261 research outputs found
GENETIC ANALYSIS IN DENT DISEASE AND FUNCTIONAL STUDIES OF CLCN5 MUTATIONS IN PATIENTS’ KIDNEY BIOPSIES
Genetics and phenotypic heterogeneity of Dent disease: the dark side of the moon
Dent disease is a rare genetic proximal tubulopathy which is under-recognized. Its phenotypic heterogeneity has led to several different classifications of the same disorder, but it is now widely accepted that the triad of symptoms low-molecular-weight proteinuria, hypercalciuria and nephrocalcinosis/nephrolithiasis are pathognomonic of Dent disease. Although mutations on the CLCN5 and OCRL genes are known to cause Dent disease, no such mutations are found in about 25-35% of cases, making diagnosis more challenging. This review outlines current knowledge regarding Dent disease from another perspective. Starting from the history of Dent disease, and reviewing the clinical details of patients with and without a genetic characterization, we discuss the phenotypic and genetic heterogeneity that typifies this disease. We focus particularly on all those confounding clinical signs and symptoms that can lead to a misdiagnosis. We also try to shed light on a concealed aspect of Dent disease. Although it is a proximal tubulopathy, its misdiagnosis may lead to patients undergoing kidney biopsy. In fact, some individuals with Dent disease have high-grade proteinuria, with or without hematuria, as in the clinical setting of glomerulopathy, or chronic kidney disease of uncertain origin. Although glomerular damage is frequently documented in Dent disease patients' biopsies, there is currently no reliable evidence of renal biopsy being of either diagnostic or prognostic value. We review published histopathology reports of tubular and glomerular damage in these patients, and discuss current knowledge regarding the role of CLCN5 and OCRL genes in glomerular function
Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation
Abstract Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype
Albumin uptake in human podocytes: a possible role for the cubilin-amnionless (CUBAM) complex
Abstract Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes
Understanding the Pathophysiology of Nephrocalcinosis
Many in vitro and in vivo studies on the mechanisms underlying calcium nephrolithiasis have provided evidence of a frequently associated condition, i.e., a microscopic renal crystal deposition that can occur within the tubular lumen (intratubular nephrocalcinosis) or in the interstitium (interstitial nephrocalcinosis). Medullary nephrocalcinosis is the typical pattern seen in 98% of cases of human nephrocalcinosis, with calcification clustering around each renal pyramid. It is common in patients with metabolic conditions that predispose them to renal calcium stones. Cortical nephrocalcinosis is rare and usually results from severe destructive disease of the cortex. It has been described in chronic glomerulonephritis, but often in association with another factor, such as an increased calcium ingestion, acute cortical necrosis, chronic pyelonephritis or trauma. The most accredited hypothesis to explain the onset of interstitial nephrocalcinosis is purely physicochemical, relating to spontaneous Ca2PO4 crystallization in the interstitium due to oversaturation of Ca2PO4salts in this milieu. The theory that nephrocalcinosis is a process driven by osteogenic cells was first proposed by our group. We review nephrocalcinosis in terms of its definition, genetic associations, and putative mechanisms, pointing out how much evidence in the literature suggests that it may have some features in common with, and pathogenic links to vascular calcification
Nephrolithiasis, kidney failure and bone disorders in Dent disease patients with and without CLCN5 mutations
open9noDent disease (DD) is a rare X-linked recessive renal tubulopathy characterised by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and/or nephrolithiasis. DD is caused by mutations in both the CLCN5 and OCRL genes. CLCN5 encodes the electrogenic chloride/proton exchanger ClC-5 which is involved in the tubular reabsorption of albumin and LMW proteins, OCRL encodes the inositol polyphosphate 5-phosphatase, and was initially associated with Lowe syndrome. In approximately 25 % of patients, no CLCN5 and OCRL mutations were detected. The aim of our study was to evaluate whether calcium phosphate metabolism disorders and their clinical complications are differently distributed among DD patients with and without CLCN5 mutations. Sixty-four male subjects were studied and classified into three groups: Group I (with CLCN5 mutations), Group II (without CLCN5 mutations) and Group III (family members with the same CLCN5 mutation). LMWP, hypercalciuria and phosphaturic tubulopathy and the consequent clinical complications nephrocalcinosis, nephrolithiasis, bone disorders, and chronic kidney disease (CKD) were considered present or absent in each patient. We found that the distribution of nephrolithiasis, bone disorders and CKD differs among patients with and without CLCN5 mutations. Only in patients harbouring CLCN5 mutations was age-independent nephrolithiasis associated with hypercalciuria, suggesting that nephrolithiasis is linked to altered proximal tubular function caused by a loss of ClC-5 function, in agreement with ClC-5 KO animal models. Similarly, only in patients harbouring CLCN5 mutations was age-independent kidney failure associated with nephrocalcinosis, suggesting that kidney failure is the consequence of a ClC-5 dysfunction, as in ClC-5 KO animal models. Bone disorders are a relevant feature of DD phenotype, as patients were mainly young males and this complication occurred independently of age. The triad of symptoms, LMWP, hypercalciuria, and nephrocalcinosis, was present in almost all patients with CLCN5 mutations but not in those without CLCN5 mutations. This lack of homogeneity of clinical manifestations suggests that the difference in phenotypes between the two groups might reflect different pathophysiological mechanisms, probably depending on the diverse genes involved. Overall, our results might suggest that in patients without CLCN5 mutations several genes instead of the prospected third DD underpin patients' phenotypes.openAnglani, Franca; D’Angelo, Angela; Bertizzolo, Luisa Maria; Tosetto, Enrica; Ceol, Monica; Cremasco, Daniela; Bonfante, Luciana; Addis, Maria Antonietta; Del Prete, DorellaAnglani, Franca; D'Angelo, Angela; Bertizzolo, Luisa Maria; Tosetto, Enrica; Ceol, Monica; Cremasco, Daniela; Bonfante, Luciana; Addis, Maria Antonietta; DEL PRETE, Dorell
Vascular endothelial growth factor (VEGF) and VEGF receptors in diabetic nephropathy: expression studies in biopsies of type 2 diabetic patients.
