|Year : 2020 | Volume
| Issue : 3 | Page : 750-754
The use of human stem cells in the treatment of experimentally induced acute kidney injury
Mahmoud A Kora1, Ahmed M Zahran1, Mahmoud M Emara1, Yahya M. N. Abdelsalam2, Mahmoud A Sobhy3
1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Physiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Internal Medicine Department, El Obour Hospital, Kafr El Sheikh, Egypt
|Date of Submission||13-Nov-2018|
|Date of Decision||02-Dec-2018|
|Date of Acceptance||15-Dec-2018|
|Date of Web Publication||30-Sep-2020|
Mahmoud A Sobhy
El Ryaid, Kafr El Sheikh
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the effect of stem cell therapy in sepsis-induced acute kidney injury (AKI) in rats.
AKI is defined as an abrupt or rapid decline in renal filtration function; thus, sepsis-induced AKI remains an important challenge in critical care medicine. The science of stem cells is a field with great potential for treating injury and disease.
Materials and methods
A total of 30 male Swiss albino rats were used in the present study. Animals were randomly divided into three groups. Group I was the control group (10 rats). Group II was the AKI group (10 rats), where sepsis-induced AKI rat model was used. Group III was the stem cell-treated AKI group (10 rats). Rats with established AKI were injected with CD34-positive stem cells. The three studied groups were assessed for serum urea, blood urea nitrogen, serum creatinine, and cystatin C. At the end, all rats were killed, and the kidneys were excised for histopathology and immunohistochemical studies.
Cystatin C level shows high significant difference between AKI group in comparison with both control group and stem cell-treated group (P = 0.001) whereas there is no significant difference between control group and stem cell-treated group (P = 0.944).
This study showed that AKI markers improved after treatment by stem cells in AKI-induced group.
Keywords: acute kidney injury, cystatin, rats, sepsis, stem cells
|How to cite this article:|
Kora MA, Zahran AM, Emara MM, Abdelsalam YM, Sobhy MA. The use of human stem cells in the treatment of experimentally induced acute kidney injury. Menoufia Med J 2020;33:750-4
|How to cite this URL:|
Kora MA, Zahran AM, Emara MM, Abdelsalam YM, Sobhy MA. The use of human stem cells in the treatment of experimentally induced acute kidney injury. Menoufia Med J [serial online] 2020 [cited 2020 Oct 29];33:750-4. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/750/296667
| Introduction|| |
Acute kidney injury (AKI), previously known as acute renal failure, is defined by the rapid onset of decreased excretory function. The stage of AKI is based on increased serum creatinine levels concurrent with decreased urine output . Patients with AKI have long struggled with diminishing health and increased mortality upon diagnosis. Risk of AKI increases with age and uncontrolled diabetes mellitus and often develops without pre-existing kidney issues. Increases in severity of AKI and number of episodes are associated with an increased risk of chronic kidney disease .
Both sepsis and AKI are diseases of major concern in critically ill patients. Severe sepsis is often complicated by AKI. The overall incidence of septic AKI among all ICU admissions ranges between 15 and 20% . The exact pathophysiology of sepsis-induced AKI is not known; however, it is generally accepted that it has a multipronged injury pathway. This form of AKI has components of ischemia-reperfusion injury, direct inflammatory injury, coagulation and endothelial cell dysfunction, and apoptosis. Moreover, based on recent evidence, we may presume that the pathophysiologic mechanisms of sepsis-induced AKI are different from nonseptic AKI .
Gram-negative sepsis is independently associated with AKI. An elevated plasma concentration of endotoxin [lipopolysaccharide (LPS)] is often found in the systemic circulation during sepsis, regardless of the type of the infecting microorganism, possibly as a result of the translocation of LPS originating from the resident gram-negative flora of the gut. During the inexorable downward spiral of sepsis, LPS, and then cytokines, and consequently nitric oxide are released. Adding to the translocation of intestinal-derived LPS that occurs during any form of sepsis, the multiplication and destruction of gram-negative bacteria results in the release of LPS into the bloodstream, and its rapid dissemination throughout the body. LPS binds with the LPS-binding protein through the biologically active component lipid A of LPS. The LPS-binding protein-LPS complex binds to the coreceptor CD14, which leads to interactions with the cell surface Toll-like receptor 4-MD-2 complex on monocytes, macrophages, and neutrophils, but this complex also binds to other cells, including renal tubular epithelial cells. These cells are then stimulated to produce cytokines through a myeloid differentiation primary response gene (MyD88)-dependent and an MyD88-independent pathway .
