Elementary Screening For Diuresis Efficiency And Pharmacological Study For Mitigating Proteinuria Of WLS In Adriamycin Rat

Literatures relating with experiment investigation had been summarized in the part of literature investigation in this article, including fare about experiment investigation of WLS recent several years, correlation study about engender mechanism of proteinuria, correlation study about connection between endothelin and kidney disease, the connection between glomerular mesangium cell (GMC) and proteinuria, development of pharmacological studying method on herbage containing serum.Experiment study had been developed on the basis of task group' s prophase work, The study included elementary filtration for diuresis efficiency and pharmacological study for mitigating proteinuria of WLS.Subgroups had been separated with method of polarity of dissolvent increasing by degrees from water extraction of WLS. These subgroups were petroleum ether extraction, chloroform extraction, ethyl acetate extraction, water-soluble of alcohol precipitate, supernatant of alcohol precipitate. Thin chromatogram layer had been used to test that respective subgroup had been separated effectively.Filter paper and metabolize cage had been used to test which subgroups could be diuretic on water loaded mice and rat. The result showed that none subgroups had diuretic effect compared with water extraction of WLS which had extinguish diuretic effect 2,3 hours after the medicine being taken. That means, the subgroups, which had same diuretic effect, as water extraction of WLS had not been found. To analysis the cause, the complication should be consider as follow:1 Two or more levels mutual cooperation between each subgroup maybeexist in Chinese traditional medicine compound, especially on the initial stage when they enter into the body(e. g. : in intestinal tract), there for , orthogonal and uniformity test design should be adopted to assemble respective subgroup to investigate if the new compounding had diuretic effect.2 The material basis for the diuretic efficacy of water extraction of WLS may be some minim or trace substance which could be lost in the cause of extracting and separating.3 Every fat-soluble subgroup presented increasing current on urine quantity in certain period (3~5h), but not statistics difference, so the sample quantity should be increased in the next experiment to investigate interaction of respective subgroup.That water extraction of WLS could mitigate proteinuria had been confirmed by prophase work of task group. This article emphasized on pharmacological study and aimed at searching WLS' s imaginable target system and target cell. In the animal experiment, the nephritic syndromed rats caused by twice intravenous of adriamycin (ADR) were adopted as proteinuria animal model. There were normal control group, model group and WLS treated group in all animal experiment.In the first instance, the amount of urinary protein was measured by method of Coomassie brilliant blue stain. Then the molecular weights of urinary protein were analyzed by sod. dodecylsulfonate-polyacrylamide gel (SDS-PAGE) electropheresis. The result showed that albumin gave priority to urinary protein of respective group rats, several bands of protein with molecular weight less than albumin also appeared. There were no obvious bands with molecular weight more than albumin in SDS-PAGE of normal group and treated group, but several these bands appeared in SDS-PAGE of model group, further more, the amount of protein with albumin and molecular weight more than albumin significantly increased. The result also showed that proteinuria of ADR rat is glomerular and WLS is likely to protect glomerular filtration barrier. The malondialdehyde(MDA) level of renal tissue wasdetected by biochemistry method which showed that WLS could not decrease ADR rat' s elevatory MDA level, which clewed that WLS' s effect of mitigating proteinuria was not by means of antagonizing oxidation of ADR.In success, the stereological method and computer image analysis system, combined light and electron microscope, were applied to estimate three-dimensional structure of the glomerular filtration barrier in the rat. The morphometric result showed that in ADR rat, the volume-weighted mean volume (Vv) and volume fraction of each amount of glomerular increased, length and volume density of epithelial foot process also increased, reversely, the surface area of glomerular basement membrane (GBM) covered with normal podocyte and the surface area of the filtration slit, slit diaphragm, the pores in the slit diaphragm, surface density and specific surface of epithelial foot process decreased. WLS could antagonize these pathological changes above all.Polyethylenemine(PEI) was used as a cationic probe to detect distribution of anionic sites on GBM. The result showed that the anionic sites arranged tightly on lamina rara externa and the PEI particles were robustus and regular. The anionic sites were also been seen on lamina rara interna but arranging rarely. There was a significant decrease of anionic sites in ADR rat and the PEI particles were very difficult to be seen. Meanwhile, the anionic sites were arranged one layer on most segments of GBM lamina rara externa in WLS treated rats, arrangement in lamina rara interna could still be seen in some segments in this group, the size of the PEI particles was similar to that in the normal rats. It indicated that the protection of WLS against nephrosis might mainly be associated with the repair of the charge selective barrier of GBM in ADR rat.Because the renal plasma flow (RPF) is also a important factor influencing the amount of urinary protein, the effect of WLS on renal hemodymatic and several correlated vascular active factors were detected. The glomerular filtration rate (GFR) and RPF were calculated by clearance rate of inulin(Cin) and para-aminohippuric acid (PHA) combined with volumeof urine in each minute. The weight of rat was used to emendate the numerical value. Renal vascular resistance (RVR) was calculated as the division of mean arterial blood pressure (MABP) by RPF, MABP was detected with a manometer. The result showed that there was no significant change of GFR in ADR rat and WLS had no effect on GFR, whereas, the RPF in ADR rat decreased and RVR increased, WLS could influence these changes reversely. Endothelin(ET) and angiotensin II (Ang II ) levels of renal tissue were measured by using radioimmunoassay method and nitric oxide(NO) level was measured by biochemistry method. The result showed that both the levels of ET and Angllin ADR rats were significantly higher than that in the normal group and WLS could decrease the levels of ET and AngIIof renal tissue in ADR rats. The level of NO in ADR rats was lower than that in the normal group. WLS took on an increasing trend in NO level of ADR rat but no statistics purport.Considering the complexity and un-definitude about the factors influencing the renal hemodymatic, experiment condition being used sufficiently, we focused on the study of effect of WLS on ET system in ADR rats.Try to orientate the effect of WLS decreasing the ET level of renal tissue in ADR rats, the protein expression of ET and the ET receptor A (ETaR) were measured semi-quantitatively with sensitive SABC immunohistochemical staining and image pattern analysis technique. The ET-positive and ETaR-positive staining were observed mostly in glomerular mesangium, glomerular capillary endothelial cell and renal tubule epithelial cell. The integral optical density of the ET-positive and ETaR-positive staining in renal tissue in ADR rats was considerable higher than that in normal rats, which could be decreased by WLS.After that, the imaginable molecule mechanism of effect of WLS on expression of ETaR in kidney tissue in ADR rats was investigated primitively with reverse transcription-polymerase chain reaction (RT-PCR). The semi-quantitative analysis result showed that WLS could attenuate the...