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Medial vascular calcification (VC) develops as a consequence of hyperphosphatemia secondary to chronic kidney disease. The role of inorganic phosphate (Pi) transporters in the pathogenesis of VC has been previously determined by interference of RNA and inhibition by phosphonoformic acid (PFA). We have found that inhibition of Pi transport by PFA in vascular smooth muscle cells (VSMC) from rat aorta showed a Ki of 2.6 mM, while the Km for Pi was 0.1 mM. VSMC express both type III Pi transporters, Pit1 and Pit2. When expressed in Xenopus laevis oocytes Pit1 and Pit2 were inhibited with PFA with Ki values of 2.7 and 4.6 mM, respectively, while they showed affinities of 0.1 and 0.2 mM. PFA at either 1 or 5 mM completely prevented calcification of VSMC induced with 2 mM phosphate for 5 days. Phase-contrast microscopy revealed that cells treated with either 1 or 5 mM PFA in either normal (1 mM) or high (2 mM) Pi concentration showed a different morphology and evidence of cell death. Cytotoxicity was analyzed as a function of cytosolic lactate dehydrogenase activity, revealing a mean lethal concentration (LC50) of 0.38 mM. Therefore the prevention calcification was not due to inhibition of Pi transport but to cytotoxicity of PFA that prevented trans-differentiation of VSMC into osteogenic-like cells. Further experiments have been developed to characterize the type of cell death, including apoptosis. [ABSTRACT FROM AUTHOR]

Sterol Regulatory Element Binding Protein-1 (SREBP-1) undergoes two proteolytic cleavages before entering the nucleus to act as a transcription factor for biosynthesis of fatty acids. This study focused on the quantitation of SREBP-1 in cultured rat renal mesangial cells (CRL-2573) exposed to either normal of high glucose concentrations that might be important in lipid accumulation as part of diabetic nephropathy. We hypothesize a significant increase of active SREBP-1 expression by cells in high glucose conditions. Rat mesangial cells were grown in either low (100 mg/dl) or high (450 mg/dl) glucose media for 48 hours. Cells were homogenized, separated into cytoplasmic and nuclear fractions and Western Blot was performed. Enzymatic Chemiluminescence was used to detect immunoreactive bands which were quantified using BioRad Quantity One 1-D analysis software. Levels of SREBP-1 were measured in arbitrary units of density and results showed that the active SREBP-1 in nuclear fraction was increased by 31.79% in the high glucose group; which was statistically significant at p<0.05. This distinctive increase may be a preliminary sign of lipid accumulation and cytotoxicity. [ABSTRACT FROM AUTHOR]

Renal reabsorption of inorganic phosphate (Pi) is mediated through the activity of high affinity, type II Na-coupled Pi cotransporters in the proximal tubular cells. The rate of reabsorption is the main target for hormonal and metabolic factors that control phosphate homeostasis, including parathyroid hormone or adaptation to changes in dietary phosphate. Pi transport is also competitively inhibited by phosphonoformic acid (PFA) and arsenate, with Ki values of 0.13 and 0.2 mM, respectively, in opossum kidney cells (OK; an in vitro model of Pi renal reabsorption). However, NaPi4, the main type II Pi transporter in OK cells, is less efficiently inhibited by PFA and arsenate, with corresponding Ki values of 0.86 and 1 mM. Preincubation of OK cells with 1 mM PFA for 1 hour mimics acute adaptation to low Pi. However, preincubation with 1 mM arsenate has the same effect on transport activity as adaptation to 2 mM Pi. These effects are specific of Pi transport, as no alterations are observed in other Na-coupled transport processes including Na-glucose. Time-course and saturation kinetics suggest that these effects are mediated through changes in the abundance of Pi transporters in the plasma membrane. While both inhibitors act in a competitive manner, only arsenate is efficiently transported and, once inside the cell, it is also recognized as Pi by the sensing mechanisms that induce the cell to adapt to high Pi concentration. [ABSTRACT FROM AUTHOR]

Recent studies indicate an important role for nuclear receptors in regulating lipid and carbohydrate metabolism, fibrosis and inflammation. Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily. High expression of FXR is restricted to liver, intestine, adrenal gland and kidney. The primary bile acids are the highest affinity endogenous ligands for FXR. The effects of FXR agonists in diabetic kidney disease, the main cause of end stage renal disease, have not been determined. To identify the effect of FXR activation in modulation of diabetic nephropathy, we have treated 1) C57BL/6J mice on low fat diet or high fat diet with FXR agonists (GW4064, 6-ECDCA or cholic acid) for one week; and 2) db/db mice, a model of obesity and type 2 diabetes mellitus, and their lean mates with cholic acid for 12 weeks. We found that FXR agonists modulate renal lipid metabolism, regulate renal expression of profibrotic growth factors, proinflammatory cytokines, and oxidative stress, and decrease glomerulosclerosis, fibrosis, and proteinuria. Overexpression of FXR or treatment with FXR agonist in mesangial cells also showed similar effects, suggesting a direct role of FXR in modulating renal lipid metabolism and modulation of fibrosis and inflammation. These results therefore indicate a new and important role for FXR in the kidney and provide new therapeutic avenues for the treatment of diabetic nephropathy. [ABSTRACT FROM AUTHOR]