Drug therapies have had limited success and sustainability in the clinical field, which highlights a dire need for curative treatment options, such as stem cell-based kidney repair. Stem cells are described by their self-renewal abilities and the capability to develop into various functional cells. There are four classes of developmental potential among stem cells. Totipotent cells are the most versatile, as they can develop into any cell of an organism, including extraembryonic tissues. Pluripotent cells, such as embryonic stem cells, can develop into all cell types in the body of an organism but not into extraembryonic tissues, such as the placenta. Multipotent cells give rise to cells of a specific lineage, for example mesenchymal stem cells give rise to skeletal tissues. Adult stem cells, umbilical cord stem cells, and mesenchymal stem cells are all examples of multipotent cell types. Unipotent stem cells are the most restricted in their potency and can only form one cell type. Sequentially treated hematopoietic stem and progenitor cells (HSPCs) with cytokines, growth factors, and a histone deacetylation inhibitor to stimulate cell cycle entry, induce chromatin modification, and promote hematopoietic-to-renal conversion. HSPCs were used because they share a common mesodermal origin with the embryonic kidney, that is, the aorta-gonad-mesonephros region contributes to early hematopoiesis . The aim of our work was to study the effect of transplanted human stem cells in experimentally induced AKI in rats.
| Materials and Methods|| |
In this study, 30 Swiss albino rats weighing ~150–250 g were used. Animals were randomly divided into three groups. Rats were maintained under controlled temperature, humidity, and 12 h light/dark cycles. The animals were fed standard rodent chow and allowed free access to water ad libitum, and were kept for 10 days before any procedure to allow proper acclimatization. Animal care and use was approved by the Ethics Committee of the Faculty of Medicine, Menoufia University, Egypt. The experiments were carried in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health .
Group I was the control group (10 rats). In this group, rats received a single intravenous injection of normal saline 0.9% in rat tail vein. These rats were left to live normally for 6 weeks.
Group II was the AKI group (10 rats). In this group, rats with sepsis-induced AKI using cecal ligation and puncture were used, which is the most widely used sepsis model .
Group III was the AKI + stem cell-treated group (10 rats). In this group, rat models of sepsis-induced AKI were generated as mentioned before, and a single dose of CD34-positive cells (in a dose of 2 × 106 cells/rat) was injected intravenously in the rat tail vein after 1 week of cecal ligation and puncture. These rats were left to live in their cages for 4 weeks.
Blood sample collection
Blood samples were collected from the retro-orbital venous plexus, using a fine heparinized capillary tube introduced into the medial epicanthus of the rat's eye . Two milliliters of blood was kept at room temperature in a water bath for 10 min, and then centrifuged at 3000 rpm for 20 min. The supernatant serum was collected in a dry clean tube and kept at 80°C for the estimation of serum urea, creatinine, blood urea nitrogen, and cystatin C.
At the end of the experiment, all rats were killed by decapitation, and the kidneys were excised for histopathology by hematoxylin–eosin stain and Masson's trichrome technique and immunohistochemical studies by labeled streptavidin–biotin horseradish peroxidase technique. Lumbar incision was done to excise the kidneys which were fixed in 4% paraformaldehyde and processed through paraffin embedding and prepared for immunohistochemical studies.
Isolation of CD34-positive stem cells: stem cell separation
Umbilical cord blood (UCB) was obtained at the end of full-term deliveries while the placenta is still in utero; after clamping and cutting of the cord using strict aseptic techniques, the umbilical vein was cleansed with alcohol followed by betadine, and then drainage of blood was done into sterile collection tubes containing the anticoagulant citrate phosphate dextrose adenine-l (~10 ml), as the total collection was ~100 ml. UCB samples were collected separately and stored at 4°C and processed within 24 h.
Separation and purification of CD34-positive hematopoietic stem cells/hematopoietic progenitor stem cells (HSC/HPC) cells was carried out according to method described by David W et al.  by immune-magnetic separation technique using Dynabeads (Dynal, Oslo, Norway). Circulating hematopoietic progenitor cells home to the damaged kidney by responding to injury signals that correspond to cognate surface receptors they express.
The UCB collection is excluded from women with a known history of hepatitis, infectious diseases, diabetes mellitus, severe hypertension, abortions, or bad obstetric history.
