Clinical and genetic characteristics of Dent's disease type 1 in Europe.

BACKGROUND: Dent's disease type 1 (DD1) is a rare X-linked nephropathy caused by CLCN5 mutations, characterized by proximal tubule dysfunction, including low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis-nephrocalcinosis, progressive chronic kidney disease (CKD) and kidney failure (KF). Current management is symptomatic and does not prevent disease progression. Here we describe the contemporary DD1 picture across Europe to highlight its unmet needs. METHODS: A physician-based anonymous international e-survey supported by several European nephrology networks/societies was conducted. Questions focused on DD1 clinical features, diagnostic procedure and mutation spectra. RESULTS: A total of 207 DD1 male patients were reported; clinical data were available for 163 with confirmed CLCN5 mutations. Proteinuria was the most common manifestation (49.1%). During follow-up, all patients showed LMWP, 66.4% nephrocalcinosis, 44.4% hypercalciuria and 26.4% nephrolithiasis. After 5.5 years, ≈50% of patients presented with renal dysfunction, 20.7% developed CKD stage ≥3 and 11.1% developed KF. At the last visit, hypercalciuria was more frequent in paediatric patients than in adults (73.4% versus 19.0%). Conversely, nephrolithiasis, nephrocalcinosis and renal dysfunction were more prominent in adults. Furthermore, CKD progressed with age. Despite no clear phenotype/genotype correlation, decreased glomerular filtration rate was more frequent in subjects with CLCN5 mutations affecting the pore or CBS domains compared with those with early-stop mutations. CONCLUSIONS: Results from this large DD1 cohort confirm previous findings and provide new insights regarding age and genotype impact on CKD progression. Our data strongly support that DD1 should be considered in male patients with CKD, nephrocalcinosis/hypercalciuria and non-nephrotic proteinuria and provide additional support for new research opportunities.

Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

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.

Genetic Analyses in Dent Disease and Characterization of CLCN5 Mutations in Kidney Biopsies.

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.

Cell Death in the Kidney.

Apoptotic cell death is usually a response to the cell's microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.

Immunization with pentraxin 3 (PTX3) leads to anti-PTX3 antibody production and delayed lupus-like nephritis in NZB/NZW F1 mice.

BACKGROUND: Anti-pentraxin 3 (PTX3) antibodies were associated with the absence of lupus glomerulonephritis in humans. AIM: To explore the effects of anti-PTX3 antibodies in New Zealand Black/White (NZB/NZW F1) mice and their inherent mechanisms of action. MATERIALS AND METHODS: 30 NZB/NZW F1 mice were subdivided into 3 groups of 10 mice each and subcutaneously injected with PTX3, alum and PBS (group 1), alum and PBS (group 2) or PBS alone (group 3), 3 times 3 weeks apart, before development of renal disease. Mice were followed until natural death. Histological analysis and immunohistochemistry were performed on harvested kidneys. Effects of anti-PTX3 antibodies on C1q binding to immobilized PTX3-anti-PTX3 immune complexes were evaluated in vitro using human SLE sera. Qualitative characterization of human IgG anti-PTX3 was performed. RESULTS: Only group 1 mice developed anti-PTX3 antibodies. Anti-dsDNA and anti-C1q antibodies appeared significantly later and at lower levels in group 1 mice vs. controls (p < 0.0001). Proteinuria-free and overall survival were significantly increased in group 1 mice vs. controls (p < 0.05 and p = 0.03, respectively). Histopathological analysis showed that glomerular and tubular PTX3 staining and renal lesions were increased in controls compared with immunized mice. Addition of human SLE sera positive for anti-PTX3 antibodies to C1q and fixed PTX3 interfered with C1q binding to PTX3-anti-PTX3 immune complexes. Qualitative characterization of human IgG anti-PTX3 showed an increased proportion of IgG4. CONCLUSIONS: Anti-PTX3 antibodies delay lupus-like nephritis and prolong survival of NZB/NZW F1 mice. In vitro observations suggest anti-PTX3 antibodies may dampen complement activation via their Fc fragment, likely hindering renal inflammation.

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, αSMA 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.

Novel INF2 mutations in an Italian cohort of patients with focal segmental glomerulosclerosis, renal failure and Charcot-Marie-Tooth neuropathy.

