli families of Iraqi origin with hereditary hypouricemia and hyperuricosuria were clinically characterized. DNA was extracted and the URAT1 gene was sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of URAT1 mutants found., Results: A missense URAT1 mutation, R406C, was detected in all three families. Two affected siblings were found to carry in addition a homozygous missense URAT1 mutation, G444R. Both mutations dramatically impaired urate uptake into X. laevis oocytes. Moreover, we demonstrate for the first time that URAT1 facilitates urate efflux, which was abolished in the mutants, indicating also a secretion defect. Homozygous patients had serum uric acid concentrations of 0.5-0.8 mg% and a fractional excretion of uric acid of 50-85%. Most individuals studied were asymptomatic, two had nephrolithiasis and none developed exercise-induced acute renal failure., Conclusions: The URAT1 R406C mutation detected in all three families is likely to be the founder mutation in Iraqi Jews. Our findings contribute to a better definition of the different types of hereditary renal hypouricemia and suggest that the phenotype of this disorder depends mainly on the degree of inhibition of uric acid transport.

Background: The rare autosomal dominant genetic disorder familial hyperkalemia and hypertension which is caused by mutations in WNK4 kinase, is characterized by childhood hyperkalemia and hypercalciuria, and appearance of hypertension in the third to fourth decade. Accompanying short stature is often described., Methods: We determined height, blood pressure and blood and urinary biochemical parameters in members of a very large family of FHHt with the WNK4 Q565E mutation., Results: The family has 57 members, 30 of whom (including 14 children) are affected. Prehypertension occurred in 7/11 affected and 1/10 unaffected children (P = 0.024). Serum potassium (SK) was ~0.5 mmol/L higher in affected children vs adults [5.98 ± 0.42 vs 5.46 ± 0.40 mmol/L, respectively (P < 0.0001)] (33 samples from 11 children and 36 samples from eight adults). SK of ≥ 6.0 mmol/L occurred in 16/33 children's samples and in 3/36 adults' samples (P = 0.0003). Hyperkalaemia in children is currently untreated. Children also had more severe hyperchloraemia and hypercalciuria. The family contains four large subfamilies, and each includes 8-10 siblings. In one subfamily, height Z-score was lower in affected vs unaffected subjects [- 2.69 ± 0.36 vs -1.05 ± 0.16, respectively (P < 0.0001)]. In the other three subfamilies, no such difference was found., Conclusions: Short stature is not part of FHHt with the WNK4 Q565E mutation. Children affected with FHHt have a high prevalence of prehypertension, and their hyperkalaemia is more severe than that of affected adults. Children may have a more severe defect in the basic mechanism that produces hyperkalaemia. We suggest that, in affected adults, the attenuation of hyperkalaemia and appearance of hypertension may be the result of a late rise in the activity of renal transporters or channels such as the epithelial sodium channel.

Background/aims: Current consensus supports the notion that proteinuria is a marker of renal disease with prognostic implications. Whereas most chronic kidney disease patients with proteinuria would often require antiproteinuric agents, there are some exceptions. Megaloblastic anemia type 1 (MGA1) is characterized by megaloblastic anemia due to congenital selective vitamin B(12) malabsorption and proteinuria. In the present study, we describe 2 Israeli Jewish patients with MGA1 and isolated proteinuria., Methods: Because of their origin, the patients were screened for the presence of the already studied Tunisian AMN mutation, by direct sequencing the corresponding region from genomic DNA. PCR products were purified and sequenced., Results: Genomic DNA sequencing of the AMN gene of both patients confirmed that the acceptor splice site in intron 3 was changed from CAG to CGG (208-2A→G)., Conclusion: We determined the molecular basis of MGA1 in both patients and discuss the involvement of the cubilin/AMN complex in this pathology and its role in the development of the proteinuria. We also discuss the questionable significance of antiproteinuric treatment for these patients., (Copyright © 2010 S. Karger AG, Basel.)

