Bone marrow derived MSCs have the ability to ameliorate renal ischemia . During renal ischemia, kidney becomes rich in reactive oxygen species, pro inflammatory cytokines and factors facilitating apoptosis . Transplanted MSCs encounter such harsh environment resulting in poor survival which limits their utility in regenerative cellular therapy for renal ischemia . In an attempt to improve the survival after transplantation, MSCs were pre-conditioned with a NO donor SNAP. NO is diverse biomolecule with a range of physiological functions and importance. NO has been found cyto-protective in vivo as well as in vitro against a variety of cyto-toxic agents [8, 22–24]. Pre-conditioning of MSCs with 100 μM SNAP has multiple effects. It significantly reduced the cyto-pathic effects induced by H2O2 by improving the cell viability and survival in vitro (Figure 1). Pre-conditioning with SNAP improved the survival and engraftment of MSCs in ischemic renal tissue (Figure 3). Pre-conditioning may stimulate endogenous gene expression which enhanced their engraftment. It has been reported that NO regulates hemodynamics during renal organogenesis . During ischemia reperfusion injury, iNOS is up-regulated and aggravates the renal injury which may be due to the production of peroxynitrites . Our in vivo data showed that iNOS expression in kidney was reduced after pre-conditioned MSCs transplantation. Consequently fibrosis in ischemic renal tissue was reduced due to decreased collagen deposition in pre-conditioned MSCs transplanted group (Figure 4).
Akt activates many signaling cascades leading to regulation of a range of critical cellular functions like glucose metabolism, cell proliferation and survival . NO mediates cyto-protection of isolated islets in serum deprivation by triggering PI3K/Akt pathway . The protection mechanism of SNAP pre-conditioned MSCs seems due to activation of Akt pathway in these cells. Akt was up-regulated in SNAP pre-conditioned MSCs resulting in improved cell viability and reduced apoptosis in vitro. It enhanced expression of Bcl-2 in kidney of SNAP pre-conditioned group of animals, inhibiting apoptosis and facilitates cellular survival in the ischemic atmosphere of kidney by maintaining mitochondrial membrane integrity as shown previously . Growth factors and cytokines play a pivotal role in development, cell differentiation, survival and proliferation. SNAP pre-conditioning induced up-regulation of an array of cyto-protective and pro-angiogenic cytokine and growth factor genes (IGF-1, SDF-1 and VEGF) in MSCs (Figure 2). IGF-1, SDF-1 and VEGF are cytoprotective, renotropic and pro-angiogenic growth factors playing an important part in vasculogenesis, angiogenesis and chemoattraction [19, 30–33]. Up-regulated expression of VEGF in many cell types like smooth muscle cells, endothelial cells and keratinocytes is under the influence of NO [34–37]. VEGF along with Akt enhances the production of NO by activating eNOS leading to relaxation in endothelium and increased endothelial permeability which causes vasodilation of blood vessels . Our results are in agreement with the previous studies as in vitro and in vivo data showed increased VEGF and eNOS expression in SNAP pre-conditioned MSCs but this response was abolished by pre-treating MSCs with methylene blue. Enhanced expression of VEGF and eNOS may result in the tissue protection by improved angiogenesis and perfusion leading to enhanced nutrient supply. Further, VEGF and SDF-1 could guide transendothelial migration of MSCs [39, 40] into the ischemic renal tissue and their proliferation, but methylene blue treatment inhibited these factors. This whole milieu of up-regulated growth factors, chemokine, Bcl-2 and Akt may favor the maintenance of mitochondrial integrity and cell survival ensuing to renal protection against ischemia.
Proliferation is one of the important aspects in cellular therapies in order to repopulate the damaged tissue. In our studies, PCNA expression was higher which shows that SNAP Pre-conditioning elicited proliferation in MSCs in vitro (Figure 2) . Concurrently, there was marked increase in Ki-67 expression in tissue sections from pre-conditioned MSCs transplanted kidneys (Figure 5A and B) compared to all other treatment groups showing early make up of cellular loss in kidney parenchyma resulting in improvement of renal function. This increase in proliferation activity was diminished by methylene blue treatment in vitro and in vivo.
Tubular epithelium is lost dramatically in ischemia reperfusion injury, resulting in impaired kidney function . Our results demonstrated that mutilation of kidney function caused by ischemia was overcome by the transplantation of SNAP pre-conditioned MSCs in a better way by increased creatinine clearance and reduced BUN (Table 2) which could be due to improved perfusion and tubulogenesis.