| Part ⅠAssociation between circulating vitamin D level and urolithiasis:a systematic review and meta-analysisBackground and objectiveMany studies compared the serum/plasma 1,25 dihydroxyvitamin D3(1,25(OH)2D3)and 25 hydroxyvitamin D3(25(OH)D)between people with and without nephrolithiasis and their results were conflicting.The aim of this study was to systematically compare the vitamin D levels between the two groups.MethodsAfter systematically searching PubMed,Web of Science,The Cochrane Library,CNKI and Wanfang Database,we conducted a meta-analysis.ResultsThirty two observational studies involving 23228 participants were included.Meta-analysis of these studies showed that stone formers(SFs),calcium SFs had significantly higher concentrations of 1,25(OH)2D3(WMD,10.19pg/ml;95%CI,4.31-16.07;p=0.0007 and WMD,11.28pg/ml;95%CI,4.07-18.50;p=0.002,respectively)than non-SFs,while the levels of 25(OH)D(WMD,0.88ng/ml;95%CI,-1.04-2.80;p=0.37 and WMD,-0.63ng/ml;95%CI,-2.72-1.47;p=0.56,respectively)were similar.Compared with controls and normocalciuria SFs,hypercalciuria SFs had increased circulating 1,25(OH)2D3(WMD,9.41pg/ml;95%CI,0.15-18.67;p=0.05 and WMD,2.75pg/ml;95%CI,-0.20-5.69;p=0.07,respectively)and markedly higher of 25(OH)D(WMD,5.02ng/ml;95%CI,0.99-9.06;p=0.01 and WMD,5.02ng/ml;95%CI,2.14-7.90;p=0.0006,respectively).Normocalciuria SFs had elevated 1,25(OH)2D3 level(WMD,6.85pg/ml;95%CI,-5.00-18.71;p=0.26)and comparable 25(OH)D(WMD,0.94ng/ml;95%CI,-3.55-5.43;p=0.68).Sensitivity analysis generated similar results.ConclusionCurrent evidence suggests that increased circulating 1,25(OH)2D3 is associated with urinary stones and a higher level of circulating 25(OH)D is significantly associated with hypercalciuria urolithiasis.Further studies are still needed to reconfirm and clarify the role of vitamin D in the pathogenesis of stones.Part IIConstruction of a hypercalciuric stone-forming rat model based on the activation of vitamin D signaling pathwayBackground and objectiveAt present,the majority of animal models of urolithiasis are hyperoxaluria animal model and the hypercalciuria stone models are less.Classic genetic hypercalciuric stone-forming rats and some gene-engineering mice with renal interstitial calcification are complex and/or expensive in cultivation.But relevant researches have demonstrated that these animal models have a common characteristic that is the abnormal activation of vitamin D signaling,and our previous studies have also confirmed the increased circulating active vitamin D in stone patients.Therefore,we try to administrate the SD rats with active vitamin D alone and hope to establish a simple,economic and repeatable hypercalciuric stone-forming rats.MethodsSix weeks old male SD rats were randomly divided into vitamin D group(24 rats),control group(18 rats)and glyoxylic acid group(6 rats),and they received normal diet and drinking water.Vitamin D group were administered with 1,25(OH)2D3 100ng/100g body weight intraperitoneally every other day.Their body weights and 24 hours urinary calcium were measured at the 2nd,4th,8th and 12th week.And at the 4th,8th and 12th week,8 rats were sacrificed and their blood samples were collected for determination of serum calcium and phosphorus,liver and renal function index.The kidneys were stained with HE and Von Kossa to assess the microstructure,calcium deposition and renal stone.The scanning electron microscope was used to observe the ultrastructure of the deposition and stones,and micro CT scan and three-dimensional reconstruction was conducted to acquire the overall observation of renal calcium deposition and stone formation.The rats in the control group received an equivalent amount of solvent intraperitoneally every two days and the other processing and detection methods were same to the rats in the vitamin D group.The rats in the glyoxylic acid model group received daily intraperitoneal injection of glyoxylic acid solution 10mg/100g body weight for 10 days.And then,they were sacrificed and their kidneys were stained with HE and Von Kossa.The distributions of calcium deposition and renal stones in the kidneys were compared between the two modelling groups.ResultsThe body weight of rats in the vitamin D group became significantly lower than that of control group since the 4th week,and the liver function and renal function were comparable between vitamin D and control group.The rats in vitamin D group had increased serum calcium at all time points,but only had significantly higher level of serum phosphorus at the 4th week.The urinary calcium concentration and 24 hours urinary calcium were significantly higher in the vitamin D group.At the 4th week,micro CT could detected occasional renal calcium deposition in the rats receiving vitamin D;at 8th week,Von Kossa staining showed calcium deposition in the renal inner medulla