Enrica Tosetto

In search of adult renal stem cells.

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidneys specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes ‐ together with a knowledge of the embryonic origin of renal cells ‐ should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.

Locus heterogeneity of Dent’s disease: OCRL1 and TMEM27 genes in patients with no CLCN5 mutations

Dent′s disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL1 gene, which is usually mutated in patients with Lowe syndrome, have recently been shown to lead to a Dent-like phenotype, called Dent’s disease 2. About 25% of Dent’s disease patients do not carry CLCN5/OCRL1 mutations. The CLCN4 and SLC9A6 genes have been investigated, but no mutations have been identified. The recent discovery of a novel mediator of renal amino acid transport, collectrin (the TMEM27 gene), may provide new insight on the pathogenesis of Dent’s disease. We studied 31 patients showing a phenotype resembling Dent’s disease but lacking any CLCN5 mutations by direct sequencing of the OCRL1 and TMEM27 genes. Five novel mutations, L88X, P161HfsX167, F270S, D506N and E720D, in the OCRL1 gene, which have not previously been reported in patients with Dent’s or Lowe disease, were identified among 11 patients with the classical Dent’s disease phenotype. No TMEM27 gene mutations were discovered among 26 patients, 20 of whom had an incomplete Dent’s disease phenotype. Our findings confirm that OCRL1 is involved in the functional defects characteristic of Dent’s disease and suggest that patients carrying missense mutations in exons where many Lowe mutations are mapped may represent a phenotypic variant of Lowe syndrome.

The renal stem cell system in kidney repair and regeneration.

The adult mammalian renal tubular epithelium exists in a relatively quiescent to slowly replicating state, but has great potential for regenerative morphogenesis following severe ischemic or toxic injury. Kidney regeneration and repair occur through three cellular and molecular mechanisms: differentiation of the somatic stem cells, recruitment of circulating stem cells and, more importantly, proliferation/dedifferentiation of mature cells. Dedifferentiation seems to represent a critical step for the recovery of tubular integrity. Dedifferentiation of tubular cells after injury is characterized by the reactivation of a mesenchymal program that is active during nephrogenesis. Epithelial-to-mesenchymal transition (EMT) of renal tubular cells is an extreme manifestation of epithelial cell plasticity. It is now widely recognized as a fundamental process that marks some physiological, such as morphogenesis, as well as pathological events, such as oncogenesis and fibrogenesis. It might be also considered as a key event in the regenerative process of the kidney. Understanding the molecular mechanisms involved in EMT might be useful for designing therapeutic strategies in order to potentiate the innate capacity of the kidney to regenerate.

An atypical Dent's disease phenotype caused by co-inheritance of mutations at CLCN5 and OCRL genes

Dent’s disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL gene, which is usually mutated in patients with Lowe syndrome, have been shown to lead to a Dent-like phenotype called Dent disease 2. However, about 20% of patients with Dent’s disease carry no CLCN5/OCRL mutations. The disease’s genetic heterogeneity is accompanied by interfamilial and intrafamilial phenotypic heterogeneity. We report on a case of Dent’s disease with a very unusual phenotype (dysmorphic features, ocular abnormalities, growth delay, rickets, mild mental retardation) in which a digenic inheritance was discovered. Two different, novel disease-causing mutations were detected, both inherited from the patient’s healthy mother, that is a truncating mutation in the CLCN5 gene (A249fs*20) and a donor splice-site alteration in the OCRL gene (c.388+3A>G). The mRNA analysis of the patient’s leukocytes revealed an aberrantly spliced OCRL mRNA caused by in-frame exon 6 skipping, leading to a shorter protein, but keeping intact the central inositol 5-phosphatase domain and the C-terminal side of the ASH-RhoGAP domain. Only wild-type mRNA was observed in the mother’s leukocytes due to a completely skewed X inactivation. Our results are the first to reveal the effect of an epistatic second modifier in Dent’s disease too, which can modulate its expressivity. We surmise that the severe Dent disease 2 phenotype of our patient might be due to an addictive interaction of the mutations at two different genes.

Family history may be misleading in the diagnosis of Dent’s disease

The rare Dent’s disease manifests with medullary nephrocalcinosis, nephrolithiasis, hypercalciuria, low molecular weight proteinuria and other tubular dysfunctions, rickets or osteomalacia, and renal failure, in various combinations. It is a recessive X-linked condition. Clinicians consider family history a fundamental pointer to its diagnosis, but this is not invariably the case as clearly pointed out by the two reported cases.

Dent’s disease and prevalence of renal stones in dialysis patients in Northeastern Italy

AbstractDents disease (DD) involves nephrocalcinosis, urolithiasis, hypercalciuria, LMW proteinuria, and renal failure in various combinations. Males are affected. It is caused by mutations in the chloride channel CLCN5 gene. It has been suggested that DD is underdiagnosed, occurring in less overt forms, apparently without family history. A possible approach to this problem is to search for CLCN5 mutations in patients who may have a high prevalence of mutations: end-stage renal disease (ESRD) patients with previous calcium, struvite, or radio-opaque (CSR) stones. We looked for CLCN5 mutations in 25 males with ESRD-CSR stones selected from all of the patients (1,901 individuals, of which 1,179 were males) of 15 dialysis units in the Veneto region. One DD patient had a new DD mutation (1070 G>T) in exon 7. The new polymorphism IVS11-67 C>T was detected in intron 11 in one patient and one control. We also found 28 females with ESRD and stone history, and seven more males with ESRD and non-CSR stones. The prevalence of stone formers among dialysis patients in our region was 3.2%, much lower than the prevalence observed in older studies. Struvite stones continue to play a major role in causing stone-associated ESRD .

