Transplantation Of Adipose-derived Stem Cells Via Renal Artery Protects Against Acute Ischemic Kidney Injury

Part oneThe isolation, culture and identification of SD rat ASCs and establishment of the model of iAKIObjective:To isolate, culture and identify SD rat ASCs in vitro and establish an model of rat iAKI.Methods:ASCs were isolated from groin subcutaneous adipose tissue by type I collagenase digestion method, whose morphology and growth process were observed under inverted microscope. The third passage of ASCs were used in research. Lipogenesis and osteogenesis capability was induced in standard condition in vitro, and surface markers(CD29, CD90, CD34, CD45)with flow cytometry. Bilateral renal pedicles were blocked for 45 minutes to established the model of iAKI by surgical clipping with noninvasive arterial clip. Serum creatinine was measured in 24 hours after reperfusion in both normal rats and iAKI rats.Results:1. The morphology of primary cells is short fusiform or small polygon, stick to the bottom of dish. After three times continuous passage, ASCs were almost in the same size and shape, and grew in spiral, radial shape.2. ASCs at its third passage was postive for CD29, CD90, and negative for CD34, CD45. which was in accordance with ASCs immune phenotype. Under the condition of standard lipogenesis induction, oil red O staining proved adipose differentiation by observation of fat droplet; under the condition of standard osteogenesis induction, Von Kossa staining confirmed that its osteoblast differentiation by mineralized nodules.3. ASCs in passage 9 can still maintain an average value-added rate of 1.33 x 104 per day.4. Serum creatinine of normal rats was 41.67±5.92μmol/L, and 261.99±5.09μmol/L in iAKI rats.Conclusion:1. High purity of rat ASCs can be obtained by type I collagenase digestion, which was positive for CD29 and CD90 and negative for CD34 and CD45, in accordance with accepted ASCs immune phenotype.2. ASCs could be induced to differentiate into osteoblasts and lipoblasts under standard conditions, this confirmed its multiple differentiation potential.3. ASCs extracted from groin subcutaneous adipose tissue has strong self-renewal ability, they could keep rapid multiplication rate during the tenth passage.4. Serum creatinine of iAKI rats was markedly higher than normal rats(P<0.05), which indicates iAKI model was successfully established.Part twoTransplantation of adipose-derived stem cells via left renal artery protects against acute ischemic kidney injuryObjective: To explore the therapeutic effect of ASCs transplanted via left renal artery on rat iAKI.Methods:iAKI model was set in male SD rats by clipping bilateral renal pedicles for 45 min. The iAKI rats were randomized into two groups (n=30):control group (renal intra-arterial administration of 500μ1 PBS) and ASCs transplantation group (renal intra-arterial administration of 5×105 ASCs). Rats were sacrificed at 12,24,48,72 hours and 1 week after reperfusion to measure renal function by serum creatinine (Scr). Renal pathology, cell apoptosis, inflammation and cell proliferation were analyzed by optical microscope.Results:1. Compared with control group, Scr in ASCs transplantation group were significantly lower at all time points (P<0.05).2. Score of left renal tubular interstitial damage degree in ASCs transplantation group were markedly lower (P< 0.05) at 12 hours,24 hours,48 hours.3. Compared with control group, TUNEL and macrophage infiltration score in ASCs transplantation group were significantly lower (P<0.05) at all time points; proliferating antigen increased at 48 hours and decrease at 72 hours and 1 week (P<0.05). Meanwhile, comparing with right kidneys in ASCs transplantation group, score of left renal tubular interstitial damage degree was markedly lower (2.40±0.50, P<0.05) at 24 hours; the number of TUNEL positive cells in 1 week was observably lower (P<0.05); macrophage infiltration score were dramatically lower at 12 hours,48 hours,72 hours and 1 week; Proliferating antigen increased at 48 hours and decrease at 72 hours and 1 week (P<0.05).Conclusion:ASCs transplantation via renal artery could significantly improve renal function and ameliorate pathological damage, relieve apoptosis and macrophage infiltration, and enhance the repair process after AKI. It may due to renal intra-arterial transplantation increasing the amounts of ASCs migrated into the kidney in AKI and reducing ASCs distribution in other organs.Part threeDistribution of ASCs after transplantation via left renal artery of rats in vivoObjective:To explore distribution of ASCs in different organs after transplantation via left renal artery of rats in vivo.Methods: ASCs was labeled by CM-Dil, frozen biopsy of ASCs which were transplanted via left renal artery distribution was measured by fluorescent microscopy. iAKI model was set according to the method of second part. Then 5×105 CM-Dil labeled ASCs was transplanted via left renal artery immediately. Frozen section of kidneys, lung, liver, spleen and heart of rats were made at 12,24,48,72 hours and 1 week after reperfusion. DAPI staining was conducted to lable cell nucleus. Distribution of different organs was observed by fluorescent microscopy, and count of different time points within a single glomerular ASCs number on average was made also.Results: 1. Distribution of ASCs in kidneys: Dil positive cells are mainly distributed in glomerulus of the left kidney, and a lot at 12 hours and 24 hours after transplantation, significantly reduced at 48 hours,72 hours and 1 week. No Dil positive cell was observed at any time points in right kidneys.2. Distribution of ASCs in lung, liver, spleen and heart:there was only a few Dil positive cells at 48 hours and 72 hours in lung and liver at 48 hours and none Dil positive cells at other time points. No Dil positive cells in spleen and heart were observed at any time points.2. Count of Dil positive cells in 12 hours,24 hours,48 hours,72 hours and 1 week within a single glomerular are:6.33±1.75,5.67±1.15,4.5±1.05,3.43±1.06,2.5±1.29.Conclusion:1. CM-Dil label to trace distribution of ASCs in vivo is feasible.2. Renal intra-arterial transplantation of ASCs may increase the amounts of ASCs migrated into the kidney in AKI and reduce ASCs distribution in other organs. So it can effectively improve the number of exogenous stem cells those are going into iAKI kidneys, and play the role of treatment.