Dent's disease: identification of a novel mutation in the renal chloride channel CLCN5

Dent's disease is an inherited tubulopathy caused by a mutation in the CLCN5 chloride channel gene. It is characterized by low-molecular weight proteinuria, hypercalciuria, nephrolithiasis or nephrocalcinosis, rickets and eventual-progressive renal failure. Onset of clinical symptoms show a great variability, making a diagnosis at an early stage of the disease often difficult. Given the variably clinical picture, genetic analysis can provide a reliable method to confirm the diagnosis. Here, we report on the case of a patient with progressive renal failure showing signs of a tubular lesion and symptoms of Dent's disease. Although this rare disease was suspected by means of the clinical features, it was genetic analysis that confirmed the diagnosis and revealed a novel mutation in the CLCN5 gene.     

Molecular analysis of the CLCN5 gene in Dent's disease: first mutation identified in a patient from South America

BACKGROUND: Dent's disease is a rare renal tubular disorder characterized by low-molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The selective loss of low-molecular weight proteins points to a defect of the proximal tubule, where filtered proteins are normally reabsorbed by endocytosis. The disease tends to present in childhood or early adult life, and males are more severely affected than females. The disease is caused by mutations in CLCN5 or OCRL1, both on the X chromosome, which code for the chloride/proton exchange transporter ClC-5 and the phosphatidylinositol-4,5-biphosphate-5-phosphatase, respectively. METHODS: Mutational analysis of CLCN5 gene from 4 unrelated patients diagnosed with Dent's disease and their relatives, 3 from Spain and 1 from Bolivia, was performed by PCR and automatic DNA sequencing. RESULTS: In the current study, we report the identification of 4 mutations in CLCN5 of 1 family from Bolivia and 3 families from Spain. Two of the mutations are novel and consist of 1 nonsense mutation, Y502X, and 1 missense mutation, L225P, affecting ClC-5alpha-helix F, one of the helices involved in formation of the chloride selectivity filter. CONCLUSIONS: Our results add to the expanding spectrum of mutations in CLCN5. This is the first report of a CLCN5 mutation in a Dent's disease patient of South American origin.     

A novel CLCN5 mutation in a boy with Bartter-like syndrome and partial growth hormone deficiency

Dent disease is an X-linked recessive disorder affecting the proximal tubule and is characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis/nephrolithiasis with a variable number of features of Fanconi syndrome. It is most often associated with mutations in CLCN5, which encodes the endosomal electrogenic chloride/proton exchanger ClC-5. Renal acidification abnormalities are only rarely seen in Dent disease, whereas the hypokalemic metabolic alkalosis associated with hyperreninemic hyperaldosteronism (Bartter-like syndrome) has been reported in only one patient so far. We report on a 5-year-old boy with Dent disease caused by mutation in CLCN5 gene, c.1073G>A, who presented with hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism persisting over the entire follow-up. No mutations were found in NKCC2, ROMK, NCCT, or ClC-Kb genes. In addition, the patient exhibited growth failure associated with partial growth hormone (GH) deficiency. Coexistence of Bartter-like syndrome features with LMWP should prompt a clinician to search for Dent disease. The Bartter syndrome phenotype seen in Dent disease patients may represent a distinct form of Bartter syndrome, the exact mechanism of which has yet to be fully elucidated. Growth delay that persists in spite of appropriate therapy should raise suspicion of other causes, such as GH deficiency.     

Enzymuria and tubular proteinuria in diabetic rats: a 12-week follow-up study.

Various biochemical parameters of renal tubular function were examined for a period of up to 12 weeks in rats rendered diabetic by an i.v. injection of streptozotocin. Except for a statistically significant decrease in the urinary excretion of gamma-glutamyl-transpeptidase to 64% of control values, the urinary excretion of beta-N-acetyl-D-glucosaminidase, beta-galactosidase, alanine aminopeptidase, and lactate dehydrogenase significantly increases in diabetic rats to between 154% and 712% of control values. This increased enzymuria is not correlated to the marked polyuria induced by diabetes (r between 0.14 and 0.35, not significant). Enzymuria is also accompanied by a 10-fold increase in the urinary excretion of the low molecular weight protein beta 2-microglobulin while the excretion of albumin is not significantly modified, indicating impairment of tubular reabsorption in diabetic animals. Clearance studies reveal that the clearance of both beta 2-microglobulin and infused egg-white lysozyme are also increased. Finally the histopathologic examination of paraffin sections of the kidney show hydropic degenerescence and pycnosis of the tubular cells. It is concluded that early-stage diabetes results in tubular impairment and that the streptozotocin-rat model appears well suited to the study of these early signs of renal dysfunction.     

