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X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

AA amyloidosis is secondary to the deposit of excess insoluble Serum Amyloid A (SAA) protein fibrils. AA amyloidosis complicates chronic inflammatory diseases, especially chronic inflammatory rheumatisms such as rheumatoid arthritis and spondyloarthritis; chronic infections such as tuberculosis, bronchectasia, chronic inflammatory bowel diseases such as Crohn's disease; and auto-inflammatory diseases including familial Mediterranean fever. This work consists of the French guidelines for the diagnosis workup and treatment of AA amyloidosis. We estimate in France between 500 and 700 cases in the whole French population, affecting both men and women. The most frequent organ impaired is kidney which usually manifests by oedemas of the lower extremities, proteinuria, and/or renal failure. Patients are usually tired and can display digestive features anf thyroid goiter. The diagnosis of AA amyloidosis is based on detection of amyloid deposits on a biopsy using Congo Red staining with a characteristic green birefringence in polarized light. Immunohistochemical analysis with an antibody directed against Serum Amyloid A protein is essential to confirm the diagnosis of AA amyloidosis. Peripheral inflammatory biomarkers can be measured such as C Reactive protein and SAA. We propose an algorithm to guide the etiological diagnosis of AA amyloidosis. The treatement relies on the etiologic treatment of the undelying chronic inflammatory disease to decrease and/or normalize Serum Amyloid A protein concentration in order to stabilize amyloidosis. In case of renal failure, dialysis or even a kidney transplant can be porposed. Nowadays, there is currently no specific treatment for AA amyloidosis deposits which constitutes a therapeutic challenge for the future., (Copyright © 2022 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Cystinuria (OMIM 220100) is an autosomal recessive hereditary disorder in which high urinary cystine excretion leads to the formation of cystine stones because of the low solubility of cystine at normal urinary pH. We developed clinical practice recommendation for diagnosis, surgical and medical treatment, and follow-up of patients with cystinuria. Elaboration of these clinical practice recommendations spanned from June 2018 to December 2019 with a consensus conference in January 2019. Selected topic areas were chosen by the co-chairs of the conference. Working groups focusing on specific topics were formed. Group members performed systematic literature review using MEDLINE, drafted the statements, and discussed them. They included geneticists, medical biochemists, pediatric and adult nephrologists, pediatric and adult urologists experts in cystinuria, and the Metabolic Nephropathy Joint Working Group of the European Reference Network for Rare Kidney Diseases (ERKNet) and eUROGEN members. Overall 20 statements were produced to provide guidance on diagnosis, genetic analysis, imaging techniques, surgical treatment (indication and modalities), conservative treatment (hydration, dietetic, alkalinization, and cystine-binding drugs), follow-up, self-monitoring, complications (renal failure and hypertension), and impact on quality of life. Because of the rarity of the disease and the poor level of evidence in the literature, these statements could not be graded. This clinical practice recommendation provides guidance on all aspects of the management of both adults and children with cystinuria, including diagnosis, surgery, and medical treatment., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Light chain cast nephropathy (LCCN) in multiple myeloma often leads to severe and poorly reversible acute kidney injury. Severe renal impairment influences the allocation of chemotherapy and its tolerability; it also affects patient survival. Whether renal biopsy findings add to the clinical assessment in predicting renal and patient outcomes in LCCN is uncertain. We retrospectively reviewed clinical presentation, chemotherapy regimens, hematologic response, and renal and patient outcomes in 178 patients with biopsy-proven LCCN from 10 centers in Europe and North America. A detailed pathology review, including assessment of the extent of cast formation, was performed to study correlations with initial presentation and outcomes. Patients presented with a mean estimated glomerular filtration rate (eGFR) of 13 ± 11 mL/min/1.73 m2, and 82% had stage 3 acute kidney injury. The mean number of casts was 3.2/mm2 in the cortex. Tubulointerstitial lesions were frequent: acute tubular injury (94%), tubulitis (82%), tubular rupture (62%), giant cell reaction (60%), and cortical and medullary inflammation (95% and 75%, respectively). Medullary inflammation, giant cell reaction, and the extent of cast formation correlated with eGFR value at LCCN diagnosis. During a median follow-up of 22 months, mean eGFR increased to 43 ± 30 mL/min/1.73 m2. Age, β2-microglobulin, best hematologic response, number of cortical casts per square millimeter, and degree of interstitial fibrosis/tubular atrophy (IFTA) were independently associated with a higher eGFR during follow-up. This eGFR value correlated with overall survival, independently of the hematologic response. This study shows that extent of cast formation and IFTA in LCCN predicts the quality of renal response, which, in turn, is associated with overall survival.

