Targeting Of Bone Marrow-Derived Mesenchymal Stem Cells To The Unilateral Obstructed Kidney In Tubulointerstitial Fibrosis

OBJECTIVE: Tubulo-interstitial fibrosis (TIF) is a major characteristic of most chronic kidney diseases leading to end-stage renal failure, regardless of the underlying pathogenesis. Except for renal replacement, TIF is considered presently incurable. The therapy aimed at complete blockade of progressive fibrosis has proven to be enormously difficult. It is now accepted that bone marrow-derived mesenchymal stem cells (BM-MSCs) have multiple differentiation potential, such as osteoblastic, chondrocytic, myogenic and adipocytic differentiation. It has been shown that systemic delivery of mesenchymal stem cells could protect mice with acute renal failure from renal function impairment and severe tubular injury, which prompted us to hypothesize that bone marrow derived mesenchymal stem cells would transdifferentiate into normal renal cells in early TIF and ameliorate chronic renal fibrosis. Here we tested whether MSCs intravenously injected into the caudal vein can engraft in the unilateral obstructed kidney in tubulo-interstitial fibrosis. METHODS: By density gradient centrifuge and adherent selecting, MSCswere isolated from SD rat bone marrow, and purified. Cellular growth curve was measured by MTT method. The phenotypes were analyzed by immunohistochemistry. MSCs were induced to differentiate into osteoblasts in DMEM containing dexamethasone, beta-sodium glycerophosphate and ascorbic acid. MSCs were genetically modified with an adenovirus vector delivered green fluorescent protein (GFP) gene. Rats were subjected to unilateral ureteral obstruction followed by intravenously transplanting GFP (+)-MSC. The rats were sacrificed at two or four weeks after transplantation randomly. Sections were stained with hematoxylin and eosin and Masson's trichrome. DAPI fluorescent staining was employed for the frozen sections. RESULTS: Isolated MSCs were homogeneous, long-fusiform-shaped or multi-angular-shaped, arranged into a fencelike structure. The growth of MSCs could be divided into three phases. They were the initial quiescent phase (day 1 to day 3), the logarithmic replication phase (day 4 to day 6) and the post-proliferation quiescent phase (after day 7). Populating doubling time of MSCs prolonged since passage 5. MSCs were positive for glycogen periodic acid Schiff reaction, CD44, CD90, and were negative for CD31, CD45. 5%-10% MSCs were positive for Alkaline Phosphatase. Cobblestone like structure and calcified nodules were observed in culture after a 2-week induction. The adenoviral transfection efficiency was >85%. GFP (+) cells were found in the context of the tubular epithelial lining in the obstructed kidney of the rats at 2 weeks after transplantation, but not in the unobstructed contralateral kidney, or in the kidneys of the rats at 4 weeks after transplantation. CONCLUSIONS: We can isolate and purify bone marrow MSCs by meansof density gradient centrifugation and adherent selecting. Identify MSCs by morphology, cell surface markers and osteoblastic differentiation. GFP is an ideal marker for MSC transplantation. Rat BM-MSCs are capable of homing to unilateral obstructed kidney following intravenously infusion. These results indicate that BM-MSCs could be the candidates for the repair of renal tubules in Tubulo-interstitial fibrosis.