and pelvic stones,and micro CT observed renal parenchyma calcification and renal stone formations;and the degree of calcification aggravated at the 12th week.Scanning electron microscopy showed that the renal interstitial calcifications were consisted of small plate-like or concentric mineral deposition,and further energy dispersive spectrometry showed that the calcification was composed primarily of calcium and phosphorus.The kidney stone had a relatively loose central structure and it was wrapped with an outer layer of dense mineral structure.Its composition also contained calcium and phosphorus.Different from those of rats in the vitamin D group,the calcium deposition of the rats receiving glyoxylic acid was mainly located at the junction of cortex and medulla.ConclusionIntraperitoneal injection of 1,25(OH)2D3 100ng/100g body weight in the SD rats could induce hypercalciuria,renal interstitial calcification and renal stone formation.This model can be used in the studies focusing on renal interstitial calcification and hypercalciuria stones.And it is worthwhile to further optimize the modeling method.Part IIIBiological mechanisms of high concentration of 1,25(OH)2D3 and Ca2+ in inducing renal interstitial calcification and stone formationBackground and objectiveRandall’s plaque,an ectopic calcification located in the renal interstitium,may be the initial nidus for kidney stones.1,25(OH)2D3 and its induced hypercalciuria can promote renal interstitial calcification and kidney formation.The aims of this study were to further confirm the association between Randall’s plaque and nephrolithiasis,and to explore the biological mechanisms of high concentration of 1,25(OH)2D3 and Ca2+ in inducing renal interstitial calcification and kidney formation.MethodsTo further validate the Randall’s plaque theory,the renal papillary density(hounsfield unit)of age and sex matched stone formers and non-stone formers were compared according to their abdominal CT scan images.The expression levels of some ectopic calcification inhibitors including matrix Gla protein(MGP),osteopontin(OPN),osteocalcin(OCN),osteoprotegerin(OPG)and Gla-rich protein(GRP)were compared between patients with and without nephrolithiasis by target analyzing microarray data,and the core molecule of this study was determined.The expression and function of the core molecule were further confirmed at animal and cellular levels,and the biological effects of high concentration of calcium and vitamin D on cellular proliferation,apoptosis,oxidative stress,wound healing and cell-crystal interaction were determined.ResultsThe mean renal papillary density(HU value)was significantly higher in stone formers(49.81±7.12 vs 40.98:± 5.68).Compared with that of non-stone formers,the expression of MGP decreased by 3.15 times(p = 0.05)in the renal papilla of the patients with stones,and the expressions of OPN,OPG,OCN and GRP were increased by 1.93(p = 0.52),1.16(p = 0.58),1.57(p = 0.06)and 2.04(p = 0.08)times,respectively.Immunochemistry staining showed that MGP was positive in the interstitial calcification region and calcification was surrounded by fibroblast specific protein 1 positive cells.Further immunohistochemistry and western blots confirmed the decreased expression of MGP in the renal medulla of the rats from vitamin D modelling group.Cell immunofluorescence and Western blot showed that high calcium and high vitamin D treatment inhibited the expression of MGP in renal tubular epithelial cells.Alizarin red staining found that high calcium and high vitamin D could induce calcium depositions around rat kidney fibroblasts and the calcium depositions were significantly reduced even disappeared after supplementation of exogenous MGP in a concentration of 0.1ug/ml or 0.5ug/ml.Malondialdehyde,a major lipid peroxidation product of renal tissue,and the apoptosis of renal tubular cells were significantly increased in the modelling group.CCK8 cell proliferation assay,flow cytometry,scratch test and crystal adhesion assay demonstrated that high calcium and high vitamin D medium could significantly inhibit proliferation,induce apoptosis and inhibit wound healing ability of renal tubular epithelial cells,and they could also promote the adhesion of renal tubular epithelial cells to calcium oxalate monohydrate crystals but not to calcium phosphate crystals.ConclusionThe calcification of renal papilla is closely related to nephrolithiasis.Active vitamin D and high calcium microenvironment might induce the formation of interstitial calcification and subsequent renal stone by down-regulating the expression of MGP,increasing the renal oxidative stress,promoting the apoptosis of renal tubular epithelial cells and inhibiting proliferation and wound repair ability of renal tubular epithelial cells. |
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