Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

An unusual patient with hypercalciuria, recurrent nephrolithiasis, hypomagnesemia and G227R mutation of Paracellin-1. An unusual patient with hypercalciuria and hypomagnesemia unresponsive to thiazide diuretics

A 19-year-old female patient with hypercalciuria and recurrent nephrolithiasis/urinary tract infection unresponsive to thiazide type diuretics is presented. The patient first experienced nephrolithiasis at the age of 4 years. Afterwards, recurrent passages of stones and urinary tract infection occurred. On diagnostic evaluation at the age of 19 years, she also had hypocitraturia and hypomagnesemia. Her serum calcium concentrations were near the lower limit of normal (8.5-8.8 mg/dl; normal range: 8.5-10.5), her serum magnesium concentrations were 1.15-1.24 mg/dl (normal range: 1.4-2.5) and urinary calcium excretion was 900 mg/24 h. PTH concentrations were increased (110-156 pg/ml; normal range: 10-65). We tried to treat the patient with hydrochlorothiazide at a dose of 50 mg/day. During treatment with thiazide diuretics, PTH concentration remained high and the patient had recurrent urinary tract infections and passages of stones. Serum magnesium concentration did not normalize even under the parenteral magnesium infusion. Her mother had a history of nephrolithiasis 20 years ago. Severe hypomagnesemia in association with hypercalciuria/urinary stones is reported as a rare autosomal recessive disorder caused by impaired reabsorption of magnesium and calcium in the thick assending limp of Henle's loop. Recent studies showed that mutations in the CLDN16 gene encoding paracellin-1 cause the disorder. In exon 4, a homozygous nucleotide exchange (G679C) was identified for the patient. This results in a point mutation at position Glycine227, which is replaced by an Arginine residue (G227R). The mother was heterozygous for this mutation. G227 is located in the fourth transmembrane domain and is highly conserved in the claudin gene family. This case indicates the pathogenetic role of paracellin-1 mutation in familial hypomagnesemia with hypercalciuria and nephrocalcinosis and further underlines the risk of stone formation in heterozygous mutation carriers.

A Spanish founder mutation in the chloride channel gene, CLCNKB, as a cause of atypical Bartter syndrome in adult age

BACKGROUND: Mutations in the chloride channel gene, CLCNKB, usually cause classic Bartter syndrome (cBS) or a mixed Bartter-Gitelman phenotype in the first years of life. METHODS: We report an adult woman with atypical BS caused by a homozygous missense mutation, A204T, in the CLCNKB gene, which has previously been described as the apparently unique cause of cBS in Spain. RESULTS: The evaluation of this patient revealed an overlap of phenotypic features ranging from severe biochemical and systemic disturbances typical of cBS to scarce symptoms and diagnosis in the adult age typical of Gitelman syndrome. The tubular disease caused a dramatic effect on mental, growth and puberal development leading to low IQ, final short stature and abnormal ovarian function. Furthermore, low serum PTH concentrations with concomitant nephrocalcinosis and normocalcaemia were observed. Both ovarian function and serum PTH levels were normalized after treatment with cyclooxygenase inhibitors. CONCLUSIONS: The present report confirms a weak genotype-phenotype correlation in patients with CLCNKB mutations and supports the founder effect of the A204T mutation in Spain. In our country, the genetic diagnosis of adult patients with hereditary hypokalaemic tubulopathies should include a screening of A204T mutation in the CLCNKB gene.

Seasonal changes in bone metabolism in sheep

There is a great need for animal models of osteoporosis and sheep are a suitable large animal that meets most requirements. Since it is known that bone mass in humans responds to seasonal changes, this study investigated natural bone metabolism in sheep in order to better define the sheep as a model for osteoporosis. Bone mineral density (BMD), trabecular structure, biochemical markers of bone formation and resorption and estrogen were analysed over a period of 18 months. The lowest BMDs, measured by peripheral quantitative computed tomography (pQCT), were observed during winter. Thereafter, a 5.1% increase in BMD was observed during spring and summer (P<0.05). Bone resorption markers showed a variable pattern, with higher values in spring compared to autumn (P<0.001). The physiological estrus phase during autumn was detected by serum estrogen levels. The findings show that it is necessary to take seasonal variations into account if sheep are used to establish an animal model for osteoporosis.