Hereditary hypouricemia may result from mutations in the renal tubular uric acid transporter URAT1. Whether mutation of other uric acid transporters produces a similar phenotype is unknown. We studied two families who had severe hereditary hypouricemia and did not have a URAT1 defect. We performed a genome-wide homozygosity screen and linkage analysis and identified the candidate gene SLC2A9, which encodes the glucose transporter 9 (GLUT9). Both families had homozygous SLC2A9 mutations: A missense mutation (L75R) in six affected members of one family and a 36-kb deletion, resulting in a truncated protein, in the other. In vitro, the L75R mutation dramatically impaired transport of uric acid. The mean concentration of serum uric acid of seven homozygous individuals was 0.17 +/- 0.2 mg/dl, and all had a fractional excretion of uric acid >150%. Three individuals had nephrolithiasis, and three had a history of exercise-induced acute renal failure. In conclusion, homozygous loss-of-function mutations of GLUT9 cause a total defect of uric acid absorption, leading to severe renal hypouricemia complicated by nephrolithiasis and exercise-induced acute renal failure. In addition to clarifying renal handling of uric acid, our findings may provide a better understanding of the pathophysiology of acute renal failure, nephrolithiasis, hyperuricemia, and gout.

Objective: We assessed hTERT mRNA levels in normal versus preeclamptic placental samples, examining hTERT expression levels in different clinical manifestations of hypertensive disorder of pregnancy., Methods: We performed a single-site, prospective case-control study of hTERT mRNA levels in placentas from term and preterm pregnancies with hypertensive disorders compared with unaffected pregnancies. Placental biopsies were collected from 61 patients (preeclamptic: 32; non-preeclamptic (control): 29). Total RNA from placenta was isolated and reversely transcribed to c-DNA. A probe-specific real-time quantitative PCR assay was employed to determine the relative expressional levels of hTERT mRNA levels in these placentas from both unaffected and affected pregnancies with different categories of hypertensive disorders including preeclampsia, severe preeclampsia, eclampsia and HELLP syndrome (Hemolysis, Elevated Liver function tests, Low Platelet)., Results: The average ratio of hTERT mRNA levels was 1.73 in the preeclamptic group and 1.02 for control group (p < 0.0001). The hTERT expression levels were elevated for each of the different categories of hypertensive disorders of pregnancy compared with control: HELLP syndrome 1.86, severe preeclampsia 1.81, eclampsia 1.71 and mild preeclampsia 1.63. In addition, hTERT levels were higher in severe than mild preeclampsia (p < 0.01)., Conclusions:  Elevated hTERT mRNA expression is observed in placentas from pregnancies with different clinical manifestations of hypertensive disorders of pregnancy. The patho-physiological significance of this finding awaits further studies.

Preeclampsia, which affects approximately 5-8% of all pregnancies and is one of the leading causes of maternal and fetal morbidity and mortality, is a pregnancy induced complex of multiple pathological changes, including elevated blood pressure, proteinuria and edema manifested after 20 weeks gestation. There is growing evidence that placental stresses during pregnancy, notably hypoxia, and an increase in circulating soluble Flt-1 (sFlt-1) are important in the etiopathogenesis of preeclampsia. How placental stress results in elevated sFlt-1 expression is currently unknown. Here we provide novel data implicating the Gadd45a stress sensor protein as an upstream modulator of pathophysiological changes observed in preeclampsia. It is shown that Gadd45a expression and activation of its downstream effector p38 kinase are elevated in preeclamptic placentas compared to non-preeclamptic controls, and correlate with elevated sFlt-1. Furthermore, a regulatory loop is demonstrated where stress, including hypoxia, IL-6 or hypertonic stress, caused induction of Gadd45a, leading to p38 activation and ultimately increasing sFlt-1 secretion in endothelial cells. These data provide a compelling working frame to further test the role of Gadd45 stress sensors in the etiology of preeclampsia, and set the stage for considering novel therapeutic regimens, including p38 inhibitors, for treatment of preeclampsia.

