The Expression Of Klotho Gene In The Rat Model Of Remnant Kidney And The Interference Effect Of Shen'an Granular

Renal osteodystrophy(ROD) is the necessary complication of CKD (chronic kidney disease), which lowers the life quality and reduces survival time of patients. With the lower activity of kidney hydroxylase due to the destruction of structure and function of kidney, the production of 1,25 (0H)2 VitD3 is relatively or absolutely in sufficient, and synthesis and release of PTH is out of control, which is the core link of ROD. Klotho gene (KL) is related to senility, and expressed in brain choroids, Parathyroid gland and kidney, especially in renal cortical expression of distal tubule strongest. KL gene defect mice could exhibit senility phenotypic variation, which is similar with mankind syndrome of deficiency of kidney essence. It is approved that the expression of KL in kidney tissues decline dramatically in CKD, and the lower of KL could result in osteoporosis, which means KL may participate in producing ROD. In addition, bone morphogenic protein-1 (BMP-1), fibroblast growth factor-23 (FGF-23), and osteoprotegerin (OPG) are treated as bone hormones which are used to regulate calcium and phosphate metabolism in bone and gut and influence the generate process of ROD. Well, whether by virtue of protein regulation KL plays an important role in production of ROD? Once the hypothesis is approved, the pathogenesis of ROD could be further clarified and a new target would be provided for clinical treatment. In this paper, Rat 5/6 nephrectomy remnant kidney model is employed, regulation mechanisms of KL gene to BMP-1, FGF-23, and OPG are discussed, intervention of Shen'an Granular, such as compound-icariin,astragalin-1,rhubarb anthraquinone, is investigated. Anticipated results not only can deepen the understanding of the pathogenesis of ROD, but also can show the action mechanism of ROD prophylaxis and treatment with Chinese medicine to a certain extent.Part I Making the rat model of remnant kidneyObjective To investigate a new kind of method of setting up animal model of remnant kidney and renal osteodstrophy.Methods 48 Wistar rats were randomly divided into normal rats group(A group,n=8), pseudo- operation group(B group,n=8),model group(C group,n=32). After one week adaptive feeding, maked intraperitoneal injection of Sodium pentobarbital,the rats of C group were 2/3 left nephrectomized,which were feeding the high-phosphorus water next day, then right nephrectomized one week later. All of the rats were given normal diet, A group and B group drinking distilled water; Acquired the blood of rats eyeball a month after the 2/3 left nephrectomized,then examined HGB, Scr,BUN, P3+, Ca2+, ALP, iPTH, Collected the urinary of all of the 24hours before acquiring blood. Examined urine creatinine and calculated creatinine clearance rate.Results Making model one month later, Both of A and B groups were lively, bright and tidy coat, weight gain; C group were apathetic, quiet and do not like to move, messy fur, body weight did not change significantly.Compared with A and B group, HGB of C group was decreased obviously (p<0.05);While, BUN and Scr, otherwise (p<0.01); Contrasted to A group, Ccr of C group was decreased dramatically (p<0.01); ALP of C group was increased dramatically (p<0.05); Compared with A group, iPTH of C group was increased obviously (p<0.01);While, Ca2+ otherwise(p<0.05); P3+ of C group was increased contrasted to A group (p<0.01). These Laboratory parameters was no significant difference between A and Bgroup(P>0.05).Conclusions 5/6 nephrectomy combined with high phosphorus water one month can lead to disfunction of calcium and phosphorus metabolism of remnant kidney rats, then PTH increase, which demonstrates renal osteodstrophy arising.Partâ…¡The influences of calcium and phosphorus metabolism of rat model of remnant kidney and the interference effect of Shen'an GranularObjective To probe the interference effect of Shen'an Granular in the calcium and phosphorus metabolism of rat model of remnant kidney.Methods Wistar rats of model group above, were divided into 4 groups:model group (C group,n=8), calcitriol group (D group,n=8), moderate-dose Shen'an Granular group (E group,n=8);high-dose Shen'an Granular group(F group,n=8). A,B group same as part-1. D group feeding with calcitriol. E group feeding