Effect of cinacalcet cessation in renal transplant recipients with persistent hyperparathyroidism

BACKGROUND: Persistent hyperparathyroidism after renal transplantation affects bone and allografts. Cinacalcet, a calcimimetic, reduces serum calcium and PTH in renal transplant recipients with persistent hyperparathyroidism. Here, we address the question whether this effect of cinacalcet persists after withdrawal. METHODS: Therefore, cinacalcet was stopped after 12 months treatment in 10 stable renal transplant patients. Serum calcium, phosphate, PTH, creatinine and cystatin C were monitored for 3 months. RESULTS: Serum calcium, normalized in nine patients before cessation of cinacalcet (2.32 +/- 0.05mmol/l, mean +/- SEM), increased after 3 months of discontinuation by 0.17 +/- 0.04mmol/l, P < 0.05, but remained within the normal range in eight patients. Compared with the time point of cessation, PTH remained unchanged or decreased further after 3 months without therapy in six patients. Measurements of cystatin C suggested an improvement of the glomerular filtration rate after cessation in 9 out of 10 patients (1.55 +/- 0.09 vs 1.33 +/- 0.12 mg/l, P < 0.01). CONCLUSION: First, a beneficial effect of cinacalcet beyond the duration of a 12-month therapy appears to be present in some patients and second, the previously suspected influence of cinacalcet therapy on renal function is reversible. Thus, it is reasonable to consider a trial of cinacalcet cessation to identify these patients. The optimal time point for such a discontinuation is unknown. The present observations are preliminary. They clearly require a prospective randomized trial for definitive confirmation.

Infliximab inhibits bone resorption by circulating osteoclast precursor cells in patients with Rheumatoid Arthritis and Ankylosing Spondylitis

OBJECTIVE: To examine the effects of infliximab on bone resorption by osteoclast precursor cells (OCPs) in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) and to compare the results with changes in disease activity. METHODS: Before and during 24 weeks of infliximab treatment peripheral blood mononuclear cells of 9 RA and 10 AS patients were seeded onto ivory wafers and adherent cells, including OCPs, were grown in medium promoting osteoclast differentiation. Bone resorption was evaluated morphometrically and correlated to disease activity. 19 healthy individuals were studied in parallel. In addition, biochemical bone markers were assessed in all patients at baseline and after 24 weeks. RESULTS: OCPs from RA patients showed a higher bone resorption at baseline when compared to AS patients. Blocking of TNFalpha with infliximab resulted in a strong reduction of bone resorption by OCPs in both cohorts and did occur faster in RA compared to AS patients. This inhibition coincided with reduction of clinical disease activity in both patient cohorts and with an increase of serum osteocalcin levels and a relative decrease of collagen crosslinks in RA compared to AS patients. CONCLUSION: These results provide an explanation on the cellular level for the anticatabolic effect of TNF neutralization on bone. The variation in the kinetics of bone resorption by the OCPs in patients with RA and AS suggests disease-specific differences in the type or in the preactivation of OCPs.

A randomized comparison of the safety and efficacy of once-daily gentamicin or thrice-daily gentamicin in combination with ticarcillin-clavulanate

PURPOSE: The primary purpose of the clinical trial was to assess the safety and efficacy of once-a-day compared with three-times-a-day gentamicin in patients with serious infections who had protocol-determined peak serum aminoglycoside concentrations. PATIENTS AND METHODS: A total of 249 hospitalized patients with suspected or proven serious infections were randomized in a 2:2:1 ratio to gentamicin given three times a day with ticarcillin-clavulanate (TC), gentamicin once a day with TC, or ticarcillin-clavulanate (TC) alone. The gentamicin once-a-day dosage for patients with estimated creatinine clearance values of > or =80 mL/min was 5.1 mg/kg. With lower creatinine clearance estimates, the mg/kg dosage of gentamicin was decreased, and the dosage intervals (once daily or three times a day) were maintained. Evaluability required documentation of achievement of protocol-defined peak serum gentamicin levels. RESULTS: Of the total 175 evaluable patients, there were no significant differences found between treatment regimens with respect to clinical or microbiologic efficacy. Bedside audiometry proved impractical due to the frequency of altered mental state in ill patients. Based on the traditional increase in serum creatinine values from baseline values, no differences in renal toxicity between the treatment groups was identified. When changes in renal function were reanalyzed based on maintaining, as opposed to worsening, of renal function, preservation of renal function was better in the gentamicin once-a-day patients as opposed to the gentamicin three-times-a-day patients, P <0.01. CONCLUSIONS: Gentamicin once a day plus TC, gentamicin three times a day plus TC, and TC alone had similar effects in seriously ill hospitalized patients. The incidence of nephrotoxicity was similar in the three treatment groups. Using a nonvalidated post-hoc analysis, renal function was better preserved in gentamicin once-a-day + TC and TC-only patients as opposed to gentamicin three-times-a-day + TC.