Introduction: Anion exchanger 1 (AE1) ( SLC4A1 gene product) is a membrane protein expressed in both kidney and red blood cells (RBCs): it exchanges extracellular bicarbonate (HCO 3 - ) for intracellular chloride (Cl - ) and participates in acid-base homeostasis. AE1 mutations in kidney α-intercalated cells can lead to distal renal tubular acidosis (dRTA). In RBC, AE1 (known as band 3) is also implicated in membrane stability: deletions can cause South Asian ovalocytosis (SAO)., Methods: We retrospectively collected clinical and biological data from patients harboring dRTA due to a SLC4A1 mutation and analyzed HCO 3 - and Cl - transports (by stopped-flow spectrophotometry) and expression (by flow cytometry, fluorescence activated cell sorting, and Coomassie blue staining) in RBCs, as well as RBC membrane stability (ektacytometry)., Results: Fifteen patients were included. All experience nephrolithiasis and/or nephrocalcinosis, 2 had SAO and dRTA (dRTA SAO+), 13 dominant dRTA (dRTA SAO-). The latter did not exert specific RBC membrane anomalies. Both HCO 3 - and Cl - transports were lower in patients with dRTA SAO+ than in those with dRTA SAO- or controls. Using 3 different extracellular probes, we report a decreased expression (by 52%, P  < 0.05) in dRTA SAO+ patients by fluorescence activated cell sorting, whereas total amount of protein was not affected., Conclusion: Band 3 transport function and expression in RBCs from dRTA SAO- patients is normal. However, in SAO RBCs, impaired conformation of AE1/band 3 corresponds to an impaired function. Thus, the driver of acid-base defect during dominant dRTA is probably an impaired membrane expression., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Monoclonal immunoglobulin deposition disease (MIDD) is a rare complication of B-cell clonal disorders, defined by Congo red negative-deposits of monoclonal light chain (LCDD), heavy chain (HCDD), or both (LHCDD). MIDD is a systemic disorder with prominent renal involvement, but little attention has been paid to the description of extrarenal manifestations. Moreover, mechanisms of pathogenic immunoglobulin deposition and factors associated with renal and patient survival are ill defined. We retrospectively studied a nationwide cohort of 255 patients, with biopsy-proven LCDD (n = 212) (including pure LCDD [n = 154], LCDD with cast nephropathy (CN) [n = 58]), HCDD (n = 23), or LHCDD (n = 20). Hematological diagnosis was monoclonal gammopathy of renal significance in 64% and symptomatic myeloma in 34%. Renal presentation was acute kidney injury in patients with LCCD and CN, and chronic glomerular disease in the other types, 35% of whom had symptomatic extrarenal (mostly hepatic and cardiac) involvement. Sequencing of 18 pathogenic LC showed high isoelectric point values of variable domain complementarity determining regions, possibly accounting for tissue deposition. Among 169 patients who received chemotherapy (bortezomib-based in 58%), 67% achieved serum free light chain (FLC) response, including very good partial response (VGPR) or above in 52%. Renal response occurred in 62 patients (36%), all of whom had achieved hematological response. FLC response ≥ VGPR and absence of severe interstitial fibrosis were independent predictors of renal response. This study highlights an unexpected frequency of extrarenal manifestations in MIDD. Rapid diagnosis and achievement of deep FLC response are key factors of prognosis., (© 2019 by The American Society of Hematology.)