Context: Familial hyperkalemia and hypertension (FHHt) is caused by mutations in WNK kinases. Its pathogenesis is not completely understood., Objective: Our objective was to investigate the mechanism of hypercalciuria in FHHt., Design and Setting: We conducted a study of a large family with FHHt and WNK4 Q565E mutation and of control subjects at a referral medical center., Subjects: Forty-six members of a family with FHHt and WNK4 Q565E mutation, 23 of them affected, and 12 control subjects participated., Main Outcome Measures: Urinary calcium and sodium concentrations, endogenous lithium clearance, age of hypertension appearance were assessed., Results: In 40 urine samples of 20 affected subjects, urinary calcium was correlated to urinary sodium (r = 0.567; P = 0.0001). In 28 urinary samples of 22 unaffected members, no correlation was found (r = 0.285; P = 0.14). Mean ratio of urinary calcium to urinary sodium was 2.7-fold higher in affected compared with unaffected members (58.7 +/- 25.9 vs. 22.1 +/- 14.0 micromol/mmol, P < 0.0001). Endogenous lithium clearance in eight affected members was about 50% lower than in 12 controls (16.2 +/- 7.7 vs. 28.8 +/- 9.8 ml/min, P = 0.0073). Hypertension was detected in males 12 yr earlier than in females (26.0 +/- 7.5 vs. 37.9 +/- 11.3 yr; P = 0.031)., Conclusions: Hypercalciuria in FHHt seems to be dependent on urinary sodium. According to molecular studies, FHHt patients are presumed to have increased distal nephron sodium reabsorption and therefore decreased proximal reabsorption of sodium, lithium, and calcium. The observed decreased lithium clearance reflects probable abnormal renal handling of lithium, i.e. distal nephron lithium reabsorption. Therefore, hypercalciuria may result from proximal nephron aberration. Finally, earlier appearance of hypertension in males may be the result of sex-hormone activity.

Purpose: Genetic causes of nephrolithiasis are underestimated. Primary hyperoxaluria type 2 is a rare autosomal recessive disease caused by mutations in the GRHPR gene, leading to an accumulation of oxalate and L-glycerate with recurrent kidney stone formation and nephrocalcinosis, and the later development of renal failure and systemic oxalate depositions. We studied the effects of a novel GRHPR mutation on GRHPR enzymatic activity and molecular modeling., Materials and Methods: Genomic DNA from a 50-year-old male with a late diagnosis of primary hyperoxaluria type 2 was extracted, analyzed and compared with the established human GRHPR gene sequence. Restriction enzyme analysis of the patient, 30 healthy controls and 30 patients with nephrolithiasis of various causes was done to confirm the presence of the mutation. GRHPR activity was analyzed by site directed mutagenesis of WT and mutant clones. We studied the effects of the mutation on enzymatic molecular modeling., Results: We found the novel homozygous single missense mutation A975G in exon 9, creating an amino acid change from asparagine to aspartic acid in position 312. No mutations were detected in restriction enzyme analysis in all 30 healthy controls and 30 patients with nephrolithiasis of various causes. Transfected cells with the mutant clone showed abolished GRHPR activity. Molecular modeling studies revealed that the mutation was likely to disrupt the correct folding of the GRHPR substrate binding domain, hence affecting the enzyme active site., Conclusions: Primary hyperoxaluria type 2 should be considered in patients at adult stone clinics who have had a history of nephrolithiasis since childhood, especially in those with consanguineous parents. Biochemical analysis followed by mutation identification should be the approach for making the definitive diagnosis of primary hyperoxaluria type 2.

Background: The successful prevention of RhD disease has brought attention to other red blood cells' antigens causing alloimmunisation including RhC/c and RhE/e. Prenatal diagnosis of fetal Rh genotype from maternal blood is in clinical use in Europe but not in the USA., Objective: To estimate the collective reported diagnostic accuracy of fetal RhCE genotyping from peripheral maternal blood and compare the results of genotyping when fetal cells and free fetal DNA (FfDNA) are used., Search Strategy: English-written literature describing fetal RhCE determination from maternal blood using fetal cells or FfDNA was performed using medical subject headings and text words. The sources included Pubmed (1966-2007), Ovid (1966-2007), CINAHL, The Cochrane Library, ACP Journal Club and OCLC. Key words were prenatal diagnosis, fetal RhCE, fetal DNA in maternal blood and alloimmunisation., Selection Criteria: A study was considered eligible if it described fetal RhCE type determination using maternal peripheral blood reported in the English literature. Abstracts were excluded., Data Collection and Analysis: From each study, we determined the number of samples tested, fetal RhCE genotype, the source of the fetal DNA, gestational age, presence of alloimmunisation and confirmation of fetal RhCE type. Exclusions and inclusions were noted. We calculated composite estimates of accuracy using a weighted random effects model. We assessed the papers against an international quality, STARD checklist which is standards for reporting studies of diagnostic accuracy., Main Results: We identified 20 protocols in six English-written publications reporting fetal RhC/c (seven protocols) and/or E/e (13 protocols) genotyping using DNA obtained from maternal blood for a total of 369 samples. For RhC/c, 176 samples were tested and for RhE/e, 193 samples were tested. Accuracy was determined for each study and for all studies. The combined accuracy of fetal genotype was 96.3% for RhC/c and 98.2% for RhE/e. Only a few samples of the sorted cells were found to be a source for accurate diagnosis, but plasma was consistently the best source of fetal RhCE genotyping in 147/147 (100%) for RhC/c and 168/168 (100%) for RhE/e., Conclusions: The combined accuracy of noninvasive fetal RhC/c or RhE/e determination using maternal peripheral blood is 96.3% and 98.2%, respectively. FfDNA in maternal plasma is a better source for genotyping reported to be 100% correct for both RHCE genotypes. Further studies and reports of accuracy from laboratories performing the tests are required before prenatal determination of fetal RhC/c or RhE/e genotypes from maternal blood can safely replace the current methods used in the management of the RhC/c or RhE alloimmunised pregnancies.

