Background
The mammalian kidney has multifarious functions, including maintenance of blood pressure, regulation of pH and other metabolic processes, such as elimination of nitrogenous wastes and synthesis and release of endocrine factors. Loss of regional or total blood flow to the kidney often happens in shock, sepsis and during renal transplantation, can lead to acute kidney injury (AKI). Inflammatory process and primary ischemic injury in vascular cells results in prolonged localized ischemia that substantially increases severity of injury in affected areas. During ischemia, the endothelium produces free radicals and secretes chemo-attractants which upon reperfusion sequester and activate neutrophils and intensification of the injury [
1]. AKI associated mortality is estimated as 50% in hospitalized patients and 70-80% in patients under intensive care [
2,
3]. Currently, the therapeutic choices are confined to supportive measures and preventive strategies. None of these have shown a decrease in mortality rate caused by AKI. Kidney transplant is an alternate option but scarcity of donor; high cost and immune rejection limits its utility. It is therefore imperative to find other therapeutic options that could regenerate the tubular epithelium, restore kidney function and reverse the effects of AKI.
MSCs are multi-potent cell population residing in bone marrow. These are capable of differentiating into cell types other than their tissue of origin. In culture, these cells are characterized by their unique ability to adhere to the plastic surface and exhibit fibroblast like morphology [
4]. Moreover MSCs secrete an array of growth factors as vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and anti-apoptotic cytokines [
5]. The therapeutic effects of MSCs after transplantation are dependent on their survival in the recipient tissue. Taking into account that prolonged cell survival may augment the usefulness of stem cell therapy. It has been reported that pre-conditioning of different tissues by a prior stimulus provides protection against injury [
6]. The most extensively studied pre-conditioning stimulus is brief sub-injurious ischemia, which induces protection against subsequent ischemic injury [
7]. Similar protective effects can also be triggered by growth factors, cytokines or with some pharmacological agents [
5,
8].
Nitric oxide (NO) is a free radical generated as a result of oxidation of L-arginine catalyzed by all three isoforms (neuronal, inducible and endothelial) of nitric oxide synthase (NOS) and performs different patho-physiological functions in different cellular environments [
9‐
13]. It protects macrophages, hepatocytes, cardiomyocytes against different injuries by modifying expression of different genes [
14]. Various donors of NO have been implicated as cyto-protective and tissue protective factors
in vivo[
1,
15].
This study was designed to evaluate the effects of pre-conditioning of bone marrow derived MSCs with a pharmacological agent S-nitroso N acetyl penicillamine (SNAP, a NO donor) on the regenerative potential of MSCs against oxidative and ischemic stress in kidney. We report that SNAP pre-conditioning of MSCs improved their potential against oxidative stress, enhanced their proliferation and reduced apoptosis in vitro. Further it augmented the homing of MSCs in ischemic renal parenchyma, induced different cytoprotective and paracrine factors and improved renal function in “rat model of renal ischemic insult”.
Discussion
Bone marrow derived MSCs have the ability to ameliorate renal ischemia [
19]. During renal ischemia, kidney becomes rich in reactive oxygen species, pro inflammatory cytokines and factors facilitating apoptosis [
20]. Transplanted MSCs encounter such harsh environment resulting in poor survival which limits their utility in regenerative cellular therapy for renal ischemia [
21]. 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 H
2O
2 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 [
25]. During ischemia reperfusion injury, iNOS is up-regulated and aggravates the renal injury which may be due to the production of peroxynitrites [
26]. 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 [
27]. NO mediates cyto-protection of isolated islets in serum deprivation by triggering PI3K/Akt pathway [
28]. 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 [
29]. 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 [
38]. 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) [
41]. 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 [
42]. 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MSM participated in the design of study, perform culturing and in vitro treatments, participated in in vivo experiments, executed statistical analysis and drafted the paper. SSA carried out polymerase chain reaction. MZA performed in vitro immunoassays and helped to draft the manuscript. AM analyzed the in vivo data and helped to draft the manuscript. SNK participated in study design and proofreading the manuscript and SR contributed in study design, funding and final approval of the manuscript. All authors read and approved the final manuscript.