Impact of changing definitions for myocardial infarction: a report from the AMIS registry

BACKGROUND: To assess the impact of the new definitions of myocardial infarction, we retrospectively analyzed 9190 patients from 63 hospitals with reported peak troponin values included between 2001 and 2007 in the Swiss AMIS (Acute Myocardial Infarction in Switzerland) Plus registry. METHODS: Patients were classified as belonging to the "classic" myocardial infarction group (peak total CK or CK-MB above the upper limit of normal, or troponin T [TnT] >0.1 microg/L or troponin I [TnI] >0.1-0.8 microg/L [depending on the assay]) or "new" myocardial infarction group (TnT >0.01 microg/L or TnI >0.01-0.07 microg/L). RESULTS: There were 489 patients in the "new" group who were similar to the 8701 "classic" patients in terms of age, sex, and prevalence of both diabetes and renal failure, but more frequently had a history of prior coronary artery disease, hypertension, and hyperlipidemia. At admission, they less frequently had ST elevation on their electrocardiogram, were more frequently in Killip class I, and received less primary percutaneous coronary intervention. Hospital mortality was 3.5% in the "new" and 6.7% in the "classic" myocardial infarction group (P=.004). In a subset of patients with a longer follow-up, mortality at 3 and 12 months was 1% and 5.6%, respectively, for "new" and 1.6% and 4%, respectively, for "classic" myocardial infarction (NS). CONCLUSIONS: Patients with minimal elevation of serum troponin have smaller infarctions, less aggressive treatment, fewer early complications, and a better early prognosis than patients with higher serum biomarker levels. After discharge, however, their prognosis currently appears no different from that of patients with a "classic" myocardial infarction event.

Infantile hypophosphatasia due to a new compound heterozygous TNSALP mutation - functional evidence for a hydrophobic side-chain?

BACKGROUND: Infantile hypophosphatasia (IH) is an inherited disorder characterized by defective bone mineralization and a deficiency of alkaline phosphatase activity. OBJECTIVE/DESIGN: The aim of the study was to evaluate a new compound heterozygous TNSALP mutation for its residual enzyme activity and localization of the comprised amino acid residues in a 3D-modeling. PATIENT: We report on a 4-week old girl with craniotabes, severe defects of ossification, and failure to thrive. Typical clinical features as low serum alkaline phosphatase, high serum calcium concentration, increased urinary calcium excretion, and nephrocalcinosis were observed. Vitamin D was withdrawn and the patient was started on calcitonin and hydrochlorothiazide. Nonetheless, the girl died at the age of 5 months from respiratory failure. RESULTS: Sequence analysis of the patient's TNSALP gene revealed two heterozygous mutations [c.653T>C (I201T), c.1171C>T (R374C)]. Transfection studies of the unique I201T variant in COS-7 cells yielded a mutant TNSALP protein with only a residual enzyme activity (3.7%) compared with wild-type, whereas the R374C variant was previously shown to reduce normal activity to 10.3%. 3D-modeling of the mutated enzyme showed that I201T resides in a region that does not belong to any known functional site. CONCLUSION: We note that I201, which has been conserved during evolution, is buried in a hydrophobic pocket and, therefore, the I>T-change should affect its functional properties. Residue R374C is located in the interface between monomers and it has been previously suggested that this mutation affects dimerization. These findings explain the patient's clinical picture and severe course.

Effect of interferon-beta and atorvastatin on Th1/Th2 cytokines in multiple sclerosis

