Brigitte Lelongt

Fluoride Ion Toxicity in Human Kidney Collecting Duct Cells

Background Several halogenated anesthetics induce a urinary concentrating defect, partly related to fluoride ion toxicity in collecting duct cells. The aim of this study was to investigate the effects of fluoride ion in human kidney cells. Methods Immortalized human collecting duct cells were used. In a first set of experiments, the toxicity threshold concentration was determined by exposing cell cultures for 24 h to increasing concentrations of fluoride ion in the medium: 0, 1, 5, and 10 mM. The second set of experiments was a time-effect study in which cells were exposed to 5 mM fluoride for 2, 6, and 24 h. Assessment of toxicity was based on several endpoints: cell number, protein content,3 Hydrogen-leucine incorporation in newly synthesized proteins, extracellularly released lactate dehydrogenase, Sodium-Potassium-ATPase pump activity, and electron microscope studies. Results After 24 h of exposure, fluoride ion decreased cell number (-23%, P < 0.05), total protein content (-30%, P < 0.05), and3 Hydrogen-leucine incorporation (-43%, P < 0.05) and increased lactate dehydrogenase release (+236%, P < 0.05) at a threshold concentration of 5 mM. Fluoride ion also inhibited Sodium-Potassium-ATPase activity at 5 mM (-58%, P < 0.05). Major morphologic alterations of mitochondria, including crystal formation, were detected from 1 mM fluoride concentration. Time-effect studies showed that, after only 6 h of exposure at 5 mM, fluoride decreased cell number (-13%, P < 0.05),3 Hydrogen-leucine incorporation (-48%, P < 0.05), and Sodium-Potassium-ATPase activity (-20%, P < 0.05) and increased lactate dehydrogenase release (+145%, P < 0.05). Crystal deposits in mitochondria again were a more sensitive marker of cell injury, detectable after only 2 h of exposure. Conclusions These results suggest that the mitochondrion is a target of fluoride toxicity in human collecting duct cells, and its alteration is partly responsible for the sodium and water disturbances observed in patients.

Do matrix metalloproteinases MMP-2 and MMP-9 (gelatinases) play a role in renal development, physiology and glomerular diseases?

Metalloproteinases MMP-2 and MMP-9 (also called gelatinases) are involved in cell invasion and in embryonic development and organogenesis. A growing number of reports suggest that MMP-2 and MMP-9 play some role in renal development, renal tubule physiology and glomerular pathophysiology. This editorial will focus on recent controversial data, especially those obtained from studies on MMP-9-deficient mice, which shed new light on the functions of gelatinases in normal and diseased kidneys.

Role of proteoglycans in renal development

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.

MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

Mannose-induced dysmorphogenesis of metanephric kidney. Role of proteoglycans and adenosine triphosphate.

Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis. 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with [35S]sulfate, [35S]-methionine, or [3H]thymidine. Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis. Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs. Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced. Biochemical studies revealed decreased synthesis of sulfated PGs. PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio. Immunoprecipitation of [35S]methionine-labeled proteins confirmed the reduction of PG core peptides. Intracellular ATP levels were reduced. The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by [3H]thymidine incorporation. The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2. Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.

Is PKC-δ a New Killer Molecule in Kidney?

The prevalence of chronic kidney diseases is increasing worldwide, and many of these nephropathies progress to renal failure. Our understanding of the pathophysiology of deteriorating renal function is still limited, and the choices for therapy are confined to a few general interventions. The degree of proteinuria correlates with the rate of progression to ESRD, suggesting that proteinuria itself could be one of the mechanisms for progression. Finding that upon therapeutic intervention the rate of decline of GFR correlates negatively with reduction in proteinuria and positively with persistent proteinuria provides further evidence for a pathogenic role of proteinuria in renal progression.1 Albumin is the major protein present in nephrotic urine, and a variety of studies have examined the toxic effect of filtered albumin in the kidney. Special attention has been paid to proximal tubule cells (PTCs), because the majority of the albumin in the glomerular filtrate is absorbed by phosphatidylinositol-3-kinase–mediated endocytosis after the binding to the multiligand receptors megalin and cubulin2 for tubular degradation by lysosomes. Gene expression profiling of PTCs from mice overloaded with BSA identified 2000 genes that are regulated differentially.1 In vitro and in vivo models of albumin overload also describe increased production of proinflammatory molecules, matrix genes, and profibrogenic cytokines such as monocyte chemoattractant protein 1, TGF-β, IL-8, endothelin 1, transcriptional factor NF-κB, and RANTES. Most of these mediators are responsible for tubulointerstitial inflammation and fibrosis through recruitment and activation of lymphocytes, macrophages, and fibroblasts. In addition, numerous studies have reported that high concentrations of albumin in vitro and in vivo induce apoptosis in PTCs, although some issues on the effect of albumin on tubular cell apoptosis are still misunderstood or controversial. First, it has been argued that tubular cell apoptosis is induced by high molecular weight proteins …

Relevance of Proteoglycans in Glomerular Matrix Pathology

Proteoglycans are one of the essential components of the extracellular matrices, i.e., glomerular basement membrane and mesangial matrix. Heparan sulfate-proteoglycan (HS-PG) appears to be the major proteoglycan present in the extracellular matrices, with small amounts of chondroitin sulfate-proteoglycans present in the mesangial matrix. The functions of the chondroitin sulfate-proteoglycans in the mesangium are unknown. The latter may play some role in glomerulo-sclerosis in various forms of nephritides, e.g., diabetes. The biologic relevance of HS-PG has been investigated extensively since its discovery a decade ago. Its biologic significance was first explored in glomerular permeability. Normally, anionic molecules such as plasma albumin are highly restricted during their passage across the glomerular capillary. With either the removal of or neutralization of the charge of HS-PG, the passage of albumin is greatly facilitated and the albumin appears in urine. Thus, the HS-PG imparts charge and size selectivity to the glomerulus. The loss of charge selectivity has been seen in several forms of glomerulonephritis, including diabetic nephropathy, where the synthesis of HS-PG has been found to be decreased. Moreover, intravenous administration of antiHS-PG antibodies induces a proteinuric response and dramatically alters the extracellular matrices, thereby indicating that the HS-PG is nephritogenic. Another important role of HS-PG has been recently elucidated in renal morphogenesis. With the perturbation of proteoglycan metabolism, one finds failure in the development of the whole kidney in general, and also delayed maturation of the glomeruli as well as their extracellular matrices. Finally, several heparin binding domains on the laminin molecule have been described in in vitro systems, but their biologic significance is unknown.