Mitochondrial diseases represent a significant clinical challenge. Substantial efforts have been devoted to identifying therapeutic strategies for mitochondrial disorders, but effective interventions have remained elusive. Recently, we reported attenuation of disease in a mouse model of the human mitochondrial disease Leigh syndrome through pharmacological inhibition of the mechanistic target of rapamycin (mTOR). The human mitochondrial disorder MELAS/MIDD (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes/Maternally Inherited Diabetes and Deafness) shares many phenotypic characteristics with Leigh syndrome. MELAS/MIDD often leads to organ failure and transplantation and there are currently no effective treatments. To examine the therapeutic potential of mTOR inhibition in human mitochondrial disease, four kidney transplant recipients with MELAS/MIDD were switched from calcineurin inhibitors to mTOR inhibitors for immunosuppression. Primary fibroblast lines were generated from patient dermal biopsies and the impact of rapamycin was studied using cell-based end points. Metabolomic profiles of the four patients were obtained before and after the switch. pS6, a measure of mTOR signaling, was significantly increased in MELAS/MIDD cells compared to controls in the absence of treatment, demonstrating mTOR overactivation. Rapamycin rescued multiple deficits in cultured cells including mitochondrial morphology, mitochondrial membrane potential, and replicative capacity. Clinical measures of health and mitochondrial disease progression were improved in all four patients following the switch to an mTOR inhibitor. Metabolomic analysis was consistent with mitochondrial function improvement in all patients., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Intracranial aneurysm rupture is a dramatic complication of autosomal dominant polycystic kidney disease (ADPKD). It remains uncertain whether screening should be widespread or only target patients with risk factors (personal or familial history of intracranial aneurysm), with an at-risk profession, or those who request screening. We evaluated this in a single-center cohort of 495 consecutive patients with ADPKD submitted to targeted intracranial aneurysm screening. Cerebral magnetic resonance angiography was proposed to 110 patients with a familial history of intracranial aneurysm (group 1), whereas it was not our intention to propose it to 385 patients without familial risk (group 2). Magnetic resonance angiography results, intracranial aneurysm prophylactic repair, rupture events, and cost-effectiveness of intracranial aneurysm screening strategies were retrospectively analyzed. During a median follow up of 5.9 years, five non-fatal intracranial aneurysm ruptures occurred (incidence rate 2.0 (0.87-4.6)/1000 patients-year). In group 1, 90% of patients were screened and an intracranial aneurysm was detected in 14, treated preventively in five, and ruptured in one patient despite surveillance. In group 2, 21% of patients were screened and an intracranial aneurysm was detected in five, and treated preventively in one. Intracranial aneurysm rupture occurred in four patients in group 2. Systematic screening was deemed cost-effective and provides a gain of 0.68 quality-adjusted life years compared to targeted screening. Thus, the intracranial aneurysm rupture rate is high in ADPKD despite targeted screening, and involves mostly patients without familial risk factors. Hence, cost-utility analysis suggests that intracranial aneurysm screening could be proposed to all ADPKD patients., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

PurposeWe aimed to identify the genetic cause to a clinical syndrome encompassing hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX syndrome), and to comprehensively delineate the phenotype.MethodsWe performed homozygosity mapping, whole-genome sequencing, gene sequencing, expression studies, functional tests, protein bioinformatics, and histological characterization in two unrelated families with HELIX syndrome.ResultsWe identified biallelic missense mutations (c.386C>T, p.S131L and c.2T>C, p.M1T) in CLDN10B in six patients from two unrelated families. CLDN10B encodes Claudin-10b, an integral tight junction (TJ) membrane-spanning protein expressed in the kidney, skin, and salivary glands. All patients had hypohidrosis, renal loss of NaCl with secondary hyperaldosteronism and hypokalemia, as well as hypolacrymia, ichthyosis, xerostomia, and severe enamel wear. Functional testing revealed that patients had a decreased NaCl absorption in the thick ascending limb of the loop of Henle and a severely decreased secretion of saliva. Both mutations resulted in reduced or absent Claudin-10 at the plasma membrane of epithelial cells.ConclusionCLDN10 mutations cause a dysfunction in TJs in several tissues and, subsequently, abnormalities in renal ion transport, ectodermal gland homeostasis, and epidermal integrity.