Dent's disease is an X-linked hereditary renal tubular disorder characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and progressive renal failure. About 60% of patients have mutations in the CLCN5 gene (Dent 1), which encodes a kidney-specific chloride/proton antiporter, and 15% of patients have mutations in the OCRL1 gene (Dent 2). The aim of the study was to identify CLCN5 mutations in Jewish Israeli families with Dent's disease and to characterize the associated clinical syndromes. We studied 17 patients from 14 unrelated Israeli families with a clinical diagnosis of Dent's disease. LMWP was detected in all patients. Most of the affected individuals had hypercalciuria and nephrocalcinosis. Renal stones were found in 1 patient, and renal insufficiency developed in 2 patients. We identified six different truncating CLCN5 mutations that were segregated with the disease in 7 families: three nonsense mutations (Arg28stop, Arg467stop and Arg637stop), one deletion mutation (505delA) and two novel mutations, consisted of one deletion mutation (1493delG) and one insertion mutation (409insC). All the mutations cause premature termination of protein translation and result in a non-functional truncated protein. The clinical characteristics of patients with different mutations were, in general, similar., (Copyright 2009 S. Karger AG, Basel.)

WNK4 kinase mutations produce the autosomal dominant disorder familial hyperkalemia and hypertension (FHH), also known as pseudohypoaldosteronism type II, by a molecular mechanism that is not completely understood. In vitro experiments in frog oocytes showed that WNK4 affects ion transport systems such as the Na-Cl cotransporter and the renal outer medullary potassium channel. Some features of FHH suggest that long-term effects are involved in WNK4 signaling. In addition, WNK1 and WNK2, paralogs of WNK4, were shown to be involved in MAP kinase signaling. We therefore investigated possible WNK4 involvement in MAP kinase signaling. We stimulated HEK 293 cells overexpressing WNK4 by hypertonicity or using EGF, and measured phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38. WNK4 augmented the phosphorylation of ERK1/2 and p38 in response to both hypertonicity and EGF. The FHH-producing and kinase-deficient mutants behaved similarly to wild-type WNK4. Hypertonicity stimulation was accompanied by cellular relocalization of WNK4 as manifested by its reversible disappearance from the supernatant fraction following extraction with a detergent-containing buffer. Live-cell microscopy showed that the cytoplasmic-soluble WNK4 redistributes rapidly to membrane-bound organelles, which, in the case of WNK1 kinase, were recently shown to represent trans-Golgi network/recycling endosomes. In contrast, EGF stimulation was not accompanied by redistribution of WNK4 as determined by cell fractionation or cell microscopy. The observation that WNK4-induced MAP kinase stimulation caused by hypertonicity, but not that caused by EGF, is associated with WNK4 subcellular redistribution suggests that this redistribution has a role in WNK4 signaling.

Background: Familial hyperkalaemia and hypertension (FHH), also termed pseudohypoaldosteronism type II, is a rare monogenic form of hypertension caused by mutations in the WNK1 or WNK4 kinases. In vitro expression of WNK4 reduces surface abundance and activity of coexpressed NaCl cotransporter (NCCT). This effect is lost in disease-producing WNK4 mutants. In two mice models of FHH, one expressing two extra copies of mutant WNK4 (Q562E) and another in which a mutant (D561A) WNK4 replaced wild-type WNK4, renal distal tubule hyperplasia with overexpression of NCCT was found. Currently no FHH human renal tissue is available to test for increased distal tubule surface abundance of NCCT. The availability of a unique large family with FHH and the Q565E WNK4 mutation enabled us to investigate this issue in an indirect manner., Methods: Assuming that shedding of NCCT to the urine reflects its abundance in the distal tubule epithelium, we measured urinary NCCT protein in eight subjects of the FHH family and in eight unrelated controls by western blotting., Results: Urinary NCCT protein was about four times higher in FHH than in controls [111.1 +/- 40.5 versus 26.1 +/- 16.4 densitometry units (P < 0.0001)]. No significant difference in urinary sodium and potassium concentrations was seen between FHH and controls., Conclusions: The increased urinary NCCT in FHH most probably reflects increased NCCT abundance in the apical membrane of distal tubule cells in patients with FHH and the WNK4 mutation and points to the pathogenetic mechanism for the clinical phenotype of FHH and the WNK4 mutation, supporting results in transgenic mice with the same mutation and in knockin mice with another mutation.