Serum urea and creatinine were estimated using kits supplied by Diamond Diagnostic (Munich, Germany). Serum blood urea nitrogen was estimated using kit supplied by Siemens Healthcare Diagnostic (Malvern, Pennsylvania, USA) according to manufactures' instructions. Cystatin C was measured by specific enzyme-linked immunosorbent assay kit (ALPCO Diagnostic, Salem, New Hampshire, USA) as described by the manufacturer.
In this work, the mean, the SD, the SE, and the P value were calculated. Post-hoc test was calculated. Data were fed to the computer using IBM statistical package for the social science (SPSS), version 20 (IBM Corporation, Chicago, Illinois, USA), for Windows (MedCalc Software BVBA, Ostend, Belgium). Quantitative data were described using mean and SD for normally distributed data. Analysis of variance test is used for differences among at least three groups.
| Results|| |
Serum creatinine level was significantly higher in AKI group (1.46 ± 0.18 mg/dl) in comparison with both control group (0.32 ± 0.03 mg/dl) and stem cell-treated group (0.41 ± 0.06 mg/dl) (P = 0.001), whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum creatinine level (P = 0.086) [Table 1].
Serum urea level was significantly higher in AKI group (126.3 ± 11.7 mg/dl) in comparison with both control group (45.2 ± 5.91 mg/dl) and stem cell-treated group (49.8 ± 6.71 mg/dl) (P = 0.001), whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum urea level (P = 0.257) [Table 1].
Blood urea nitrogen was significantly higher in AKI group (60.1 ± 4.91 mg/dl) in comparison with both control group (22.7 ± 2.10 mg/dl) and stem cell-treated group (32.3 ± 5.31 mg/dl) (P = 0.001), and also high significant difference was found between control group and stem cell-treated group regarding blood urea nitrogen level (P = 0.001) [Table 1].
Cystatin C level was significantly higher in AKI group (3.28 ± 0.28 mg/l) in comparison with both control group (1.87 ± 0.10 mg/l) and stem cell-treated group (1.88 ± 0.26 mg/l) (P = 0.001), whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum cystatin C level (P = 0.944) [Table 1].
| Discussion|| |
AKI is a syndrome characterized by a rapid (hours to days) deterioration of kidney function. It is often diagnosed in the context of other acute illnesses and is particularly common in critically ill patients. The clinical consequences of AKI include the accumulation of waste products, electrolytes, and fluid, but also less obvious effects, including reduced immunity and dysfunction of nonrenal organs (organ cross-talk) . The present study showed that serum creatinine level was significantly higher in AKI group in comparison with both control group and stem cell-treated group, whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum creatinine level.
Serum urea level was significantly higher in AKI group in comparison with both control group and stem cell-treated group, whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum urea level.
Blood urea nitrogen was significantly higher in AKI group in comparison with both control group and stem cell-treated group, and also highly significant difference was found between control group and stem cell-treated group regarding blood urea nitrogen level.
Cystatin C level was significantly higher in AKI group in comparison with both control group and stem cell-treated group, whereas no statistically significant difference was found between control group and stem cell-treated group regarding serum cystatin C level. This demonstrated that CD34-positive HSC/HPC cells have been reported to maintain renal functions within normal levels in several AKI models including sepsis-induced AKI.
Ashwani K et al.  induced mouse HSPC to differentiate into cells that partially resemble a renal cell phenotype and tested their therapeutic potential. They sequentially treated HSPC with a combination of protein factors for 1 week to generate a large number of cells that expressed renal developmentally regulated genes and protein. Cell fate conversion was associated with increased histone acetylation on promoters of renal-related genes. Further treatment of the cells with a histone deacetylase inhibitor improved the efficiency of cell conversion by sixfold. Treated cells formed tubular structures in three-dimensional cultures and were integrated into tubules of embryonic kidney organ cultures. When injected under the renal capsule, they integrated into renal tubules of postischemic kidneys and expressed the epithelial marker E-cadherin. No teratoma formation was detected 2 and 6 months after cell injection, supporting the safety of using these cells. Furthermore, intravenous injection of the cells into mice with renal ischemic injury improved kidney function and morphology by increasing endogenous renal repair and decreasing tubular cell death. The cells produced biologically effective concentrations of renotrophic factors including vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, and human growth factor (HGF) to stimulate epithelial proliferation and tubular repair. This study indicates that HSPC can be converted to a large number of renal-like cells within a short period for potential treatment of AKI.