Monoclonal gammopathy of renal significance (MGRS) regroups renal disorders caused by a monoclonal immunoglobulin without overt hematological malignancy. MGRS includes tubular disorders, glomerular disorders with organized deposits, and glomerular disorders with non-organized deposits, such as proliferative glomerulonephritis with monoclonal IgG deposits. Since glomerular involvement related to monotypic IgA deposits is poorly described we performed retrospective analysis and defined clinico-biological characteristics, renal pathology, and outcome in 19 referred patients. This analysis allowed distinction between 2 types of glomerulopathies, α-heavy chain deposition disease (5 patients) and glomerulonephritis with monotypic IgA deposits (14 patients) suggestive of IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits in 12 cases. Clinicopathologic characteristics of α-heavy chain deposition disease resemble those of the γ-heavy chain disease, except for a higher frequency of extra-capillary proliferation and extra-renal involvement. IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits should be differentiated from diseases with polytypic IgA deposits, given distinct clinical, histological, and pathophysiological features. Similarly to IgG-proliferative glomerulonephritis with monoclonal immunoglobulin deposits, overt hematological malignancy was infrequent, but sensitive serum and bone marrow studies revealed a subtle plasma cell proliferation in most patients with IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits. Anti-myeloma agents appeared to favorably influence renal prognosis. Thus, potential progression towards symptomatic IgA multiple myeloma suggests that careful hematological follow-up is mandatory. This series expands the spectrum of renal disease in MGRS., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Dent disease classically combines low-molecular-weight proteinuria, hypercalciuria with nephrocalcinosis, and renal failure. Nephrotic range proteinuria, normal calciuria, and hypokalemia have been rarely reported. It is unknown whether the changes in phenotype observed over time are explained by a decrease in glomerular filtration rate (GFR) or whether there is any phenotype-genotype relationship. To answer this we retrospectively analyzed data from 109 male patients with CLCN5 mutations (Dent-1) and 9 patients with mutation of the OCRL gene (Dent-2). In Dent-1 disease, the estimated GFR decreased with age, by 1.0 to 1.6 ml/min per 1.73 m(2)/yr in the absence and presence of nephrocalcinosis, respectively, with no significant difference. Median values of low-molecular-weight proteinuria were in the nephrotic range and remained at the same level even in late renal disease. End-stage renal disease occurred in 12 patients, at a median age of 40 years. Hypercalciuria decreased with glomerular filtration and was absent in 40% of the patients under 30 and 85% of those over the age of 30. Hypophosphatemia did not resolve with age and calcitriol concentrations were in the upper normal range. Kalemia decreased with age, with half of the patients over the age of 18 presenting with hypokalemia. Thus, no phenotype/genotype correlation was observed in this cohort of patients with Dent disease., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Monoclonal immunoglobulin deposition disease (MIDD) is a rare complication of plasma cell disorders, defined by linear Congo red-negative deposits of monoclonal light chain, heavy chain, or both along basement membranes. While renal involvement is prominent, treatment strategies, such as the impact of novel anti-myeloma agents, remain poorly defined. Here we retrospectively studied 49 patients with MIDD who received a median of 4.5 cycles of intravenous bortezomib plus dexamethasone. Of these, 25 received no additional treatment, 18 also received cyclophosphamide, while 6 also received thalidomide or lenalidomide. The hematological diagnoses identified 38 patients with monoclonal gammopathy of renal significance, 10 with symptomatic multiple myeloma, and 1 with Waldenstrom macroglobulinemia. The overall hematologic response rate, based on the difference between involved and uninvolved serum-free light chains (dFLCs), was 91%. After median follow-up of 54 months, 5 patients died and 10 had reached end-stage renal disease. Renal response was achieved in 26 patients, with a 35% increase in median eGFR and an 86% decrease in median 24-h proteinuria. Predictive factors were pre-treatment eGFR over 30 ml/min per 1.73 m(2) and post-treatment dFLC under 40 mg/l; the latter was the sole predictive factor of renal response by multivariable analysis. Thus, bortezomib-based therapy is a promising treatment strategy in MIDD, mainly when used early in the disease course. dFLC response is a favorable prognostic factor for renal survival.