Inherited hypercalciuric syndromes: Dent's disease (CLC-5) and familial hypomagnesemia with hypercalciuria (paracellin-1)

Dent's disease and familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) are inherited diseases in which hypercalciuria, nephrocalcinosis, and renal failure are prominent features. Dent's disease resembles a Fanconi syndrome, with impaired reabsorption in the proximal tubule; FHHNC, with urinary loss of magnesium and calcium, is associated with impaired cation transport in the thick ascending limb of Henle's loop. Gene mapping in families and positional cloning led in both cases to identification of the responsible gene. Dent's disease is associated with mutations that disrupt function of a voltage-gated chloride channel, CLC-5, expressed in subapical endosomes of the proximal tubule and in other nephron segments. Impaired function of this channel disturbs reabsorption of filtered proteins, as well as other transport functions of the proximal tubule, and leads, apparently indirectly, to hypercalciuria and renal failure. FHHNC results from mutations in paracellin-1, a tight-junction protein that appears to be important in conducting or regulating paracellular cation transport. Impaired function of paracellin-1 leads specifically to urinary losses of magnesium and calcium, but because transcellular transport is intact these patients do not have hypokalemia or salt wasting. Identification of both genes represent triumphs of a genetic approach to solving problems of pathophysiology.     

Risk for tumor and other disease transmission by transplantation: a population-based study of unrecognized malignancies and other diseases in organ donors

BACKGROUND: Organ donation may involve the risk of transmittal of unwanted host factors such as infections and malignancy. These may be concealed in the emergent donation process. It may be unavoidable if first observed in a donor postmortem. A number of reports on transferred cancers have been published, but quantification of the risk has never been reliably performed. We report here the first population-based analysis of unrecognized malignancies and other diseases in cadaveric or living-related donors and the possible consequences for the recipients. METHODS: We compiled a cohort of all organ donors through 27 years (1969-1996) in one single kidney transplant center covering a population on one million people. This cohort was linked to the Danish Cancer Registry, the Danish National Hospital Register, and the Danish Register of Causes of Death by means of the unique personal identification number, and all cancers, diagnosis from hospital admissions, and causes of death were identified. Follow-up was to the end of 1996. RESULTS: A total of 626 donors (491 cadaveric and 135 living-related donors) was included in the study. Ten carcinoma in situ or dysplasia cervix uteri (by definition nonmalignant), and 13 malignant tumors (5 of these were detected in living-related donors after donation) were detected by linkage to the cancer registry. All together, 17 recipients received organs from donors with carcinoma in situ or dysplasia cervix uteri and 20 from donors with malignancies. Two recipients from organ donors with carcinoma in situ or dysplasia of the cervix uteri and two recipients from donors with malignancies had a cancer detected; however, these were likely unrelated. One died 1 year after transplantation from a melanoma transmitted from the donor. Two cadaveric donors had previous admissions for glomerulonephritis, five for pyelonephritis, five for nephrolithiasis or ureterolithiasis, four for cystitis, and one for hydronephrosis. CONCLUSION: Despite all efforts to secure a safe organ for transplantation, transmission of donor malignancy and other diseases nevertheless can happen, as is recorded many times in the literature. We have quantified the risk using the population-based cancer registry and found a risk of 8 in 626 (1.3%) for having a donor with undetected malignancy and a risk of 1 in 626 (0.2%) for transmitting a cancer. The risk for getting some transmitted glomerulonephritis is 2 in 626 (0.3%). None of the donors with cerebral malignancies transmitted any tumors to the recipients. Compared with the benefits of organ transplantation, these risks are small; however, if time allows, a search for additional medical information from registries could further minimize the risk of transmission of malignancies or other diseases. However, this requires updated, accurate, and accessible registries and legislation that allows access to personal data and transmission of such data across administrative borders.     