Beneficial effects by both interferon-beta and statin treatment in patients with multiple sclerosis (MS) may be linked to interference with the Th1/Th2 cytokine balance. We determined patterns of Th1/Th2 cytokines (interleukin (IL)-1beta, IL-2, IL-6, IL-12p70, tumor-necrosis factor (TNF)-alpha and interferon-gamma, and IL-4, IL-5 and IL-10, respectively) in the serum of patients with relapsing-remitting MS treated with 250microg interferon-beta 1b or with interferon-beta plus 40mg atorvastatin. In treatment naïve patients with MS, a trend for lower TNF-alpha serum levels compared to controls was detected (P=0.08). Interferon-beta treatment increased TNF-alpha levels, while a trend for lowering of IL-5 serum levels was found (P=0.07). Addition of atorvastatin raised IL-12p70 serum levels (P<0.05). Mean levels of two Th2 cytokines (IL-4, IL-10) showed a non-significant increase after addition of atorvastatin. We conclude that interferon-beta and atorvastatin exert divergent action on Th1/Th2 serum cytokines levels in MS. Supplemental atorvastatin might promote a Th1-type response by raising IL-12p70. Further studies are required to support a Th2 cytokine shift by atorvastatin in patients with MS.

Copper availability contributes to iron perturbations in human nonalcoholic fatty liver disease

BACKGROUND ; AIMS: Iron perturbations are frequently observed in nonalcoholic fatty liver disease (NAFLD). We aimed to investigate a potential association of copper status with disturbances of iron homeostasis in NAFLD. METHODS: We retrospectively studied 140 NAFLD patients and 25 control subjects. Biochemical and hepatic iron and copper parameters were analyzed. Hepatic expression of iron regulatory molecules was investigated in liver biopsy specimens by reverse-transcription polymerase chain reaction and Western blot analysis. RESULTS: NAFLD patients had lower hepatic copper concentrations than control subjects (21.9 +/- 9.8 vs 29.6 +/- 5.1 microg/g; P = .002). NAFLD patients with low serum and liver copper concentrations presented with higher serum ferritin levels (606.7 +/- 265.8 vs 224.2 +/- 176.0 mg/L; P < .001), increased prevalence of siderosis in liver biopsy specimens (36/46 vs 10/47 patients; P < .001), and with elevated hepatic iron concentrations (1184.4 +/- 842.7 vs 319.9 +/- 451.3 microg/g; P = .020). Lower serum concentrations of the copper-dependent ferroxidase ceruloplasmin (21.7 +/- 4.1 vs 30.4 +/- 6.4 mg/dL; P < .001) and decreased liver ferroportin (FP-1; P = .009) messenger RNA expression were found in these patients compared with NAFLD patients with high liver or serum copper concentrations. Accordingly, in rats, a reduced dietary copper intake was paralleled by a decreased hepatic FP-1 protein expression. CONCLUSIONS: A significant proportion of NAFLD patients should be considered copper deficient. Our results indicate that copper status is linked to iron homeostasis in NAFLD, suggesting that low copper bioavailability causes increased hepatic iron stores via decreased FP-1 expression and ceruloplasmin ferroxidase activity thus blocking liver iron export in copper-deficient subjects.

Increased risk of hepatocellular carcinoma among patients with hepatitis C cirrhosis and diabetes mellitus

Recent studies suggest that diabetes mellitus increases the risk of developing hepatocellular carcinoma (HCC). The aim of this study is to quantify the risk of HCC among patients with both diabetes mellitus and hepatitis C in a large cohort of patients with chronic hepatitis C and advanced fibrosis. We included 541 patients of whom 85 (16%) had diabetes mellitus. The median age at inclusion was 50 years. The prevalence of diabetes mellitus was 10.5% for patients with Ishak fibrosis score 4, 12.5% for Ishak score 5, and 19.1% for Ishak score 6. Multiple logistic regression analysis showed an increased risk of diabetes mellitus for patients with an elevated body mass index (BMI) (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.00-1.11; P = 0.060) and a decreased risk of diabetes mellitus for patients with higher serum albumin levels (OR, 0.81; 95% CI, 0.63-1.04; P = 0.095). During a median follow-up of 4.0 years (interquartile range, 2.0-6.7), 11 patients (13%) with diabetes mellitus versus 27 patients (5.9%) without diabetes mellitus developed HCC, the 5-year occurrence of HCC being 11.4% (95% CI, 3.0-19.8) and 5.0% (95% CI, 2.2-7.8), respectively (P = 0.013). Multivariate Cox regression analysis of patients with Ishak 6 cirrhosis showed that diabetes mellitus was independently associated with the development of HCC (hazard ratio, 3.28; 95% CI, 1.35-7.97; P = 0.009). CONCLUSION: For patients with chronic hepatitis C and advanced cirrhosis, diabetes mellitus increases the risk of developing HCC.

