Razvoj animalnog modela nefrotoksične tubulointersticijalne lezije

Abstract

U eksperimantalnom postupku disertacije miševi NMRI soja su tretirani infuzom biljke Aristolochia clematitis. Sasušeni listovi, grane i plodovi biljke potopljeni su u ključalu vodu i ostavljeni 3-5 sati da stoje, a potom su profiltrirani kroz filter papir. Pravljen je rastvor biljke/vode od 10g/ 1000ml (1%), 20g/ 1000ml (2%) i 40g/ 1000ml (4%). Različite koncentracije infuza su date miševima da piju u neograničenoj količini u periodu od 7 nedelja. Tako su formirane tri ispitne grupe, prva koja je primala 1% infuz, druga 2% infuz i treća 4% infuz i kontrolna grupa koja je dobijala samo vodu da pije. U svakoj grupi je bilo 20 životinja. Tako je razvijen animalni model hronične toksičnosti. Na kraju eksperimenta je urađena patohistološka analiza bubrega, makroskopski pregled organa i merenje diureze tokom trajanja eksperimenta. Urađena je kompletna analiza urina koja podrazumeva utvrđivanje: boje, izgleda, pH, specifične težine, proteina i sedimenta urina. Analize urina ponavljane su na svakih 7 dana u toku 7 nedelja istraživanja. Na kraju eksperimenta urađena je analiza biohemijskih parametara (glukoza, urea, kreatinin, mokraćna kiselina, ukupni bilirubin, direktni bilirubin, ukupni tj. totalni proteini, natrijum i kalijum) i analiza kompletne krvne slike. Utvrđeno je da je Aristolochia clematitis izrazito nefrotoksična biljka. Utvrđene su patohistološke promene tubula i intersitcijuma NMRI miša, koje su bile najveće u ispitnoj grupi koja je primala najaču dozu. Ustanovljene patohistološke promene su slične opisanim patohistološkim promenama tubulointersticijuma bolesnika obolelih od Balkanske endemske nefropatije. Nije ustanovljeno postojanja karcinoma gornjeg urotrakta. Makroskopskim pregledom prilikom obdukcije eksperimentalnih životinja nisu ustanovljene značajnije promene bubrega. Došlo je prvo do izrazitog porasta diureze u prvoj, odnosno drugoj nedelji praćenja, kod druge i treće eksperimentalne grupe, da bi nakon 7 nedelja istaživanja diureza u svim ispitnim grupama bila manja od kontrolne grupe. Postoji porast ureje na kraju istraživanja, koji je dvostruko veći u trećoj eksperimentalnoj grupi u odnosu na kontrolnu. Postoji izrazit pad mokraćne kiseline na kraju istraživanja kod eksperimentalne grupe 3. Postoji izrazit pad granulocita u leukocitarnoj formuli u svim ispitnim grupama, a najveći je u trećoj ispitnoj grupi. Kako je došlo do pada relativnih vrednosti granulocita, tako je došlo do porasta relativnih vrednosti limfocita u prvoj i drugoj ispitnoj grupi. U trećoj ispitnoj grupi je pad granulocita praćen izrazito velikim povećanjem relativnog broja bazofilnih granulocita. Postoji značajan pad specifične težine urina na kraju istraživanja u drugoj i trećoj eksperimentalnoj grupi. Proteinurija je bila čest nalaz svim eksperimentalnim grupama, dok je bila odsutna ili samo u tragu u kontrolnoj grupi. Na kraju eksperimenta je utvrđen znatni porast broja kristala fosfata u eksperimentalnim grupama. Cilindri su se pojavljivali samo u nalazu urina u trećoj ispitnoj grupi. Najveći broj promena urina je utvrđen u trećoj eksperimentlanoj grupi.

In the experimental procedure of dissertation, NMRI strain mice were treated with infusion of plants Aristolochia clematitis. Dried leaves, branches and fruit plants are submerged in boiling water and left to stand for 3-5 hours, and then filtered through filter paper. It was made a solution of the plant / water of 10g / 1000ml (1%), 20g / 1000ml (2%) and 40g / 1000ml (4%). Different concentrations of infusions were given to mice to drink an unlimited amount for a period of 7 weeks. So we formed the three test groups, the first who received 1% infusion, the second received 2% infusion and third received 4% infusion and a control group that received only water to drink. In each group there were 20 animals. Thus, developed an animal model of chronic toxicity. At the end of the experiment was performed histopathological analysis of kidneys, macroscopic examination of organs and measuring urine output during the experiment. We performed a complete analysis of urine, which is the determination of: color, appearance, pH, specific gravity, protein and urine sediment. Urinalysis were repeated every 7 days during the 7 weeks of the study. At the end of the experiment were analyzed for biochemical parameters (glucose, urea, creatinine, uric acid, total bilirubin, direct bilirubin, total proteins, sodium and potassium) and analysis of the complete blood count. It has been found that Aristolochia clematitis is extremely nephrotoxic plant. Identified histopathological changes of tubules and interstitium of NMRI mouse, which were the biggest in the test group receiving biggest dose. Established histopathological changes are similar to those described by pathological changes of tubulointerstitial injury of patients with Balkan endemic nephropathy. Not established the existence of cancer of the upper urinary tract. Macroscopic examination at autopsy of experimental animals, did not determine significant changes in the kidneys. There is first an enormous increase in diuresis in the first and second week of follow-up, in the second and third experimental groups retrospectively, that after 7 weeks of research, diuresis in all test groups was lower than the control group. There is an increase of urea at the end of the research, which is twice higher in the third experimental group compared to the control. There is a marked decrease in uric acid at the end of the research in the experimental group 3. There is a marked decrease in granulocytes in the leukocyte formula in all test groups, and the highest in the third test group. As the decline in the relative values of granulocytes, so there has been a rise in the relative values of lymphocite in the first and second test group. In the third test group, granulocyte drop was accompanied by a extremely large increase in the relative number of basophils. There is a significant drop in specific gravity of urine at the end of the research in the second and third experimental group. Proteinuria is a common finding to all experimental groups, while it was absent or only in traces in the control group. At the end of the experiment was determined to increase significantly the number of phosphate crystals in the experimental groups. The cylinders have appeared only in the urine in the third test group. The greatest number of changes in the urine is determined in the third experimental group.