Novel mutations of the CLCN5 gene including a complex allele and A 5′ UTR mutation in Dent disease 1

To the Editor : Dent disease (DD) 1 (OMIM 300009) is an X-linked disorder of renal tubular function, characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and progressive renal failure, and associated with mutations in the ClC-5 Cl−/H+ endosomal exchanger all located in the coding region of the CLCN5 gene (1, 2). We report here the identification of 15 novel mutations including a complex allele and a nucleotide substitution in the 5′UTR region of the CLCN5 gene. Thirty patients with clinical suspicion of DD were screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing for the presence of mutations in the coding sequence and the exon-intron boundaries as well as in the 5′untranslated exons of the CLCN5 gene. All the patients were of Italian origin, except 1 from the United Kingdom. Sixteen of the 30 patients presented CLCN5 mutations of which 15 were novel and 1 recurrent (S244L). The phenotypic characteristics of patients with and without CLCN5 mutations are reported in Table 1. The table shows that LMWP, hypercalciuria and nephrocalcinosis, represent the triad of manifestations most relevant for the diagnosis of DD 1. Table 2 reports the 15 novel CLCN5 mutations identified in DD patients. Each of the missense and deletion–insertion mutations predicts a structurally significant alteration to the ClC-5 Cl−/H+ exchanger, and is thus likely to be of importance in the aetiology of the disease. The three frameshift mutations, leading to premature termination codons (PTC), can induce either a truncated ClC-5 product or mRNA degradation through nonsense-mediated decay which are likely to result in a loss of antiporter function and chloride conductance. The two in-frame codon deletion and the insertion are thought to alter the ClC-5 α-helices H and I, which are two of the four major helices involved in the formation of dimer interface (3). All the missense mutations, except one located in the C-terminal domain, are at or near the ClC-5 dimer interface. Moreover they involved amino acids conserved among different species or present in all known ClCs and were predicted with a high confidence to affect protein function by both the web program SIFT (4) and PolyPhen (5). For four out of six missense mutations (W58L, G512D, V519D, P621L) and for T277_L278 ins S mutation it was possible to establish that they segregated with DD trait in family members. Three intronic mutations affecting the canonical consensus splicing donor site of intron 4, 8 and 11 were detected in three patients. In two of them, the presence of an aberrantly spliced ClC-5 mRNA in leukocytes, leading to a truncated or absent protein, confirmed the functional significance of the mutations (Fig. 1). The presence of an aberrantly spliced mRNA due to the IVS4 +4 A>G could not be proved by transcription polymerase chain reaction (RT-PCR) analysis because the patient’s RNA was not available. The Automated Splice-Site Analysis program (ASSA; (6)), however, predicts that this variant would lower the strength of the donor splice-site leading probably to exon 4 skipping. We identified a complex allele (S386F and S388fsX434) in one patient from North Italy, inherited from his mother and grandmother. Since both the web program SIFT and PolyPhen predicted that the missense mutation S386F did not affect protein function, and the search for potential exonic splicing enhancer (ESE) motifs using ESE finder (7), RESCUE-ESE (8) and ASSA did not evidence any modification, we wondered if the mutation was indeed a polymorphism. The substitution was not found in our series of 69 patients affected by DD and in 311 X chromosomes from umbilical cord DNA samples thus

Urine proteome analysis in Dent's disease shows high selective changes potentially involved in chronic renal damage

UNLABELLED Definition of the urinary protein composition would represent a potential tool for diagnosis in many clinical conditions. The use of new proteomic technologies allows detection of genetic and post-trasductional variants that increase sensitivity of the approach but complicates comparison within a heterogeneous patient population. Overall, this limits research of urinary biomarkers. Studying monogenic diseases are useful models to address this issue since genetic variability is reduced among first- and second-degree relatives of the same family. We applied this concept to Dents disease, a monogenic condition characterised by low-molecular-weight proteinuria that is inherited following an X-linked trait. Results are presented here on a combined proteomic approach (LC-mass spectrometry, Western blot and zymograms for proteases and inhibitors) to characterise urine proteins in a large family (18 members, 6 hemizygous patients, 6 carrier females, and 6 normals) with Dents diseases due to the 1070G>T mutation of the CLCN5. Gene ontology analysis on more than 1000 proteins showed that several clusters of proteins characterised urine of affected patients compared to carrier females and normal subjects: proteins involved in extracellular matrix remodelling were the major group. Specific analysis on metalloproteases and their inhibitors underscored unexpected mechanisms potentially involved in renal fibrosis. BIOLOGICAL SIGNIFICANCE Studying with new-generation techniques for proteomic analysis of the members of a large family with Dents disease sharing the same molecular defect allowed highly repetitive results that justify conclusions. Identification in urine of proteins actively involved in interstitial matrix remodelling poses the question of active anti-fibrotic drugs in Dents patients.

Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

BackgroundDent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5’UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5’UTR region has not yet been fully characterized. To date 6 different 5’ alternatively used exons - 1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb) - have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5’UTR region in the human kidney and other tissues.MethodsTo clone more of the 5’ end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5’ ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real -Time RT/PCR.ResultsEleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5’UTR isoforms were confirmed, but their ends were extended. Six additional 5’UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.ConclusionsOur results confirm the structural complexity of the CLCN5 5’UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-β1 expression in mesangial cells: biochemical and structural considerations

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-β1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-β1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-β1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-β1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-β1 overexpression.