Incomplete distal renal tubular acidosis coinherited with a mutation in the band 3 (AE1) gene

Background. Band 3 (anion exchanger 1, AE1) is one of the most abundant proteins of the erythrocyte membrane. We have previously characterized twenty AE1 gene defects underlying spherocytic haemolytic anaemia with band 3 deficiency. Since AE1 is also expressed in the intercalated cells of renal cortical collecting ducts where it is thought to participate in urine acidification, we asked whether the spherocytogenic AE1 mutations also affect the regulation of urine acidity. Methods. We examined 10 patients from seven unrelated families with hereditary spherocytosis with band 3 deficiency using the short urine acidification test with CaCl2 administration at a dose of 0.2 g/g b.w. To assess the ability of the nephron to secrete protons, 400 mol of NaHCO3 were infused over a period of 2 h. Results. While we detected no significant abnormalities in eight patients, we have diagnosed incomplete distal renal tubular acidosis (dRTA) in two patients from one family whose urinary pH 5 h after a CaCl2 administration were 6.65 and 6.89. Administration of bicarbonate in these two patients resulted in high urinary HCO3^- concentration. The patients carry the previously characterized mutation band 3^PRIBRAM that encodes a C-terminally truncated band 3 containing only the cytoplasmic domain and the first three putative transmembrane segments. Conclusions. This finding shows an association of a band 3 defect with abnormal urinary acidification perhaps secondary to Cl^-/HCO3 exchange in the basolateral membrane of &agr;-intercalated cells of cortical collecting ducts.     

Lipoprotein(a) and apolipoprotein(a) isoforms and proteinuria in patients with moderate renal failure.

BACKGROUND: Atherosclerotic diseases are a major cause of death in patients with renal failure. Increased serum concentrations of lipoprotein(a) [Lp(a)] have been established as a genetically controlled risk factor for these diseases and have been demonstrated in patients with moderate renal failure, suggesting that this lipoprotein contributes to the increased cardiovascular risk seen in these patients. Variable alleles at the apolipoprotein(a) [apo(a)] gene locus are the main determinants of the serum Lp(a) level in the general population. The purpose of this study was to investigate apo(a) isoforms in patients with moderate renal failure and mild proteinuria (less than 1.0 g/day). METHODS: In 250 consecutive subjects recruited at a hypertension clinic, we assessed the renal function by 24-hour creatinine clearance, proteinuria, and microalbuminuria, as well as the prevalence of atherosclerotic disease, and we also measured apo(a) isoforms, serum albumin, and Lp(a) concentrations. RESULTS: Moderate impairment of renal function (creatinine clearance, 30 to 89 ml/min per 1.73 m2 of body surface area) was found in 97 patients. Lp(a) levels were significantly greater in patients with moderate renal failure (21.7+/-23.9 mg/dl) as compared with patients with normal renal function (15.6+/-16.4 mg/dl, P<0.001), and an inverse correlation was observed between log Lp(a) and creatinine clearance (r = -0.181, P <0.01). However, no difference was found in the frequency of low molecular weight apo(a) isoforms between patients with normal (25.5%) and impaired (27.8%) renal function. Only patients with the smallest size apo(a) isoforms exhibited significantly elevated levels of Lp(a), whereas the large-size isoforms had similar concentrations in patients with normal and impaired renal function. No significant relationship was found between serum Lp(a) and proteinuria. Clinical and laboratory evidence of one or more events attributed to atherosclerosis was found in 9.8% of patients with normal renal function and 25.8% of patients with moderate renal failure (P<0.001). CONCLUSIONS: These results indicate that renal failure per se or other genes beside the apo(a) gene locus are responsible for the elevation of serum Lp(a) levels in patients with moderate impairment of renal function. The elevation of Lp(a) levels occurs independently of the level of proteinuria and may contribute to the risk for atherosclerotic disease in these patients.     

