Research On The Differential Potency And Therapeutical Effect Of Bone Marrow Stem Cell Transplantation To Renal Tubule Epithelial Cells Of The Chronic Aristolochic Acid Nephropathy Rats

Aristolochic acid nephropathy (AAN) is a renal disease reported after the introduction of Chinese herbs contained aristolochic acid. The pathology changes major is renal tubule atrophy,renal tubular basement membrane became thickening, flexion; Renal interstitium is lamellar or diffuse fibrosis; Arteriole wall thickening, lumens stenosis; Glomcrulus is ischemic crenation, sclerosis. The majority is chronicAAN (CAAN). It's not easy to be detected in the beginning, renal disfunction always occurrt after kept taking medicine for several years. And chronic renal disease is a inreversible course, it'll lead to renal failure(RF) in the end.Pathogenesis of CAAN is not clear yet currently, so it still hasn't a effective treatment way. It's one of important researches that finding pathogenesis of AAN, searching new and effective treatment and precaution method, controlling and delaying RF.Stem cell transplantation has became focus in medical researches. Investigation has proved that bone marrow derived stem cell can differentiate to other functional tissue ,including differentiate to renal cell, except differentiating to blood system. This provide a new way for the terapy of difficult renal disease such as CAAN. Our investigation is to explore the differential potentiality of stem cell into renal cell and the therapeutical effect of stem cell to CAAN through make the model of CAAN and transplant bone marrow stem cell to CAAN rat, and then to deduce the pathogenesy of CAAN.Our investigation included three parts:â‘ The differential potentiality study of bone marrow stem cells into renal tubular epithelial cell of the chronic aristolochic acid nephropathy rats.â‘¡The therapeutic action of stem cell transplantation on the chronic aristolochic acid nephropathy rats.â‘¢The effect of stem cell transplantation to the express of theα-SMA,CK18,TGF-β₁,HGF in the kidney of chronic aristolochic acid nephropathy rats.Materials and MethodsPart 1: All animal procedures were conducted after approval of protocol by the Chinese Medical University Animal Care Committee. Female Wistar rats (190 to 210g, 6 to 8 weeks ) were kept under standard conditions with unrestricted access to food and water in a 12: 12-h light-dark cycle. The Transplantation Group(n= 16) were established by administering with Caulis Aristolochiae Manshuriensis(CAM) decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then transplant bone marrow stem cells (1×10⁸ or 0.35ml per rat) of the homologous series male rats through the caudal vein after irradiated with sublethal dose ofâ…©ray, then sacrifice rats at 30 and 60 days after transplantation. The Nontransplantation Group(n=21) were established by administering with Caulis Aristolochiae Manshuriensis(CAM) decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then intravenous injection of Sodium Chloride (0.35ml per rat)through the caudal vein, then sacrifice rat at 30 and 60 days after injection. Otherwise, we have selected 5 rats in random in this group to accept irradiation before intravenous injection of Sodium Chloride, together with the Transplantation Group rats, and then sacrifice rats at 30 days after injection. Another we setted up Nomal Control Group (n=5), all were male Wistar rats, freedom to take food and water, then sacrifice rats at the end of our experiment. At death, the kidneys were rapidly removed and transferred to a metal plate cooled on ice for sectioning, then the kidney was sectioned coronally and immersed in 4% neutral-buffered formalin for 24h, dehydrated in grated alcohols, and embedded in paraffin. In this part, we major to observe the differentiation of stem cells to renal cell through Y chromosome fluorescence in situ hybridization combined with immunofluorescence by serial sections. Otherwise, HE staining of the Nontransplantation Group rats' kidney section was made to observe the influence ofâ…©irradiation to kidney.Part 2: The Transplantation Group(n=16) were established by administering with CAM decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then transplant bone marrow stem cells (1×10⁸ or 0.35ml per rat) of the homologous series male rat through the caudal vein after irradiated with sublethal dose ofâ…©ray, then sacrifice rat at 30 and 60 days after transplantation. The Nontransplantation Group(n=16) were established by administering with CAM decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then intravenous injection of Sodium Chloride (0.35ml per rat)through the caudal vein, then sacrifice rats at 30 and 60 days after injection. The Nomal Control Group (n=8) were administered with drinking water by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then intravenous injection of Sodium Chloride (0.35ml per rat)through the caudal vein, then sacrifice rats at 60 days after intravenous injection. Urine was collected for 24h in metabolic cages before death. At death, sample of serum was collected. Blood urea nitrogen (BUN) and serum creatinine (Scr) levels were determined with a Keysys analysator. The hemoglobin (Hb) were determined with photoelectric colorimetry. Urineβ₂-MG were determined with radioimmunity. Urine NAG were determined with enzyme-substrate. 24h urinary protein excretion rates were measured using the sulfosalicylic acid method.Part 3: The Transplantation Group(n=16) were established by administering with CAM decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then transplant bone marrow stem cells (1×10⁸ or 0.35ml per rat) of the homologous series male rat through the caudal vein after irradiated with sublethal dose ofâ…©ray, then sacrifice rat at 30 and 60 days after transplantation. The Nontransplantation Group(n=16) were established by administering with CAM decoction by gavage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then intravenous injection of Sodium Chloride (0.35ml per rat)through the caudal vein, then sacrifice rat at 30 and 60 days after injection. The Nomal Control Group (n=8) were administered with drinking water by garage at a dose of 10ml kg^-1 d^-1(twice a day, continuous 5 days per week) for 12 weeks, then intravenous injection of Sodium Chloride (0.35ml per rat)through the caudal vein, then sacrifice rat at 60 days after intravenous injection. At death, the kidneys were rapidly removed and transferred to a metal plate cooled on ice for sectioning. One of the kidneys was divided into two parts and snap frozen in liquid nitrogen and stored at -70℃. The other kidney was sectioned coronally and immersed in 4% neutral-buffered formalin for 24h, dehydrated in grated alcohols, and embedded in paraffin. Pathological examination were made to the renal specimens. The electron microscope was used to evaluate the ultramicrostructure injury of renal tubular epithelial cell.α-SMA,CK18,TGF-β₁,HGF were detected in the CAAN rat model by immunohistochemistry analysis, western-lotting and RT-PCR.Statistical Comparisons: We used SPSS12.0 software to conduct statistical analyses. Values are reported as mean±SD. All comparisons were one-way ANOVA. Significance was defined as P＜0.05.ResultsPart 1: We could detect the Y chromatin-positive hybrid cells(Y⁺, flavo-green fluorescence brightened dot) cell in the renal section of the Nomal Control Group rat and couldn't detect the Y⁺ cell in the Nontransplantation Group through Y chromosome fluorescence in situ hybridization. Then we detect the Transplantation Group, also found the Y⁺ cell, and at the same time part of the Y⁺ cell expressed renal tubular epithelial cell phenotype, CK18 and RCAI (CK18⁺, RCAâ… ⁺), with immunofluorescence by serial sections. HE staining of the Nontransplantation Group rats kidney didn't found obviously differences betweenâ…©irradiation and non-irradiation rats.Part 2: A increase in BUN and Scr levels was observed for the Nontransplantation Group rats from the 30th day, compared with the Normal Control Group, P＜0.01; BUN and Scr levels of the Transplantation Group was significant lower than the Nontransplantation Group rats, P＜0.01, but still super to the Nomal Control Group rat P＜0.01. The Nontransplantation Group rats were anaemia at the 30th day, and anaemia became worse at the 60th day. The level of Hb of the Nontransplantation Group rats compared with the Normal Control Group, P＜0.01; The anaemia obviously amelioration in the Transplantation Group rats, compared with the Normal Control Group and the Nontransplantation Group, P＜0.01. Pathologic proteinuria (defined as＞25mg/24h) was present in the Nontransplantation Group rats from the 30th day and then exhibited a continous increase up to day 60, compared with the Normal Control Group, P＜0.01; The pathologic proteinuria was manifestly degraded in the Transplantation Group rats, compared with the Nontransplantation Group P＜0.01, Otherwise, compared with the Normal Control Group, P＜0.01 at the 30th day and P＞0.05 at the 60th day. The level of urineβ₂-MG