| OBJECTIVE:To address the effect of bone-related factors and1,25(OH)2D3/VDR on renal stone formation in idiopathic hypercalciuria (IH) using genetic hypercalciuric rats.METHODS:Experiment1:Ten20-generation genetic hypercalciuric rats with a body weight of200-270g were used for the study. A total of8Sprague-Dawley rats matched with genetic hypercalciuric rats on body weight and age were served as NC rats. The kidney tissues were removed from genetic hypercalciuric and NC rats for analysis using real-time reverse transcription polymerase chain reaction and Western blot to quantify the mRNA and protein expression levels of BMP2, Runx2, Osterix, MSX2, osteopontin (OPN) and alkaline phosphatase (ALP), respectively.Experiment2:Forty five22-and thirty23-generation genetic hypercalciuric rats were randomly divided into3groups. Group1(n=25) was injected with1mL normal saline (NS) through the rat tail vein, group2(n=25) with1mL VDR-RNAi-Lentivirus, and group3(n=25) with1mL GFP-Lentivirus. On days 0,3,7,14and28of the experiment,5rats in each group were killed. Expression of VDR-RNAi observed by delivered green fluorescent protein (GFP) fluorescence using fluorescence microscopy.Experiment3:The RTECs were seeded into six-well plates at a density of2x105cells/well. Primary RTECs were transduced with Lenti-VDR-sh at1×106TU per ml at37℃, and classified as group2. As control, the cells were incubated with Lenti-VDR-nc was served as group3. In group1, the RTECs were treated with normal saline (NS). Cells were harvested at0,6,12,24,48and96hr of infection and cells expressing green fluorescence were identified using a fluorescence microscope. Both gene and protein levels of BMP2, Runx2and Osterix were detected using real-time PCR and Western blot assays, respectively.Experiment4:The RTECs were seeded into six-well plates at a density of5x105cells per well. Primary RTECs treated with1,25(OH)2D3at10-8mol/L per well were collected at6,12,24,48and96hours. Both gene and protein levels of BMP2, Runx2and Osterix were detected using real-time PCR and Western blot assays, respectively. The calcium content of cells was also measured.RESULTSExpression levels of bone-related factors in vivoAs shown in the results of RT-PCR and Western blot, both mRNA and protein expression levels of BMP2, Runx2, Osterix and OPN in the kidney tissue were significantly increased in genetic hypercalciuric rat group compared with NC group. Conversely, there were no significant differences in mRNA and protein expression levels of MSX2and ALP between the genetic hypercalciuric and NC rats.Effect of VDR knockdown on expression of bone-related factors in genetic hypercalciuric rat kidney tissueVDR knockdown reduced the mRNA levels of BMP2, Runx2, Osterix and OPN in genetic hypercalciuric rat kidney tissue. Compared to NS and GFP-Lentivirus groups, the mRNA expression levels of BMP2, Runx2, Osterix and OPN in VDR-RNAi-Lentivirus group were markedly decreased on days7,14and28. In contrast, no significant effects were detected on the mRNA expression levels of BMP2, Runx2, Osterix and OPN between the NS and GFP-Lentivirus groups. Western blot analysis confirmed the constant change in protein expression level as in mRNA expression level after silencing the VDR gene in genetic hypercalciuric rat kidney tissue. After VDR-RNAi-Lentivirus transfection, the protein levels of BMP2, Runx2, Osterix and OPN were evidently decreased on days7,14and28. Conversely, BMP2, Runx2, Osterix and OPN protein levels were not altered after treatment of NS or GFP control lentivirus.Immunohistochemical staining showed that OPN protein was present in the cytoplasm of renal tubule cells of the cortex (d0) and medulla (d3) in genetic hypercalciuric rats. There was a reduction in OPN expression along with reduction in calcification. The reduction in OPN expression was present on days7,14and28in VDR-RNAi-Lentivirus group. However, in GFP-Lentivirus group, there was still a vivid expression of OPN protein on day28. Representative pictures were shown with a magnification of100.Effect of VDR Depletion on calcium phosphate deposits in genetic hypercalciuric rat kidney tissueAbundant calcium phosphate deposits were revealed in the renal tubules of the cortex and medulla in genetic hypercalciuric rat kidney tissue stained with von Kossa method. Tubular calcium phosphate deposits were decreased on days7,14and28after VDR-RNAi-Lentivirus transfection. However, at28day of infection, tubular calcium phosphate deposits in GFP-Lentivirus group were as rich as in VDR-RNAi-Lentivirus group on days0and3.Effect of VDR knockdown on expression of bone-related factors in primary RTECsNo significant differences in mRNA expression levels of BMP2, Runx2and Osterix among NS, Lenti-VDR-sh and Lenti-VDR-nc were deteced at the beginning of experiment (0hr). After24,48and96hr of infection, however, the mRNA expression levels of BMP2, Runx2and Osterix were markedly decreased (P<0.01) in Lenti-VDR-sh, and in the latter more less than in the former. In contrast, there were no significant differences in the mRNA expression levels of BMP2, Runx2and Osterix between the NS and Lenti-VDR-nc. Western blot analysis confirmed the similar change in protein expression level as in mRNA expression level after silencing the VDR gene in primary RTECs. Effect of VDR Depletion on calcium depositions in primary RTECsThe quantify of calcium deposition in primary RTECs was determined colorimetrically by the o-cresolphthalein complexone method. There were no significant differences in calcium deposition in primary RTECs among NS, Lenti-VDR-sh and Lenti-VDR-nc were deteced at the beginning of experiment. The decrease in the calcium content of primary RTECs could be observed as early as12hr after exposure to Lenti-VDR-sh, and the lowest level was reached at48hr. However, The total amounts of calcium deposition in cells between the NS and Lenti-VDR-nc were unchanged during the same periods.Effect of elevated1,25(OH)2D3on expression of bone-related factors in primary RTECsThe mRNA expression levels of BMP2, Runx2and Osterix mRNA in the treated cells began to increase in12h and continued to increase. At96h, the up-regulation of1,25(OH)2D3induced a double increase in mRNA levels of BMP2, Runx2and Osterix in primary RTECs. Western blot analysis confirmed the similar protein expression change as mRNA expression after the up-regulation of1,25(OH)2D3in primary RTECs.Effect of elevated1,25(OH)2D3on calcium levels in primary RTECsWe cultured cells for the indicated times in NS (control), VD3-1(10-7mol/L), VD3-2(10-8mol/L), VD3-3(10-9mol/L) and VD3-4(10-10mol/L).1,25(OH)2D3at10-8mol/L had the best efficiency in primary RTECs determined by real-time PCR (P<0.01).Primary RTECs were divided into treated and untreated cells. The treated cells were incubated with1,25(OH)2D3and collected at96h.The untreated cells were served as control group. Von Kossa staining indicated that calcium phosphate deposits were located in the cytoplasm of primary RTECs and the intensity of staining in treated cells at96h was significantly greater than in untreated cells.To determine the relationship between1,25(OH)2D3and RTEC mineralization, calcium levels were examined in primary RTECs treated with elevated1,25(OH)2D3. Treatment of primary RTECs with1,25(OH)2D3caused time-dependent upregulation of calcium levels and upregulated calcium levels by2.14-fold at96hr.CONCLUSIONSOur findings indicate that bone-related factors, including BMP2, Runx2, Osterix and OPN may play an important role in renal stone formation in IH, while MSX2and ALP are not associated with that.1,25(OH)2D3/VDR may be an important regulator on renal stone formation in IH. |
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