Characterization of proteinuria and tubular protein uptake in a new model of oral L-lysine administration in rats

Intravenous infusion of basic amino acids is used experimentally and pharmacologically to prevent renal proximal tubular uptake of filtered proteins. Intravenously injected L-lysine is rapidly cleared from plasma and the effect on tubular protein reabsorption is transient. To obtain a more sustained effect, we developed a model of oral L-lysine administration and characterized this model by analyzing urinary protein excretion and proximal tubule uptake of filtered proteins. Rats placed in metabolic cages were treated with 20 mmol/kg/6 h of L-lysine, glycine, or water. Urines were analyzed for proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and radioimmunoassay. Proximal tubule uptake of proteins and expression of apical membrane receptors were investigated by immunocytochemistry. In vitro uptake and receptor expression were studied using a yolk sac cell line. L-lysine administration produced increased urinary excretion of a large number of proteins while the effect on tubular accumulation of selected proteins was variable. L-lysine treatment induced changes in the localization of two receptors responsible for tubular endocytosis of filtered proteins. In conclusion, oral L-lysine treatment induced proteinuria, in particular albuminuria, as efficiently as previous reports on intravenous infusion. The effect on tubular protein accumulation was variable suggesting differential effects on tubular reabsorption and degradation of filtered proteins. Changes in tubular protein handling were accompanied by changes in the localization of the endocytic receptors, megalin, and cubilin. In vitro experiments supported the in vivo observations. The findings suggest that L-lysine may affect receptor trafficking in addition to possible effects on the direct binding of ligands to the receptors.     

Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex.

The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM2 and GM1 by immunostaining of thin layer chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of growth by calcitriol in a proximal tubular cell line (OK)

Calcitriol has been shown to inhibit (i) cell proliferationof renal carcinoma cell lines and of cultured adult human mesangialcells in vitro, and (ii) renal compensatory growth in vivo.In the present study we examined the effects of calcitriol onDNA synthesis and cell replication in an immortalized cell lineshowing the phenotypic characteristics of proximal tubular cells(opossum kidney, OK cells). The viability of OK cells was notaffected by calcitriol (Trypan-blue exclusion, LDH and K⁺ release),but the cells did not convert ³H-25(OH)₂ to 3H-1,25(OH)₂D3.In the log growth phase, calcitriol (but not alternative vitaminD metabolites) caused dose-dependent (1012 to 10^–6 M)inhibition of radiothymidine incorporation. Inhibition was calciumdependent, i.e. it was more pronounced at the lower nominalcalcium concentration in tissue culture media (0.9 versus 1.8mmol/l) and amplified by coincubation with nifedipine (I µM).Inhibition of DNA synthesis was paralleled by inhibition ofcell replication (growth curve) under basal conditions and afterstimulation with EGF (10 ng/ml). In conclusion, calcitriol inhibitsproliferation of proximal tubular cells which normally express1-alpha- hydroxylase activity.     

The frequency of kidney and urinary tract diseases in a defined population

The frequencies of kidney and urinary tract diseases (KUTD) in a defined population are presented. Rates of first hospitalizations with KUTD in the 5-year period, 1971 through 1975, were determined among over 1 million members of the Kaiser Foundation Health Plan (KFHP) of Northern California. Cases were identified from computer-stored ICDA-coded final discharge diagnoses for hospitalizations. Kidney and other urinary tract diseases were final diagnoses in 35.7 first hospitalizations per 10,000 members per year in the period 1971 through 1975. Disease-specific rates were highest for urinary tract infections, benign prostatic hypertrophy, and renal and ureteral calculi. The frequency of KUTD in this population is compared to available rates from other sources.     

Clinicopathological study of patients presenting hematuria and proteinuria by renal biopsy

We performed 308 series renal biopsies during 4 years (1985-1989) and 289 cases were examined by light microscopic, electron microscopic, or immunofluorescent study. Clinically, chronic nephritic syndrome was most frequent (55.4%), followed by nephrotic syndrome (15.1%), and recurrent or persistent hematuria (12.8%). Pathologically, IgA nephropathy was most popular (39.3%), followed by normal glomerulus (9.1%), and thin basement membrane disease (8.7%). Glomerulonephritis clinically recognized with recurrent or persistent hematuria, hardly showing proteinuria, in 81.6% of the cases, consisted of normal glomerulus, or thin basement membrane disease by electron microscopic and immunofluorescent examinations. The remainder (18.4%) was with IgA nephropathy, which was histologically mild. On the other hand, cases of chronic nephritic syndrome (latent type) with persistent proteinuria and hematuria were with glomerulonephritis of various types including IgA nephropathy in 78.8% of the total cases. Therefore, proteinuria is an important sign of glomerulonephritis. In investigation in different age groups, IgA nephropathy was seen in about 40% of both pediatric and adult cases, whereas minor glomerular abnormalities and thin basement membrane disease were more frequent in pediatric cases. Tubulo-interstitial lesions and glomerular lesions in vascular or metabolic diseases were recognized more in adults than in children. Membranous glomerulonephritis (17 cases including 4 pediatric cases), complicated with malignant tumors such as bladder or rectal cancers and hepatoma was found in 3 aged patients. Examination for malignant tumor would be necessary for aged patients with membranous glomerulonephritis. As for the prognosis of IgA nephropathy, because histological changes of IgA nephropathy varied widely from very mild state to severe state, the prognosis is not always good.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of proteinuria after conversion from calcineurin inhibitors (CNI) to sirolimus (SRL) in renal transplant patients: a multicenter study