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).

Familial hyperkalemia and hypertension (FHH; pseudohypoaldosteronism type II) is an autosomal dominant disorder characterized by hyperkalemia, hypertension, and low renin. WNK1 kinase overexpression and WNK4 kinase inactivating missense mutations cause FHH. When expressed in frog oocyte, WNK4 inhibits Na-Cl cotransporter surface expression, and WNK1 relieves this inhibition. We have reported hypercalciuria in subjects with the WNK4 Q565E mutation. In contrast, in subjects with WNK1 overexpression, normocalciuria was found. Here we report a major extension of our previously described kindred that contains 34 subjects, 18 of them affected by the mutation. Hypertension was diagnosed in 13 affected subjects at the age of 31 +/- 12 yr. Five of the affected or obligatory affected subjects had stroke, in four at the age of 50-62 yr. Seven subjects with FHH were diagnosed 27 yr previously. All four subjects who were normotensive at diagnosis became hypertensive during follow-up. The mean time between detection of hyperkalemia and appearance of hypertension was 13 yr. In the extended kindred, compared with the unaffected subjects, affected subjects had hyperkalemia, low transtubular potassium gradient, hyperchloremia, low bicarbonate, higher aldosterone, and marked suppression of renin. Urinary calcium levels in affected and unaffected subjects were 0.85 +/- 0.27 and 0.28 +/- 0.12 mmol/mmol creatinine, respectively (P < 0.0001). Hypercalciuria was accompanied by lower serum calcium levels [9.44 +/- 0.15 vs. 9.81 +/- 0.31 mg/dl (2.36 +/- 0.04 vs. 2.45 +/- 0.08 mmol/liter); P = 0.01], supporting a mechanism of renal calcium leak. The six affected, currently normotensive subjects had the same degree of hyperkalemia, hypercalciuria, and low renin as the affected hypertensive subjects. We conclude that in FHH with WNK4 mutations, with time all affected subjects will apparently develop hypertension. Hypercalciuria accompanies hyperkalemia, and both precede hypertension. Based on the recent findings that WNK4 regulates the renal outer medullary potassium channel as well as epithelial Cl(-)/base exchanger and the Na(+)-K(+)-2Cl(-) cotransporter, we suggest that WNK4 interacts with a calcium channel or transporter.

Mitochondrial DNA plays a crucial role in oxidative production of energy. Thus, defects in mitochondrial DNA can affect virtually all organ systems. The point mutation A --> G at position 3243 in the mitochondrial tRNAleu(UUR) gene is the cause of several distinct types of mitochondrial cytopathy and several clinical phenotypes, including encephalomyopathy with lactic acidosis and stroke-like episodes and maternally inherited diabetes and deafness. This mutation has been recently described also in association with kidney disease, mainly focal and segmental glomerulosclerosis. At present, little is known about the prevalence of this mitochondrial nephropathy, its clinical course and the pathogenesis of glomerular damage. We describe 2 unrelated patients, who presented with proteinuria and progressed to end-stage renal failure. Other clinical features were short stature, severe headache, hearing loss, diabetes mellitus and hypertrophic cardiomyopathy. The main histological finding was an increased number of abnormal mitochondria in tubular cells and podocytes. Analysis of mitochondrial DNA from leukocytes and urine sediment revealed heteroplasmy for the A3243G mutation in tRNAleu(UUR) gene in both patients. Recognition of the characteristic clinical and histological features of the mitochondrial A3243G mutation-associated glomerulopathy will enable correct diagnosis and better management of a disease which is likely to be underdiagnosed.