BACKGROUND: Mutations of INF2 represent the major cause of familial autosomal dominant (AD) focal segmental glomerulosclerosis (FSGS). A few patients present neurological symptoms of Charcot-Marie-Tooth (CMT) disease but the prevalence of the association has not been assessed yet. METHODS: We screened 28 families with AD FSGS and identified 8 INF2 mutations in 9 families (32 patients overall), 3 of which were new. Mutations were in all cases localized in the diaphanous-inhibitory domain (DID) of the protein. RESULTS: Clinical features associated with INF2 mutations in our patient cohort included mild proteinuria (1.55 g/L; range 1-2.5) and haematuria as a unique symptom that was recognized at a median age of 21.75 years (range 8-30). Eighteen patients developed end-stage renal disease during their third decade of life; 12 patients presented a creatinine range between 1.2 and 1.5 mg/dL and 2 were healthy at 45 and 54 years of age. CMT was diagnosed in four cases (12.5%); one of these patients presented an already known mutation on exon 2 of INF2, whereas the other patients presented the same mutation on exon 4, a region that was not previously associated with CMT. CONCLUSIONS: We confirmed the high incidence of INF2 mutations in families with AD FSGS. The clinical phenotype was mild at the onset of the disease, but evolution to ESRD was frequent. The incidence of CMT has, for the first time, been calculated here to be 12.5% of mutation carriers. Our findings support INF2 gene analysis in families in which renal failure and/or neuro-sensorial defects are inherited following an AD model.

When Waldenström macroglobulinemia hits the kidney: Description of a case series and management of a "rare in rare" scenario.

BACKGROUND: Renal injury related to Waldenström macroglobulinemia (WM) occurs in approximately 3% of patients. Kidney biopsy is crucial to discriminate between distinct histopathological entities such as glomerular (amyloidotic and non-amyloidotic), tubulo-interstitial and non-paraprotein mediated renal damage. In this context, disease characterization, management, relationship between renal, and hematological response have been poorly explored. We collected clinical, genetic and laboratory data of seven cases of biopsy-proven renal involvement by WM managed at our academic center and focused on three cases we judged paradigmatic discussing their histopathological patterns, clinical features, and therapeutic options. CASE: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. In our series AL Amyloidosis (n = 3/7) and tubulo-interstitial infiltration by lymphoma cells (n = 3/7) were the two more represented entities. BTKi did not seem to improve renal function (Case 1), while bortezomib-based regimens demonstrated a beneficial activity on the hematological and organ response, even when used as second-line therapy after chemoimmunotherapy (Case 3) and also with coexistence of anti-MAG neuropathy (Case 2). In case of poor response to bortezomib, standard chemoimmunotherapy (CIT), such as rituximab-bendamustine, represents an effective option (Case 1, 6, and 7). In our series, CIT generates durable responses more frequently in cases with amyloidogenic renal damage (Case 1, 5, and 7). CONCLUSION: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. Studies with higher numerosity are needed to better clarify the pathological and clinical features of renal involvement during WM and to determine the potential benefit of different therapeutic regimens according to the histopathological subtypes.

Human parietal epithelial cells (PECs) and proteinuria in lupus nephritis: a role for ClC-5, megalin, and cubilin?

BACKGROUND: Parietal epithelial cells are a heterogeneous population of cells located on Bowman's capsule. These cells are known to internalize albumin with a still undetermined mechanism, although albumin has been shown to induce phenotypic changes in parietal epithelial cells. Proximal tubular cells are the main actors in albumin handling via the macromolecular complex composed by ClC-5, megalin, and cubilin. This study investigated the role of ClC-5, megalin, and cubilin in the parietal epithelial cells of kidney biopsies from proteinuric lupus nephritis patients and control subjects and identified phenotypical changes occurring in the pathological milieu. METHODS: Immunohistochemistry and immunofluorescence analyses for ClC-5, megalin, cubilin, ANXA3, podocalyxin, CD24, CD44, HSA, and LTA marker were performed on 23 kidney biopsies from patients with Lupus Nephritis and 9 control biopsies (obtained from nephrectomies for renal cancer). RESULTS: Two sub-populations of hypertrophic parietal epithelial cells ANXA3+/Podocalyxin-/CD44-, both expressing ClC-5, megalin, and cubilin and located at the tubular pole, were identified and characterized: the first one, CD24+/HSA-/LTA- had characteristics of human adult parietal epithelial multipotent progenitors, the second one, CD24-/LTA+/HSA+ committed to become phenotypically proximal tubular cells. The number of glomeruli presenting hypertrophic parietal epithelial cells positive for ClC-5, megalin, and cubilin were significantly higher in lupus nephritis patients than in controls. CONCLUSIONS: Our results may provide further insight into the role of hypertrophic parietal epithelial cells located at the tubular pole and their possible involvement in protein endocytosis in lupus nephritis patients. These data also suggest that the presence of hypertrophic parietal epithelial cells in Bowman's capsule represents a potential resource for responding to protein overload observed in other glomerulonephritis.