Liu Y et al.  found that AKI is frequently characterized by damage and the subsequent loss of glomerular podocytes. These complex and specialized cells play an important role in glomerular filtration, so the destruction of such cells quickly results in impaired renal function. Podocytes are derived from metanephric mesenchyme, and therefore require different factors to drive correct specification. Induced pluripotent stem cell (iPSCs) can be directed to a podocyte progenitor fate with activin A, BMP-7, and retinoic acid in a suspension culture. After a 10-day incubation, the iPSCs successfully differentiated into podocyte progenitors with cell morphology of primary human podocytes but also a capacity for proliferation. The podocytes derived from iPSCs exhibit the complex cell morphology of adult podocytes, including not only the primary pedicels but secondary and tertiary foot projections as well. Beyond the histological appearance of the iPSC-derived podocytes, the cells generated by Song and colleagues also expressed podocyte-specific cell markers such as podocin and synapodin. In contrast to primary podocytes that are quiescent in culture, iPSC-derived podocytes maintained proliferation for 3 months, a promising finding for potential organ regeneration. The podocytes generated from iPSCs also exhibited a contractile response to angiotensin II and albumin uptake, similar to normally functioning human podocytes. Using murine embryonic kidneys, iPSC podocytes incorporated into the developing kidney particularly in regions of nephrogenesis. The results of this study show the potential for iPSC-derived kidney cells to both attain the same functionality as primary renal cells as well as integrate into previously existing renal tissue.
Patschan D et al.  investigated the efficacy of iPSCs without exogenous c-Myc as a mechanism for repair and regeneration of renal tissue in AKI. Ischemia and inflammation are two of the most common mechanisms that lead to AKI. iPSCs may be a safe and effective means to repair renal tissue damage in such a way because iPSCs have been found to suppress intracellular reactive oxygen species and reduce inflammatory cytokines. Lee and colleagues found that iPSCs created without c-Myc both improved renal function and renal tubular injury as well as eliminated post-transplantation tumorigenesis. The success of iPSC generation was confirmed by the expression of pluripotency cell markers and the cells' ability to differentiate into endodermal, mesodermal, and ectodermal lineages. The ability of the iPSCs to attenuate the damaging effects of ischemia-induced AKI was tested by measuring the blood urea nitrogen and creatinine levels of rats with AKI following intrarterial, intraperitoneal, and intravenous administration of the iPSCs. Furthermore, after iPSC transplantation, there was a reduction in macrophage proliferation and oxidative stress in the renal tissue. Overall, the survival rates in rats with AKI was significantly increased and renal function improved, indicating iPSC transplantation as a potential therapeutic agent for AKI.
Bruno et al.  investigated the therapeutic effects of microvesicles derived from mesenchymal stem cells (MSCs) in severe combined immunologic deficiency (SCID) mice upon injection of cisplatin, a chemotherapy drug that is toxic to the kidney and thus induces AKI. Cisplatin-injected SCID mice received a single injection of microvesicles (siMV), multiple injections of microvesicles (miMV), and a control group with no MSC-derived microvesicles injected. Microvesicles derived from MSCs reduced mortality of cisplatin-injected mice. By day 21, the survival rate in cisplatin-injected mice with siMV was 40% and from miMV was 80% with respect to the control group. Treatment with miMV was significantly more effective than siMVs by day 21, indicating that microvesicles derived from MSC aid in the AKI repair process, perhaps in a dose-dependent manner.
Tsuda et al.  verified pathway by introducing an anti-IL-10 antibody to MSC transplanted rats, which decreased the therapeutic ability of MSC following ischemia-reperfusion related injury. Other factors of the inflammatory response at play in ischemia-reperfusion conditions include monocyte chemotactic protein (MCP-1 or CCL2) and IL-6. When treated with fetal membrane MSC (FM-MSC), macrophage infiltration has been shown to decrease, and this may be owing to FM-MSC ability to decrease MCP-1 and IL-6 activity. FM-MSC treatment in animal models with myocarditis has also been shown to involve the adaptive immune response, by reducing activation, proliferation, and infiltration of T cells upon injury.
| Conclusion|| |
In conclusion, this study showed that AKI markers improved in sepsis-induced AKI group after treatment with human HSCs by stem cell transplantation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rodríguez E, Arias-Cabrales C, Bermejo S, Sierra A, Burballa C, