SVF (Adipose Tissue Derived MSC) Based Therapy for CKD.

Introduction: Chronic kidney disease (CKD) is a disease of alarmingly increasing prevalence (8 - 16%) associated with mortality \[1\]. CKD can progress towards end-stage renal disease (ESRD), requiring renal replacement therapy. ESRD currently accounts for 6.3% of Medicare spending in the United States and is projected to increase by 85% by 2015 \[2\]. In a study conducted among the rural populations in Bangladesh overall CKD prevalence was found about 19% \[3\]. Furthermore, ESRD has a major impact on quality of life and life expectancy \[4\]. Therefore, it is very important to develop therapeutic interventions to prevent, alleviate, or decelerate the progression of renal failure. Diabetes mellitus and hypertension represent major causes of CKD and initiation of dialysis \[5\]. In addition, glomerular diseases, malnutrition, infectious diseases, and acute kidney injury may lead to ESRD, contributing to the increased global burden of death \[6\]. Current treatment modalities often fail to target the major underlying contributors to the progression of renal disease \[7\]. Management of CKD at present mostly aims at control of the predisposing factors and supplementation of kidney homeostatic functions but not at the treatment of the diseased kidney itself. Again due to lack of adequate facilities or financial constraints people of a developing country like Bangladesh are unable to continue long-term or lifelong dialysis. Chronic glomerular and tubule-interstitial fibrosis is a common pathway to ESRD, often associated with apoptosis, oxidative damage, fibrosis, and microvascular rarefaction. Unfortunately, the regenerative potential of kidneys is limited under chronic conditions and inefficient to prevent progressive glomerulosclerosis and tubule-interstitial fibrosis \[8\]. Treatment strategies that boost cellular regeneration might therefore offer good alternatives for patients with CKD. Stromal vascular fraction (SVF): SVF of adipose tissue is a rich source of pre-adipocytes, mesenchymal stem cells (MSC), endothelial progenitor cells, T cells, B cells, mast cells as well as adipose tissue macrophages \[9,10\]. SVF is a component of the lipo-aspirate obtained from liposuction of subcutaneous tissue. Lipo-aspirate contains a large population of stem cells called adipose-derived stem cells (ADSCs), which share a number of similarities with bone marrow stem cells, including the capacity for multilineage differentiation. Stem Cells: A stem cell is a generic term referring to any unspecialized cell that is capable of long-term self-renewal through cell division but that can be induced to differentiate into a specialized, functional cell. Stem cells are generally two types, embryonic stem cells, and adult stem cells. Adult stem cells can be obtained from many differentiated tissues including bone marrow, bone, fat, muscle etc. Obtaining adult stem cells also does not raise any ethical concerns \[11\]. For most studies, the adult stem cell in question is actually a mesenchymal stem cell (MSC) or mesenchymal stromal cell. They are multipotent but not pluripotent, which means they can differentiate into some, or "multiple," but not all tissue types \[11\]. Stem cells that are harvested from the patient with the intention of administering them back to the same patient are termed autologous MSCs. MSC can also be isolated from the bone marrow (bmMSC), peripheral blood, connective tissue, skeletal muscle, the dental pulp (dpMSC), umbilical cord wall (ucMSC), umbilical cord blood (cbMSC), amniotic fluid (afMSC) and all have been used in experimental settings to treat various types of renal diseases. An important feature of MSCs is their capacity to induce the proliferation of renal glomerular and tubular cells, increasing cellular survival \[12\]. Advantages of adipose tissue-derived stem cells (ADSCs): ADSCs are somatic stem cell populations contained in fat tissue and have been shown to possess stem cell properties such as trans-differentiation and self-renewal \[13\]. Similar to other types of MSCs, ADSCs express multiple CD marker antigens (CD73+CD90+CD105+ CD34+/- CD11b- CD104b- CD19- CD31- CD45- SMA-) \[14,15\]. Additionally, utilizing ADSCs is advantageous in that large quantities of stem cells are easily isolated using minimally invasive surgical procedures \[16\]. ADSCs are vascular precursor cells. Many studies have shown that SVF contains progenitor cells that are able to differentiate into endothelial cells and participate in blood vessel formation \[17\]. Additionally, a recent study demonstrated that SVF cells expressing both pericyte and mesenchymal markers reside in a peri-endothelial location and stabilize endothelial networks \[17\] Another study showed that ADSCs were transplanted into an ischemic renal cortex preferentially migrate toward microvessels where they differentiate into vascular smooth muscle cells \[18\]. Some trials on kidney transplant recipients as well as the one on FSGS and 2 on CKD patients include in their protocol the utilization of adMSC. Adipose tissue is an important source of MSC, with a frequency 100 to 1000 times higher than bmMSC. They also seem to possess a higher potential for angiogenesis or vasculogenesis \[19\]. Kidney disease and mesenchymal stem cells: A number of different types of cells from the bone marrow have been tested in animals and in clinical studies for potential use in kidney disease. Amongst all the cells under investigation, MSCs have shown the most promising results to date as they help kidney cells to grow, inhibit cell death, and encourage the kidney's own stem cells to repair kidney damage \[20\]. Few clinical trials have tested the safety and efficacy of MSCs for renal disease. Reinders and colleagues studied safety and feasibility in six kidney allograft recipients who received two intravenous infusions of expanded autologous bone marrow-derived MSCs \[21\]. Importantly, delivery of autologous MSCs was not associated with adverse events, nor did it compromise graft survival. Several clinical trials are currently underway to evaluate the therapeutic potential of autologous and allogeneic MSCs for the treatment of renal diseases \[22\] Administration of both bmMSC and adMSC has demonstrated significant renoprotective effects including reduction of intrarenal inflammatory infiltrate, decreased fibrosis, and glomerulosclerosis \[12\] MSCs possess unique immunomodulatory properties that ameliorate inflammation and immune responses, constituting a promising tool to facilitate renal repair. In recent years, experimental studies have uncovered the potential of MSCs to improve renal function in several models of CKD, and several clinical studies have indicated their safety and efficacy in CKD \[22\]. ADSCs could be incorporated into damaged tissues or organs which could give rise to new functional components and also exert potent anti-inflammatory, anti-fibrotic, or immunomodulation effects through paracrine or autocrine routes (via vascular endothelial growth factor, granulocyte/macrophage colony-stimulating factor, stromal-derived factor-1alpha, and hepatocyte growth factor) \[23,24\]. Interestingly, it is proposed that even apoptotic or dying ADSCs exhibit distinctive immunosuppressive properties \[25\]. ADSCs have been shown to possess stronger anti-inflammatory and immuno-modulating functions than bone marrow-derived MSCs \[26\]. Villanueva et al. explored the effect of ADSCs on CKD by a single intravenous infusion of ADSCs on a nephrectomy-induced CKD model of rats \[27\]. ADSC treatment was associated with reduced plasma creatinine, higher levels of epitheliogenic and angiogenic proteins, and improved renal function. Work by Hyun et al \[28\] illustrated the beneficial effects of ADSCs on improving renal function in an IgAN mouse model. Zhang et al. \[30\] found that repeated systemic administration of ADSCs attenuated proteinuria, glomerulus hypertrophy, and tubular interstitial injury in a DN rat model \[29\]. Currently, several clinical trials have been uploaded to the NIH database, all aiming to test mainly the safety of using MSC and their efficacy in treating CKD. Two of them propose the use of autologous bmMSC and two adMSC. A study conducted in Tehran, the Islamic Republic of Iran, was designed to provide confirmation of Mesenchymal stem cell therapy in CKD. There 18 months of safety and efficacy of autologous MSC as a therapy for CKD total of 10 patients were conducted with I/V injection of a high dose of 2x106/kg of autologous MSC. Assessments were performed at 1,3,6,12 and 18 months after cell injections \[30\]. Another study was conducted in Birmingham, Alabama, Rochester, Minnesota, and Jackson, Mississippi, the USA where a stem cell product called "Mesenchymal stem cell" grown from a person's own fat tissue was infused back into the patient's own kidney and primary outcome measured after 3 months where renal tissue oxygenation increased and decrease in kidney inflammation was seen as a secondary outcome \[31\]. Route of delivery: Various routes for delivery of ADSCs, ADSC-induced cells, or ADSCs combined with compound materials have been developed for the treatment of different diseases or damaged tissue. These routes can be classified into two categories: systemic delivery through blood vessels (intravenous injection or intra-arterial injection) or local delivery directly into injured tissues or organs \[32\] The route of MSC delivery may influence the cells' capacity to home and engraft the damaged tissue, and thereby their efficacy for renal repair. Commonly used experimental methods to deliver MSCs include systemic intravenous, intra-arterial, or intra-parenchymal delivery. In nonhuman primates, the cells distribute broadly into the kidneys, skin, lung, thymus, and liver with estimated levels of engraftment ranging from 0.1 to 2.7% \[33\]. The route of MSC delivery, intravenous, intra-arterial, or intra-parenchymal, may affect their efficiency for kidney repair. When labeled MSC intravenously infused into baboons were observed for 9-21 months, estimated levels of engraftment in the kidney, lung, liver, thymus, and skin ranged from 0.1-2.7% \[34\] Indeed, the intravenous route lags in delivery efficiency, because MSC may initially be trapped in the lungs. Intra-arterial infusion of MSC was the most effective route to achieve immunomodulation in rat kidney transplantation, possibly by avoiding lodging in the pulmonary circulation, allowing MSC to home to the injured kidney \[35\]. An important feature of MSCs is their capacity to induce the proliferation of renal glomerular and tubular cells, increasing cellular survival. By secreting proangiogenic and trophic factors, injected MSCs not only can enhance proliferation but also can decrease the apoptosis of tubular cells \[36\]. Several routes of administration (intra-parenchymal, sub-capsular, intravenous) have been explored and all seem to be effective. Multiple, repeated injections of MSCs appear to be even more effective than single injections \[37,38\]. Methodology: A Prospective study from April 2019 onwards (Approximately Five years or till completion of sample requirements with a minimum of one year of follow-up) will be conducted in Bangladesh LASER \& Cell Surgery Institute \& Hospital, Dhaka. Thirty-One patients with CKD who fulfill the selection criteria and are admitted to the selected health care facility for treatment will be included in the study. SVF (stromal vascular fraction) will be collected from the abdominal subcutaneous adipose tissue. Subsequent processing ( Incubation, centrifugation, mixing, washing \& neutralization ) will produce the final viable \& active SVF (ADSCs) cell which will be transfused intravenously. Considering the possibility of further damage to the already damaging or damaged kidney during angiography and placement of a catheter into the renal artery, the investigators opt for intravenous transfusion of the SVF. Before transfusion samples will be collected \& cell counting will be done using an automated fluorescent cell counter.