Autosomal dominant polycystic kidney disease is the most common inherited kidney disease and accounts for 7-10% of all patients on renal replacement therapy worldwide. Although first reported 500 years ago, this disorder is still regarded as untreatable and its pathogenesis is poorly understood despite much study. During the past 40 years, however, remarkable advances have transformed our understanding of how the disease develops and have led to rapid changes in diagnosis, prognosis, and treatment, especially during the past decade. This Review will summarise the key findings, highlight recent developments, and look ahead to the changes in clinical practice that will likely arise from the adoption of a new management framework for this major kidney disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive enzyme defect of purine metabolism that usually manifests as 2,8-dihydroxyadenine (2,8-DHA) nephrolithiasis and more rarely chronic kidney disease. The disease is most often misdiagnosed and can recur in the renal allograft. We analyzed nine patients with recurrent 2,8-DHA crystalline nephropathy, in all of whom the diagnosis had been missed prior to renal transplantation. The diagnosis was established at a median of 5 (range 1.5-312) weeks following the transplant procedure. Patients had delayed graft function (n=2), acute-on-chronic (n=5) or acute (n=1) allograft dysfunction, whereas one patient had normal graft function at the time of diagnosis. Analysis of allograft biopsies showed birefringent 2,8-DHA crystals in renal tubular lumens, within tubular epithelial cells and interstitium. Fourier transformed infrared microscopy confirmed the diagnosis in all cases, which was further supported by 2,8-DHA crystalluria, undetectable erythrocyte APRT enzyme activity, and genetic testing. With allopurinol therapy, the allograft function improved (n=7), remained stable (n=1) or worsened (n=1). At last follow-up, two patients had experienced allograft loss and five had persistent chronic allograft dysfunction. 2,8-DHA nephropathy is a rare but underdiagnosed and preventable disorder that can recur in the renal allograft and may lead to allograft loss., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Alport syndrome inevitably leads to end-stage renal disease and there are no therapies known to improve outcome. Here we determined whether angiotensin-converting enzyme inhibitors can delay time to dialysis and improve life expectancy in three generations of Alport families. Patients were categorized by renal function at the initiation of therapy and included 33 with hematuria or microalbuminuria, 115 with proteinuria, 26 with impaired renal function, and 109 untreated relatives. Patients were followed for a period whose mean duration exceeded two decades. Untreated relatives started dialysis at a median age of 22 years. Treatment of those with impaired renal function significantly delayed dialysis to a median age of 25, while treatment of those with proteinuria delayed dialysis to a median age of 40. Significantly, no patient with hematuria or microalbuminuria advanced to renal failure so far. Sibling pairs confirmed these results, showing that earlier therapy in younger patients significantly delayed dialysis by 13 years compared to later or no therapy in older siblings. Therapy significantly improved life expectancy beyond the median age of 55 years of the no-treatment cohort. Thus, Alport syndrome is treatable with angiotensin-converting enzyme inhibition to delay renal failure and therapy improves life expectancy in a time-dependent manner. This supports the need for early diagnosis and early nephroprotective therapy in oligosymptomatic patients.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease, and sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to significantly retard cyst expansion in animal models. The optimal therapeutic dose of sirolimus is not yet defined. Here, we report the history of a previously unknown ADPKD deceased donor whose kidneys were engrafted in two different recipients. One of the two received an immunosuppressive regimen based on sirolimus for 5 years while the other did not. After transplantation, both patients developed severe transplant cystic disease. Donor DNA sequence identified a new hypomorphic mutation in PKD1. The rate of cyst growth was identical in the two patients regardless of the treatment. While sirolimus treatment reduced the activation of mTOR in peripheral blood mononuclear cells, it failed to prevent mTOR activation in kidney tubular cells, this could account for the inefficiency of treatment on cyst growth. Together, our results suggest that the dose of sirolimus required to inhibit mTOR varies according to the tissue.

