The Expression of Tamm-Horsfall Protein in the Kidneys of Rats Models of Nephrolithiasis Treated by Potassium Citrate
|Keywords||Tamm-Horsfall protein Potassium citrate Kidney stones Rat model Experimental study|
Purpose and background: kidney stones Urology common disease, and a high incidence of, and with the improvement of living standards, the upward trend in its incidence. Mechanism and prevention of kidney stones has been the urologists concerned about the problem. In recent years, the international study found that Tamm-Horsfall protein (Tamm-Horsfall protein, THP) may be an organic macromolecules inhibiting the formation of urinary stones. But THP inhibit stone formation mechanism and the role played by the different studies give different results, there is considerable controversy. Although foreign researchers have reported the results of the the kidney stones rat kidney tissue THP expression, but also treatment factors reported intervention under the the kidney stones rat kidney tissue THP expression. Citrate is one of the inhibitors to study the earliest and most fully stones, but the exact mechanism of anti-stone, and other macromolecules relationship between the suppression factor also needs further study. Therefore, we examined the potassium citrate intervention under the kidney stones rat kidney tissue THP expression, and to understand the impact of the of potassium citrate intervention on THP expression of renal tissue, potassium citrate inhibits further explore the mechanism of stone formation, and citrate relationship between potassium and THP, which not only provide the experimental basis for parsing the pathogenesis of kidney stones, to seek kidney stone prevention and treatment pathways also have certain guiding significance for clinical. Methods: 1, animals and tissue preparation: select 20 male Sprague-Dawley rats (Run Run Shaw Hospital, Zhejiang University animal laboratory), weighs about 350g mixed solution containing 0.5% ethylene glycol and 0.5% ammonium chloride lithogenic agent. First rats reared under normal conditions one week to fully adapt to the environment, were randomly divided into three groups: control group 4: Hello ordinary purification of drinking water; stones group of eight, instead of drinking water so that the stone agent; citrate 8 potassium intervention group, in addition to that the stone agent instead of drinking water, while 25% potassium citrate solution 1ml/100g weight orally once a day. Experimental condition 1ml/100g body weight with purified water in order to maintain a consistent, stones and control groups gavage once daily for the three groups of rats. The rats were killed after two weeks, anatomical taken kidney, renal surface morphology observed under the naked eye, and then cut open along the long axis of the renal hilum, part of the kidney tissue with 10% formalin-fixed, part of the tissue under a dissecting microscope observation of the renal stone formation profile. 2 stone formation observed: first visual observation of the the kidney surface morphology, and then cut the kidney is placed under a dissecting microscope to observe the renal profile crystal stone formation. Kidney sections with or without obvious crystalline precipitate free stones as a standard to determine whether the stones. Into four categories according to the extent of the amount of crystalline stone formation and kidney damage, and statistical analysis. 3, renal tissue THP expression intensity measurement: the relative expression using immunohistochemical determination of kidney tissue THP amount of the SANTA CRUZ rabbit anti-human Tamm-Horsfall protein 291-425 sequence of amino acids polyclonal antibody and goat anti-rabbit ABC staining system reagents box. The first formalin-fixed kidney tissue dehydration Baptist wax-embedded 3um thick slices, then hydration after dewaxing, 1% H closed endogenous 2 O 2 -PBS solution peroxidase, 1.5% serum in blocking one hour, THP-a antibodies (concentration: 2ug/ml) or PBS negative control at room temperature and incubated for 30 minutes, incubated for 30 minutes in biotinylated goat anti-rabbit secondary antibody, avidin, and The biotinylated horseradish peroxidase incubated for 30 minutes, DAB color 5 minutes, then hematoxylin, dehydrated ethanol xylene neutral resin Fengpian. Last observed under an optical microscope, \4: data processing professional statistical software SPSS13.0 for windows using Kruskal-Walls non-parametric tests on three groups of rats kidney into stone the extent and THP expression intensity for statistical analysis using the Mann-Whitney nonparametric test three groups the degree of rat kidney into stone and THP expression intensity pairwise comparison. Results: 1, the formation of kidney stones: visual observation of gross morphology of the surface of the three groups of kidney, the kidney shape of the control rats no obvious abnormalities, soft texture, no calcified plaque forming; stones rat kidney markedly swollen, hard texture, kidney visible surface of a large number of light yellow calcified plaque, the partially calcified plaques fusion; the potassium citrate intervention group rat kidney slightly swollen, pale yellow calcified plaque surface is also visible, but fusion is not obvious. Observed under a dissecting microscope, the the rat renal profile of the control group had no visible crystalline stones precipitate; stones, renal profile embedded a large number of crystallization, and visible free stone formation; citrate intervention group rat kidney profile shows a small amount of crystalline precipitation, no free stone formation. Classification of three groups of rats into the stone of using non-parametric Kruskal-Walls test three groups of rats kidney stone the extent of statistical analysis into stone the extent there are significant differences (P <0.01) between the three groups, using Mann-Whitney nonparametric test for three groups of rats kidney stone degree of pairwise comparison found between the control group and the gallstone group, the control group with potassium citrate between the intervention group and gallstone group citrate bell intervention group the between into stone the degree were significantly different. Kidney tissue THP expression intensity: three groups of rats kidney tissue immunohistochemical staining of THP THP mainly located in the tubular the Heinz loop and the distal convoluted tubule, but at the same dilution as the primary antibody and the same experimental conditions, THP expression intensity difference in the three groups of rat kidney tissue. Control group THP staining deep gallstone group staining is very shallow, potassium citrate intervention group in between. Classification statistics of the three groups in accordance with the above criteria, the use of non-parametric tests Kruskal-Walls THP expression strength of the three groups of renal tissue, found among the three groups THP expression intensity there are significant differences (P <0.01) using the Mann- Whitney nonparametric test three groups of renal tissue THP expression strength of pairwise comparisons between the control group and the gallstone group, the control group with citrate bell intervention in the intervention group of the group and between the the gallstone group with potassium citrate between THP renal tissue expression intensity were significantly different. Kidney into stone the degree of renal tissue THP expression of the strength of the relationship: the THP expression into the stone, each rat kidney degree of renal tissue corresponding to the intensity, found in stone formation strength \expression was significantly weakened, mostly \Kidney stones form the more obvious, THP expression in renal tissue, the weaker the contrary, the less kidney stone formation, THP expression in renal tissue is stronger. Conclusion: We reported intervention treatment factors THP expression changes in kidney stones renal tissue. We found that the intervention of potassium citrate, kidney stones rats. The kidney tissue THP expression has been restored to some extent, potassium citrate intervention led to the specific mechanism of this change, still need further study.