Proliferation and apoptosis in mammary epithelium during the rat oestrous cycle

AIM: During each oestrous cycle, the mammary gland is subject to changes in ovarian hormone levels. It responds with limited proliferation, differentiation and regression. To understand the processes resulting in these changes, particularly the regulation of cell death, we examined protein levels in mammary epithelium during the oestrous cycle of the Sprague-Dawley rat. METHODS: Studies of serum hormone levels, vaginal smears, uterine weight and morphology, mammary gland morphology, proliferation and apoptotic indices, and protein levels during the stages of the Sprague-Dawley rat oestrous cycle were used to examine the response of mammary epithelium to the oestrous cycle. RESULTS: Proliferation of mammary epithelium was greater in diestrus and proestrus, while apoptosis was increased in metestrus and diestrus. Growth factor-, hormone- and anchorage-mediated cell survival signalling, indicated by activation of Stat5A, FAK and Akt 1 and expression of anti-apoptotic Bcl-2 family members, was greater in proestrus and reduced in metestrus. In contrast, the levels of pro-apoptotic Bcl-2 family members and proteins associated with apoptosis in mammary epithelium (TGFbeta3, pStat3) were increased during metestrus and diestrus. CONCLUSION: Decreases in growth factor, hormone and cell attachment survival signals corresponded with increased apoptosis during the second half of the oestrous cycle. The protein levels detected during oestrus suggest parallels to apoptosis in mammary involution.

Photodynamic therapy for cholangiocarcinoma: overview and new developments

PURPOSE OF REVIEW: Photodynamic therapy (PDT) with hematoporphyrins has emerged as promising treatment for nonresectable cholangiocarcinoma in several prospective observational studies and two randomized studies. This review describes the mechanism of action of PDT, gives an overview of clinical experience in cholangiocarcinoma and summarizes the results published in 2007 and 2008. RECENT FINDINGS: The mechanism of action of PDT has been further elucidated. PDT induces an apoptotic, antiangiogenic as well as an immunomodulatory response. Interleukin-6, a bile duct epithelium growth factor correlating with tumor burden, decreases after PDT. The efficacy of PDT was confirmed in a comparative study in the United States. Patients with no visible mass on imaging studies, high serum albumin levels and treatment immediately after diagnosis seem to benefit most from PDT. Although it is recommended to perform PDT in bile ducts without stents in place, illumination through metal stents is possible if the light dose is adjusted. Meso-tetrahydroxyphenyl chlorine is a new potent photosensitizer for PDT of cholangiocarcinoma. SUMMARY: In advanced nonresectable cholangiocarcinoma, PDT is the only evidence-based treatment that improves survival when compared with stenting. Therefore, PDT should be offered to those who are unsuitable for surgery.

Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood

We report on a female who is compound heterozygote for two new point mutations in the CYP19 gene. The allele inherited from her mother presented a base pair deletion (C) occurring at P408 (CCC, exon 9), causing a frameshift that results in a nonsense codon 111 bp (37 aa) further down in the CYP19 gene. The allele inherited from her father showed a point mutation from G-->A at the splicing point (canonical GT to mutational AT) between exon and intron 3. This mutation ignores the splice site and a stop codon 3 bp downstream occurs. Aromatase deficiency was already suspected because of the marked virilization occurring prepartum in the mother, and the diagnosis was confirmed shortly after birth. Extremely low levels of serum estrogens were found in contrast to high levels of androgens. Ultrasonographic follow-up studies revealed persistently enlarged ovaries (19.5-22 mL) during early childhood (2 to 4 yr) which contained numerous large cysts up to 4.8 x 3.7 cm and normal-appearing large tertiary follicles already at the age of 2 yr. In addition, both basal and GnRH-induced FSH levels remained consistently strikingly elevated. Low-dose estradiol (E2) (0.4 mg/day) given for 50 days at the age of 3 6/12 yr resulted in normalization of serum gonadotropin levels, regression of ovarian size, and increase of whole body and lumbar spine (L1-L4) bone mineral density. The FSH concentration and ovarian size returned to pretreatment levels shortly (150 days) after cessation of E2 therapy. Therefore, we recommend that affected females be treated with low-dose E2 in amounts sufficient to result in physiological prepubertal E2 concentrations using an ultrasensitive estrogen assay. However, E2 replacement needs to be adjusted throughout childhood and puberty to ensure normal skeletal maturation and adequate adolescent growth spurt, normal accretion of bone mineral density, and, at the appropriate age, female secondary sex maturation.