The HELLP syndrome, defined by the existence of hemolysis, elevated liver enzymes, and low platelet count, is a serious complication of pregnancy-related hypertensive disorders and shares several clinical and biologic features with thrombotic microangiopathy (TMA). Several recent studies have clearly shown that an abnormal control of the complement alternative pathway is a major risk for the occurrence of a peculiar type of TMA involving mainly the kidney. The aim of this study was to screen for complement abnormalities in 11 patients with HELLP syndrome and renal involvement. We identified 4 patients with a mutation in one of the genes coding for proteins involved in the regulation of the alternative pathway of complement. Our results suggest that an abnormal control of the complement alternative pathway is a risk factor for the occurrence of HELLP syndrome.

Alport syndrome is a collagen type IV disease caused by mutations in the COL4A5 gene with the X-linked form being most prevalent. The resultant alpha5(IV) collagen chain is a component of the glomerular and skin basement membranes (SBMs). Immunofluorescent determination of the alpha5(IV) chain in skin biopsies is the procedure of choice to identify patients. In 30% of patients, however, the mutant protein is still found in the SBM resulting in a normal staining pattern. In order to minimize or eliminate false results, we compared the distribution of the alpha2(IV) chain (another SBM component) and the alpha5(IV) chain by standard double label immunofluorescence (IF) and by confocal laser scanning microscopy. The study was performed on 55 skin biopsies of patients suspected of Alports and five normal control specimens. In normal skin, IF showed the classical linear pattern for both collagens along the basement membrane. Additionally, decreased alpha5(IV) was found in the bottom of the dermal papillary basement membrane. Confocal analysis confirmed the results and show alpha5(IV) focal interruptions. In suspected patients, both techniques showed the same rate of abnormal alpha5(IV) expression: segmental in women and absent in men. Our results show a physiological variation of alpha5(IV) location with focal interruptions and decreased expression in the bottom of the dermal basement membrane. Comparison of alpha5(IV) with alpha2(IV) expression is simple and eliminates technical artifacts.

Background: Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation., Methods: Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection., Results: Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001)., Conclusion: Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.

Extracellular matrix alterations have been suggested to be part of the early events occurring in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a disease characterized by formation of renal cysts and progressive renal failure. Here we report that cDNA array analysis identified beta(4) integrin aberrant expression in ADPKD cells. Furthermore, laminin 5 (Ln-5), the main alpha(6)beta(4) integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin alpha(6)beta(4)-Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through alpha(6)beta(4) interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin alpha(3)beta(1) cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated beta(4) integrin ligation, through an alpha(3)beta(1) integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and alpha(6)beta(4) integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.

VHL is the causative gene for both von Hippel-Lindau (VHL) disease and sporadic clear-cell renal cancer. We showed earlier that VHL downregulates vascular endothelial growth factor transcription by directly binding and inhibiting the transcriptional activator Sp1. We have now mapped the VHL Sp1-binding domain to amino acids 96-122. The 96-122 domain is disproportionately affected by substitution mutations, which interfere with the VHL-Sp1 interaction. Deletion of the 96-122 domain prevents VHL effects on Sp1 DNA binding and on VHL target gene expression, indicating the domain contributes importantly to VHL tumor suppressor activity. Nevertheless, prevention of the VHL-Sp1 interaction only partially abrogates VHL's transcriptional repressor activity, supporting the existence of VHL transcriptional effectors in addition to Sp1. VHL also directly interacts with the Sp1 zinc fingers and self-associates via the 96-122 domain, which furthermore suggest the domain may bind other metalloproteins and contribute to VHL dominant-negative effects., (Copyright 1999 Academic Press.)