Downregulation of megalin, cubilin, ClC-5 and podocin in Fabry nephropathy: potential implications in the decreased effectiveness of enzyme replacement therapy.

Fabry disease is an X-linked disorder due to mutations in α-galactosidase A, resulting in the accumulation of enzyme substrates and cell malfunction. Kidney involvement is frequent, affecting all native kidney cell types. Podocyte damage results in proteinuria and chronic kidney disease. End-stage kidney disease is the rule in middle-aged males and some females with the classic phenotype. In podocytes and kidney proximal tubular cells, megalin is one of the molecules involved in enzyme replacement therapy (ERT) cellular absorption. After podocyte damage, podocin concentration is decreased and contributes to progressive proteinuria. We report in a male and a female patient the decreased expression of megalin, cubilin, ClC-5 and podocin compared to controls and chronic kidney disease (CKD) biopsies. Moreover, the decrease in ClC-5, a molecule engaged in endosomal-lysosomal acidification, could also affect ERT. These findings may partially explain some of the dysfunctions described in Fabry nephropathy and could highlight possible alterations in the pharmacokinetics of the delivered enzyme.

Genotype Phenotype Correlation in Dent Disease 2 and Review of the Literature: OCRL Gene Pleiotropism or Extreme Phenotypic Variability of Lowe Syndrome?

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

From protein uptake to Dent disease: An overview of the CLCN5 gene.

Proteinuria is a well-known risk factor, not only for renal disorders, but also for several other problems such as cardiovascular diseases and overall mortality. In the kidney, the chloride channel Cl-/H+ exchanger ClC-5 encoded by the CLCN5 gene is actively involved in preventing protein loss. This action becomes evident in patients suffering from the rare proximal tubulopathy Dent disease because they carry a defective ClC-5 due to CLCN5 mutations. In fact, proteinuria is the distinctive clinical sign of Dent disease, and mainly involves the loss of low-molecular-weight proteins. The identification of CLCN5 disease-causing mutations has greatly improved our understanding of ClC-5 function and of the ClC-5-related physiological processes in the kidney. This review outlines current knowledge regarding the CLCN5 gene and its protein product, providing an update on ClC-5 function in tubular and glomerular cells, and focusing on its relationship with proteinuria and Dent disease.

CLCN5 5'UTR isoforms in human kidneys: differential expression analysis between controls and patients with glomerulonephritis.

ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed in glomeruli of glomerulonephritis and in cultured human podocytes under albumin overload. The transcriptional regulation of human ClC-5 is not fully understood. Three functional promoters of various strengths and 11 different 5' untranslated region (5'UTR) isoforms of CLCN5 messenger RNA (mRNA) were detected in the human kidney (variants 1-11). The aim of this study was to investigate the expression pattern of CLCN5 5'UTR variants and the CLCN5 common translated region in glomerulonephritis. The 5'UTR ends and the translated region of CLCN5 mRNA were analyzed using quantitative relative real-time PCR or quantitative comparative endpoint PCR with GAPDH as housekeeping gene in 8 normal kidneys and 12 renal biopsies from patients with glomerulonephritis. The expression profile for all variants in normal and glomerulonephritis biopsies was similar, and variant 3 and alternative variant 4 were the most abundantly expressed in both sets. In glomerulonephritis biopsies, isoforms under the control of a weak promoter (variants 4, 6 and 7) showed an increased expression leading to an increase in the CLCN5 translated region, underscoring their importance in kidney pathophysiology. Since weak promoters can be turned on by different stimuli, these data support the hypothesis that proteinuria could be one of the stimuli capable of starting a signaling pathway that induces an increase in CLCN5 transcription.

