Biochemical Evaluation to Mesenchymal Stem Cells Therapy of Renal Tubulointerstitial Injury

Background Acute renal failure (ARF) remains a frequent clinical complication, Ischemia is the major cause of acute kidney injury (AKI), associated with high mortality and morbidity. Mesenchymal stem cells(MSCs) have multilineage differentiation potential and can be a potent therapeutic option for the cure of AKI. The aim of this study was, therefore, to evaluate the therapeutic effectiveness of bone marrow–derived mesenchymal stem cellsin a rat model of ischemia/reperfusion (I/R) ARF. Materials and Methods MSCs were cultured and rats were used as a common I/R model to induce ARF by clamping both renal pedicles for 45 minutes then stem cells were transplanted. Ki-67 was used as marker for regeneration of renal tubulointerstitial injury. Results This study reports that Rat MSCs decreased cell apoptosis and increased proliferation and reduced fibrosis in common I/R model in rats. Conclusion MSCs may be useful in regeneration of renal tubulointerstitial injury. INTRODUCTION The kidney is comprised of heterogeneous cell populations that function together to perform a number of tightly controlled and complex processes. Renal ischemia\ reperfusion (I\R) injury is a common cause of acute renal failure and contributed to consider able morbidity associated with surgery and anesthesia (Chander et al., 2006). One cause of acute kidney injury (AKI) is ischemia which can occur for a number of reasons, for example with the use of the vasoconstrictive drugs or radiocontrast agents; hypotension linked to loss of blood after surgery (Asif and Bruce, 2011). Stem cells play fundamental roles in the self-renewal of adult tissues throughout life. Bone marrow–derived hematopoietic stem cells have been discovered to transdifferentiateinto cells of different germ layers(Krause et al., 2001). Physiologically, mesenchymal stem cellsgive rise to osteocytes, chondrocytes, and adipocytes, but were recently found to differentiate into endothelial, myocardial (Nagaya et al., 2004), liver (Shu et al., 2004), renal (Morigi et al., 2004), and pulmonary epithelial cells (Ortiz et al., 2003). Based on this background, the objective of the present study was to test the therapeutic potential of mesenchymal stem cells, administered to rats following induction of ARF by ischemia/reperfusion (I/R) and showed the regeneration of renal tubulointerstitial injury that marked by the pathological change and uses of marker Ki-67. Materials and Methods: Animals were approved by the animal house, faculty of science, Zagazig University, Egypt. I/R (ARF) were induced in anesthetized (ketamine/valpam) adult male Sprague Dawley rats, weighing 200 to 250 g by clamping both renal pedicles for 45 minutes. Then animals were injected with 4 ml warmed normal saline gave intraperitoneally before abdominal closure. (Claudia et al., 2005) Cell culture and transplantation Rat mesenchymal stem cellswere generated from the bone marrow of adult Sprague-Dawley rats by standard procedures (Javazon et al., 2001, Lange et al., 2003). Mesenchymal stem cellsused in this study were cultured for more than 3 passages, which practically excludes hematopoietic cell contamination. After 3 days of isolation non-adherent cells were removed by two to three washes with PBS and adherent cells further cultured in complete medium. The medium was changed every 3 days until the monolayer of adherent cells reach 7080% confluence. Trypsinization was made for cell splitting by Trypsin/EDTA solution (0. 25%, lonza, USA) for passage 1 .Number of cells were evaluated by Homocytometer and cellular viability by the Trypan Blue exclusion test. Each 250-300 × 103 cells were inoculated in 75 Cm2 culture flask that were incubated at 37oC and 5% CO2. Cell cultivation was maintained up to the 3rd passage. After reflow, 2 × 106 mesenchymal stem cellsin 0.5 mL of complete medium were infused into the renal vein. (Uta et al., 2007). Biochemical study: Renal function was monitored by measurement of serum creatinine according to the method of (Young., 2001) and blood urea nitrogen (BUN) according to the method of (Henry et al., 1974) Histopathology study: a) Histology The kidneys were fixed in a 10% neutral-buffered formalin solution, embedded in paraffin and 5μ thick sections were stained with Hematoxylin and Eosin for injury scoring according to a previously published guideline. (Solezk et al., 1979) b) Imunohistochemstry Other sections were stained with Ki-67 imunohistochemstry