with moderate-dose Shen'an Granular.F group feeding with high-dose Shen'an Granular. A,B,C feeding with same dose normal saline. The total course of treatment was 8 weeks. Respectively examined HGB, Scr,BUN, P3+, Ca2+, ALP, iPTH of each group before and after medication, Collected the urinary of all of the 24hours before acquiring blood. Examined urine creatinine and calculated creatinine clearance rate.Results Survival of the rats:each of C and D group had one died during the medication, the remaining were survival. Before medication,such as Scr, P3+ and BUN of C, D, E, F group were higher than A,B group, While Ca2+ and Ccr otherwise (p<0.05);After 8 weeks of medication, there was no significant difference between before and after treatment about the Laboratory parameters, which were HGB,BUN,Scr,Ccr,P3+,Ca2+,ALP and iPTH in A,B group (p>0.05); In D group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.05); In E group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.01);In F group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.01); Compared with C group, Ca2+ of D group was increased obviously after medication (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with C group, Ca2+ of E group was increased obviously (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with C group, Ca2+ of Fgroup was increased obviously (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with D group, the P3+, Ca2+, ALP, iPTH of E and F group (p<0.01) had no significant difference (P>0.05).After 8 weeks mediction, Compared with D group, HGB,Ccr of E,F group were to be better dramatically(p<0.05); While, Scr otherwise (p<0.05); as well as BUN, Ca2+, ALP, iPTH(P> 0.05);and P3+ decreased significantly(p<0.05); These Laboratory parameters were no significant difference between E and F group(P>0.05).Conclusions Shen'an Granular can stabilize renal function, increase HGB,and also improve calcium and phosphorus metabolism of rat model of remnant kidney and inhibit hyper-parathyroidism. Partâ…¢The expression of Klotho gene in the rat model of remnant kidney and the interference effect of Shen'an GranularObjective To probe the influences of the expression of Klotho gene in the rat model of remnant kidney and the interference effect of Shen'an GranularMethods The drug and grouping method was same as part-2, After treatment, all rats were executed, then acquired renal and femoral bone, routine embedded sections of kidney stained line of optical microscopy, measured the density of the femoral head by dual energy X-ray. Examine the expression of Klotho mRNA of Wistar rats by RT-PCR,and Western blot was administered to measure the expression of kindey's Klotho and the thighbone'FGF-23,BMP-7,OPG, in the level of protein. Immunohistochemistry was used to detect the expression of thighbone' BMP-7 and OPG.Detected the expression of thighbone'FGF-23 by immunofluorescence.Results Compared with C group, the expression of bone density in E,F,D group was to be better dramatically(p<0.05), E group was slightly higher than D group,but had no significant difference (P>0.05),as well as the comparison between F group and D group(P>0.05); Contrasted to A group and B group, KlothomRNA of C group and D group were obviously decreased (P<0.01 or P<0.05), KlothomRNA of E group and F group were obviously inecreased, compared with C group(P<0.01),as well as D group(p<0.05), the expression of Klotho protein of renal cortex,which was highest in A group (P<0.05),and significantly higher compared with C group (P<0.01), between E group and F groups showed no difference (P> 0.05). By Western blot and immunofluorescence detection of FGF23, the expression of FGF-23 in the E and F groups and D group were lower than C group (P< 0.05) otherwise of BMP-7 (P<0.05), the E and F group'BMP-7 was above D group in the level of protein (P<0.05). the expression of OPG in C group, was lower than the A group (P<0.05),while,each durg group was above it (P<0.05). between D and E,F group had no significant difference (P>0.05).Conclusions The expression of Klotho gene in the rat model of renal osteodstrophy is suppressed, the low Klotho gene is closely related to FGF-23,BMP-7,OPG,Shen'an granular can produce favorable effects on the high-expression of Klotho gene and osteoblast factor, regulating calcium and phosphorus metabolism, improving renal osteodystrophy.