Conversion from calcineurin inhibitors (CNI) to sirolimus (SRL) has become an option in patients with chronic allograft nephropathy or other conditions. However, in some cases an increase of proteinuria has been reported after such therapeutic intervention. The aim of this study was to characterize the clinical course of this so far unexplained proteinuria after conversion. We performed a retrospective analysis evaluating 94 renal transplant patients from various Spanish centers. Proteinuria (629 determinations) and clinical developments were analyzed between 6 months before and 6 months after conversion. Patients were divided into three groups according to mean proteinuria before conversion (group A: <300 mg/d; group B: 300 to 2000 mg/d; and group C: >2 g/d). The mean proteinuria level was 1.69 g/24 h (n = 312 determinations) before and 2.36 g/24 h after conversion (n = 317 determinations; P = .006), which corresponds to an overall increase of 25% (1.55 to 1.69 g/24 h considering only determinations of 1 month before and 1 month after conversion; P = NS). We could not detect any clear correlation between proteinuria and serum creatinine nor between changes of proteinuria and changes of serum creatinine. A variance analysis for repeated measures showed an increase in proteinuria compared to the preconversion values (P = .003), and when the three groups of preconversion proteinuria were evaluated separately it could be observed that this change in the evolution of proteinuria was almost completely dependent of an increase in the group C (preconversion proteinuria greater than 2 g/d; P = .03), whereas in the other two groups changes were almost irrelevant. Finally, the switch to SRL in renal transplant recipients is followed by an increase in the level of proteinuria predominantly dependent of an increase in patients with high levels of preconversion proteinuria, whereas it seems to be irrelevant in patients without or with light or moderate proteinuria. These results suggest that this might not be a direct effect of SRL.     

The product of duration and amount of proteinuria (proteinuria index) is a possible marker for glomerular and tubulointerstitial damage in IgA nephropathy

BACKGROUND/AIMS: IgA nephropathy (IgAN) is one of the major causes for chronic renal failure (CRF). Presence of massive proteinuria, hypertension, increased serum creatinine level and sclerotic histopathological changes of the glomerulus are known to be determinants for the progression of CRF. However, the relationships between duration of proteinuria/hematuria and histopathological changes, which may be correlated with the renal prognosis, have not been clarified. METHODS: A cross-sectional, univariate analysis of clinical parameters on the four glomerular and three tubulointerstitial histopathological grades in 57 untreated biopsy-proven IgAN patients (M/F = 32/25) was performed. RESULTS: The age at the time of renal biopsy (35.2 +/- 13.0 years; mean +/- SD), average duration of proteinuria (5.3 +/- 5.8 years), mean urinary protein excretion (0.99 +/- 1.22 g/day), serum creatinine (Cr 0.97 +/- 0.28 mg/dl), Cr clearance (Ccr 75.5 +/- 29.4 ml/min), and blood urea nitrogen (BUN 15.4 +/- 3.9 mg/dl) were well correlated with both histopathological grades. The product of duration (years) and urinary protein excretion (g/day) at the time of renal biopsy was more significantly correlated with glomerular and tubulointerstitial histopathological grades and serum Cr. CONCLUSION: The natural course of IgAN is steadily progressive depending on the duration and amount of proteinuria. The product of these two factors (proteinuria index) may be a useful predictor for glomerular and interstitial histopathological changes and the fate of renal function in IgAN.