was increase in the Nontransplantation Group rats from the 30th day, compared with the Normal Control Group, P＜0.05; The level of urineβ₂-MG was decrease in the Transplantation Group rats, compared with the Nontransplantation Group and the Normal Control Group, all of P values were less than 0.05. A significantly increase in urine NAG levels was observed for the Nontransplantation Group rats from the 30th day, but decreased to some extent at the 60th day, compared with the Normal Control Group, P values were less than 0.05; compared between the 30th day and the 60th day in the Nontransplantation Group p＞0.05; Urine NAG levels of the Transplantation Group was markedly lower than the Nontransplantation Group rats, P＜0.05, but still super to the Nomal Control Group rat P＜0.05.Part 3: In the Nomal Control Group, no significant abnormality was observed in the renal tissue samples, but severe injury was found in the Nontransplantation Group and renal interstitial fibrosis was obvious. On the contrary, in the Transplantation Group rats, there was moderate interstitial edema, no significant interstitial fibrosis. Interstitial fibrosis areas of the Nontransplantation Group was increased, compared with the Normal Control Group and the Transplantation Group, (P value was less than 0.01). Compared to the Nontransplantation group, ultramicrostructure of renal tubular epithelial cell of the Transplantation Group rats was obviously improved through electron microscope. At the 60th day in the Transplantation Group, the cell structure was nearly nomal, there was a little edema with mitochondria and slightly irregularity with brush border microvillus; But in the Nontransplantation Group, the mitochondria was markedly edema and partially dissolved and the brush border microvillus was almost disappear, the cell membrane also partially dissolved. In the nontransplantation Group, the protein and mRNA expression ofα-SMA and TGF-β₁ was increased, but that of CK18 decreased gradually, and the expression of HGF increased firstly and decreased later. In the transplantation Group, the protein and mRNA expression ofα-SMA and TGF-β₁ was decreased, the expression of CK18 was increased gradually, and the expression of HGF was further increased, significantly superior to the nontransplantation Group, P values were less than 0.01. At the 60th day, theα-SMA-IOD of the Transplantation Group was 11.51±3.41 compared to that of the Nontransplantation group rats 38.04±6.5, P＜0.01; The CK18-IOD was 24.67±3.86 compared to that of the Nontransplantation group rats7.62±1.96, P＜0.01; The TGF-β1-IOD was 13.44±2.70 compared to that of the Nontransplantation group rats 77.53±7.66 P＜0.01; The HGF-IOD was 42.80±3.84 compared to that of the Nontransplantation group rats 11.94±3.14, P values were also less than 0.01.ConclusionThese data suggested that:(1) There have Y chromatin-positive hybrid cells(flavo-green fluorescence brightened dot) in the tissue section of the Transplantation Group and the Normal Control Group rats' kidney through Y-FISH detection, but it didn't be found in the section of the Nontransplantation group rats. Meanwhile there were partial Y⁺ cells which also expressed the phenotype characteristic of renal tubular epithelial cell, CK18and RCA. Demonstrate that the bone marrow derived stem cells have the ability to differentiate into renal tubules cells of the chronic aristolochic acid nephropathy rats.(2) Bone marrow derived stem cell transplantation can significantly improve the renal function of CAAN rats, increase the level of Hb, lessen the level of 24h Urine protein and the loss ofβ₂-MG, lower the activity of Urine NAG. It expanded a new therapeutic way to CAAN, even to the other renal failure.(3) Bone marrow derived stem cell transplantation can makedly remission renal pathological lesion of CAAN rats, lighten interstitial fibrosis; Decrease the protein and mRNA expression ofα-SMA and TGF-β₁, and increase the expression of CK18 and HGF. There by stem cell transplantation can lessen renal damage and interstitial fibrosis from the two aspects of cell and cytokine. It will provide a beneficial renfernece to the therapy of CAAN, even to that of other renal tubule interstitial fibrosis.(4) At the same time, we can deduce that there probably has a injury or insufficient in the stem cells of CAAN rats, through the therapeutic action to CAAN with bone marrow derived stem cell transplantation, it will also provide a new research direction and theory renfernece to the pathogenetic investigate of CAAN.