Early activation of fibrogenesis in transplanted kidneys: a study on serial renal biopsies.

BACKGROUND/AIMS: In kidney transplants, the renin-angiotensin system (RAS) is involved in systemic and local changes that may induce fibrosis. Our aim was to use gene expression and immunohistochemical analysis to investigate the RAS and several factors involved in the fibrogenic cascade in allograft biopsies. METHODS: We considered 43 donor biopsies (T0), 18 biopsies obtained for diagnostic purposes (Td) and 24 protocol biopsies (Tp) taken 2 months after transplantation in patients with stable renal function. Morphometric alpha SMA and TGF beta 1 analysis, and Masson's Trichrome staining were performed. mRNA levels of angiotensinogen, renin, ACE, AT1-R, AT2-R, TGF beta 1, BMP-7, Coll III, fibronectin and alpha SMA were analyzed by real-time RT/PCR. MDRD a year after the transplant was also considered. RESULTS: Significantly higher levels of AT1-R and alpha SMA transcripts were found in Tp than in T0. Regression analysis showed significant TGF beta 1-independent positive correlations between RAS and matrix components in T0 and Tp, but more evident in Tp, where a positive correlation between TGF beta 1 and Masson's Trichrome stained areas was also seen. CONCLUSION: Our results suggest that RAS and TGF beta 1-related fibrogenic loops are activated as early as 2 months after kidney transplantation.

Primary IgA nephropathy is more severe in TGF-beta1 high secretor patients.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and is characterized by extremely variable clinical and morphological features and outcome. TGF-beta1 has a key role in fibrogenesis and the progression of renal damage. Its production is under genetic control. METHODS: We recruited 105 Italian biopsy-proven IgAN patients for genotyping for the TGF-beta1 C-509T, T869C (COD 10) and G915C (COD 25) polymorphisms; 200 healthy blood donors were used as normal controls. Glomerular and interstitial mRNA levels of TGF-beta1 were assessed by real-time PCR in 34 patients to seek relationships with clinical, renal histopathological features and outcome. RESULTS: The genotype distributions in the IgAN population were not statistically different from the controls. The COD 10 TT genotype was associated with more severe histological damage as assessed by Lee's classification (CC 50%, CT 39.6% and TT 17.2% were graded as mild; CC 35.7%, CT 43.7% and TT 44.8% as moderate, and CC 14.3%, CT 16.7% and TT 37.9% as severe [p=0.0049]) and with severe interstitial infiltrates (CC 10.4%, CT 35.2% and TT 54.2% [p=0.03]). A higher interstitial immunodeposition was observed for TGF-beta1, collagen IV and alpha-SMA in patients with the COD 10 T allele (p=0.045, p=0.049, p=0.032, respectively). The T allele was associated with significantly higher TGF-beta1 mRNA levels in the interstitium (TT+CT vs. CC: 0.52 +/- 0.16 vs. 0.18 +/- 0.10 copies/mL, respectively; p=0.000). The T allele was also associated with higher mRNA levels in glomeruli, though the difference was not statistically significant. Finally, the T allele was significantly associated with a worse prognosis, the end points being reached by 40% of TT+CT and 32% of CC patients (p=0.009). CONCLUSIONS: In primary IgA nephropathy, the T allele of the TGF-beta1 COD 10 C/T polymorphism seems to be associated with more severe histological lesions, higher renal TGF-beta1 mRNA levels and a worse prognosis. This polymorphism seems to be functionally relevant and to have a prognostic impact.

The origin of nephrocalcinosis, Randall's plaque and renal stones: a cell biology viewpoint.

Medullary nephrocalcinosis is a rare condition typically observed in metabolic conditions prone to renal calcium stones. Randall's plaques are very frequently observed in the common idiopathic calcium-oxalate nephrolithiasis. These plaques are apatite mineral structures, and we propose they also are an example of nephrocalcinosis. While these calcium deposits are generally considered to be the consequence of purely physico-chemical phenomena, we advance the hypothesis that they form because of a true ectopic biomineralization in the renal tissue. Henle's loop epithelial cells, or pericyte-like interstitial cells, or papillary stem-cells differentiating along a bone lineage could be involved.

Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation.

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.