The KCl cotransporter (KCC) plays a significant role in the ionic and osmotic homeostasis of many cell types. Four KCC isoforms have been cloned. KCC1 and KCC4 activity is osmolality-sensitive and involved in volume regulation. KCC2, a neuronal-specific isoform, can lower intracellular Cl(-) and is critical for inhibitory GABA responses in the mature central nervous system. KCC3, initially cloned from vascular endothelial cells, is widely but not universally distributed and has an unknown physiological significance. Here we show a tight link between the expression and activity of KCC3 and cell growth by a NIH/3T3 fibroblast expression system. KCC3 activity is sensitive to [(dihydroindenyl)oxy] alkanoic acid (DIOA) and N-ethylmaleimide and is regulated by tyrosine phosphorylation. Osmotic swelling does not activate KCC3, and the process of regulatory volume decrease is refractory to DIOA, indicating that KCC3 is not involved in volume regulation. KCC3 expression enhances cell proliferation, and this growth advantage can be abolished by the inhibition of KCC3 by DIOA. Fluorescence-activated cell sorting measurements and Western blot analysis show DIOA caused a significant reduction of the cell fraction in proliferative phase and a change in phosphorylation of retinoblastoma protein (Rb) and cdc2, suggesting that KCC3 activity is important for cell cycle progression. Insulin-like growth factor-1 up-regulates KCC3 expression and stimulates cell growth. Tumor necrotic factor-alpha down-regulates KCC3 expression and causes growth arrest. These data indicate that KCC3 is an important KCC isoform that may be involved in cell proliferation.

Objective: This study was undertaken to determine the fetal E/e or e/e Rh genotype prenatally from peripheral maternal blood by examining sorted fetal cells from alloimmunized and nonalloimmunized pregnancies., Study Design: Eighteen maternal peripheral venous blood samples were obtained before amniocentesis from 15 pregnant women who were homozygous for the e allele. Five were not alloimmunized and 10 were alloimmunized. The mononuclear cell layer was isolated from the maternal blood and enriched for fetal nucleated red blood cells by flow cytometry with monoclonal antibodies to CD36 or CD71 and to glycophorin A. Eight samples were treated with CD45 monoclonal antibody-coated magnetic beads before they were sorted to deplete the maternal sample of leukocytes (CD45(+) cells). We defined the positive fetal cell fractions as the monoclonal antibody positive-sorted cells derived from the maternal samples. These included sorted cells that were CD36(+)/glycophorin A(+), CD71(+)/glycophorin A(+) and CD45(-) cells that were sorted to become CD45(-)/CD36(+)/glycophorin A(+) or CD45(-)/CD71(+)/glycophorin A(+). The negative fractions were the cells that were negative for either CD36/glycophorin A or CD71/glycophorin A or were the CD45(+) cells. Deoxyribonucleic acid was isolated from all fractions and amplified by polymerase chain reaction with allele-specific primers for the E or e Rh genes. Gel electrophoresis was performed to detect fetal E/e or e/e Rh genotype. The fetal E/e or e/e Rh genotype was confirmed by serologic and deoxyribonucleic acid testing. The accuracy of E/e or e/e Rh genotype determination from the positive cell fractions was compared with that of E/e or e/e Rh genotype determination from the negative fractions., Results: Fetal E/e or e/e Rh genotype was determined correctly in 17 of 18 of the fetal cell enriched positive fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 11 of 14 of the maternal samples in the negative unselected cell fractions (79%). Fetal E/e or e/e Rh genotype was determined correctly in 15 of 16 sample fractions that underwent magnetic bead separation with CD45 and were subsequently sorted into positive and negative fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 13 of 13 of the samples obtained from the alloimmunized pregnancies (100%)., Conclusions: The use of monoclonal antibodies for cell sorting or for magnetic separation predicted fetal E/e or e/e Rh genotype from peripheral maternal blood correctly in as many as 100% of alloimmunized pregnancies. Thus noninvasive fetal E/e or e/e Rh genotyping can be performed by polymerase chain reaction amplification of the rare fetal cells in maternal blood. The correct prediction of fetal E/e or e/e Rh genotype from the cell population not selected by the monoclonal antibodies suggests that there are fetal cell types other than fetal nucleated erythrocytes that can also be used as a source of fetal deoxyribonucleic acid for noninvasive genetic diagnosis. Improved technology may provide methods less laborious than cell sorting to accurately determine fetal Rh type from different fetal cell types that circulate in maternal blood.

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75-76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and approximately 20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH(2) and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide., (Copyright 1999 Academic Press.)