Angiostatin which contains the first four kringle domains of plasminogen has been documented to be a potent inhibitor of angiogenesis. More recently, another kringle structure within plasminogen but outside angiostatin, known as kringle 5 (K5), was found to inhibit endothelial cell proliferation and migration. Here, we report the cloning and expression of mouse kringle 5 (rK5) in a bacterial expression system. The protein was purified to homogeneity using a Ni-NTA column. rK5 inhibited both proliferation and migration of endothelial cells with ED50's of 10 nM and < 500 nM, respectively. In addition, we show for the first time that rK5 causes cell cycle arrest and apoptosis, shedding further insight into rK5's mechanism of action. Finally, we show that these actions are endothelial cell specific., (Copyright 1999 Academic Press.)

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model., (Copyright 1999 Academic Press.)

Alport syndrome (AS) is an hereditary disease of basement membrane collagen. It is mainly transmitted as a dominant X-linked trait and caused by mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen. However, autosomal recessive AS due to mutations in the COL4A3 or COL4A4 genes could represent up to 15% of AS. Using the immunofluorescence technique, we analyzed the distribution of the different chains of type IV collagen in renal (12 specimens) and skin (4 specimens) basement membranes of 12 AS patients belonging to 11 unrelated kindreds in which autosomal recessive inheritance had been demonstrated (3 kindreds) or was suggested by clinical and genealogic data (8 kindreds). The renal and skin distribution was normal in one patient with COL4A4 mutations. A peculiar pattern of distribution of the alpha 3-alpha 5(IV) chains was observed in the other patients. It was characterized the co-absence of the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains in the glomerular basement membrane, and the presence of the alpha 5(IV) chain in a series of extraglomerular basement membranes including capsular, collecting ducts and epidermal basement membranes, a combination never observed in X-linked AS. This immunohistochemical pattern is correlated with the specific distribution of the alpha 3-alpha 5 chains of type IV collagen chains within extraglomerular basement membranes. It could be a useful marker for the identification of autosomal recessive AS.

Alport syndrome (AS) is an hereditary glomerulonephritis that is mainly inherited as a dominant X-linked trait. Structural abnormalities in the type IV collagen alpha 5 chain gene (COL4A5), which maps to Xq22, have recently been detected in several patients with AS. The association of AS with diffuse esophageal leiomyomatosis (DL) has been reported in 24 patients, most of them also suffering from congenital cataract. The mode of transmission and the location of the gene(s) involved in this association have not been elucidated. Southern blotting using cDNA probes spanning the whole COL4A5 and a 5' end COL4A5 genomic probe showed that three out of three patients with the DL-AS association had a deletion in the 5' part of the COL4A5 gene extending beyond its 5' end. This indicates that the same gene, COL4A5, is involved in classical AS and in DL-AS and that the transmission of DL-AS is X-linked dominant. These results also suggest that leiomyomatosis might be due to the alteration of a second gene involved in smooth muscle cell proliferation, which is located upstream of the COL4A5 gene, and that there might be a contiguous gene deletion syndrome, involving at least the genes coding for congenital cataract, DL and AS.

Anti-Müllerian hormone, responsible for Müllerian regression in male fetuses, is a glycoprotein dimer with two 72 kD subunits. The AMH gene is a small (2,800 bp) gene with 5 exons, localized on the tip of the short arm of chromosome 19, band p 133, and transcribed in a 2,000 kbp mRNA. Persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism characterized by the presence of uterus and Fallopian tubes in patients with normally virilized genitalia, may result from defective AMH gene or from target-organ insensitivity. Four mutations were identified in the AMH gene, 3 are point mutations (2 stop codons, the third altering the secondary structure of the molecule), the last is a 14 bp deletion, leading to alteration of the reading frame of the mRNA.