Gadd45 in Preeclampsia.

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.

Treatment of Severe Renal Artery Stenosis with Acute Kidney Injury Requiring Hemodialysis by Percutaneous Transluminal Renal Angioplasty and Stent Implantation.

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.

Familial Hyperkalemia and Hypertension (FHHt) and KLHL3: Description of a Family with a New Recessive Mutation (S553L) Compared to a Family with a Dominant Mutation, Q309R, with Analysis of Urinary Sodium Chloride Cotransporter.

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.

Normal arterial stiffness in familial Mediterranean fever. Evidence for a possible cardiovascular protective role of colchicine.

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.

High Normal Uric Acid Levels Are Associated with an Increased Risk of Diabetes in Lean, Normoglycemic Healthy Women.

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.

Uric acid levels within the normal range predict increased risk of hypertension: a cohort study.

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.

Hypercalciuria in familial hyperkalemia and hypertension with KLHL3 mutations.

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.

Wild-type uromodulin prevents NFkB activation in kidney cells, while mutant uromodulin, causing FJHU nephropathy, does not.

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)-1ß 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.

Gadd45 stress sensors in preeclampsia.

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.

Preeclampsia-associated stresses activate Gadd45a signaling and sFlt-1 in placental explants.

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.

Two novel homozygous SLC2A9 mutations cause renal hypouricemia type 2.

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.

Cyclosporine metabolic side effects: association with the WNK4 system.

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.

URAT1 mutations cause renal hypouricemia type 1 in Iraqi Jews.

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.

Effect of age and affection status on blood pressure, serum potassium and stature in familial hyperkalaemia and hypertension.

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.

Increased placental telomerase mRNA in hypertensive disorders of pregnancy.

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.

Homozygous SLC2A9 mutations cause severe renal hypouricemia.

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.

Familial hyperkalemia and hypertension: pathogenetic insights based on lithium clearance.

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.

Gadd45a stress signaling regulates sFlt-1 expression in preeclampsia.

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.

Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF.

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.

Increased urinary Na-Cl cotransporter protein in familial hyperkalaemia and hypertension.

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.