Vascular endothelial growth factor (VEGF) is involved in the pathogenesis of diabetic retinopathy but its role in diabetic nephropathy is only speculative so far. It has been shown that in renal cortex of normal kidneys, glomerular and tubular epithelial cells express VEGF and that VEGF 165 is the predominant isoform. Two VEGF receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase) are co-expressed by glomerular and peritubular capillary endothelial cells. However, VEGF and VEGF receptors are predominantly expressed at glomerular level. We recently demonstrated that in type 2 diabetic patients glomerular qualitative and quantitative changes of VEGF mRNA expression are associated with functional and structural renal changes. In the present work we focused on the tubulo-interstitial compartment; by reverse transcription/polymerase chain reaction (RT/PCR) we evaluated the expression of VEGF, KDR, Flt-1 and the relationship between the two main type of VEGF isoforms, VEGF121 and VEGF165 in the tubulo-interstitium of type 2 diabetic patients. Patients were divided in three category on the basis of renal structure pattern: CI, with normal or near normal renal structure; CII, with glomerular and tubulo-interstitial lesions occurring in parallel (typical diabetic nephropathology); CIII, with atypical pattern of renal injury, i.e., more severe tubulo-interstitial and vascular than glomerular changes. Comparison between the two cortical compartments revealed that, both in glomeruli and in tubulo-interstitium. VEGF121 isoform exceed VEGF165 while Flt-1 was significantly lower in glomeruli. CIII patients had the lowest tubulo-interstitial level of VEGF and Flt-1 mRNAs. These results suggest that the transcriptional shifting from VEGF165 to VEGF121 isoform and the unbalanced FIt-1 expression between tubulo-interstitium and glomeruli could be involved in the pathogenesis of diabetic nephropathy. Furthermore, at least in CIII patients, down-regulation of the VEGF-Flt-1 system could be involved in the mechanisms leading to tubulointerstitial diabetic lesions
Genes involved in TGFβ1-driven epithelial-mesenchymal transition of renal epithelial cells are topologically related in the human interactome map
<p>Abstract</p> <p>Background</p> <p>Understanding how mesenchymal cells arise from epithelial cells could have a strong impact in unveiling mechanisms of epithelial cell plasticity underlying kidney regeneration and repair.</p> <p>In primary human tubular epithelial cells (HUTEC) under different TGFβ1 concentrations we had observed epithelial-to-mesenchymal transition (EMT) but not epithelial-myofibroblast transdifferentiation. We hypothesized that the process triggered by TGFβ1 could be a dedifferentiation event. The purpose of this study is to comprehensively delineate genetic programs associated with TGFβ1-driven EMT in our in vitro model using gene expression profile on large-scale oligonucleotide microarrays.</p> <p>Results</p> <p>In HUTEC under TGFβ1 stimulus, 977 genes were found differentially expressed. Thirty genes were identified whose expression depended directly on TGFβ1 concentration. By mapping the differentially expressed genes in the Human Interactome Map using Cytoscape software, we identified a single scale-free network consisting of 2630 interacting proteins and containing 449 differentially expressed proteins. We identified 27 hub proteins in the interactome with more than 29 edges incident on them and encoded by differentially expressed genes. The Gene Ontology analysis showed an excess of up-regulated proteins involved in biological processes, such as "morphogenesis", "cell fate determination" and "regulation of development", and the most up-regulated genes belonged to these categories. In addition, 267 genes were mapped to the KEGG pathways and 14 pathways with more than nine differentially expressed genes were identified. In our model, Smad signaling was not the TGFβ1 action effector; instead, the engagement of RAS/MAPK signaling pathway seems mainly to regulate genes involved in the cell cycle and proliferation/apoptosis.</p> <p>Conclusion</p> <p>Our present findings support the hypothesis that context-dependent EMT generated in our model by TGFβ1 might be the outcome of a dedifferentiation. In fact: 1) the principal biological categories involved in the process concern morphogenesis and development; 2) the most up-regulated genes belong to these categories; and, finally, 3) some intracellular pathways are involved, whose engagement during kidney development and nephrogenesis is well known. These long-term effects of TGFβ1 in HUTEC involve genes that are highly interconnected, thereby generating a scale-free network that we named the "TGFβ1 interactome", whose hubs represent proteins that may have a crucial role for HUTEC in response to TGFβ1.</p
Spontaneous calcification process in primary renal cells from a medullary sponge kidney patient harbouring a GDNF mutation.
Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process
in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding
glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two
patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was
studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using
immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry
and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes;
however, Ca2PO4 was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes
or stromal stem cells and were positive for vimentin, ZO1, aSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin,
indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown
was established in the HK2 cell line and was found to promote Ca2PO4 deposition when the cells were incubated with calcifying medium by regulating
the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which
pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have
influenced the observed phenomenon
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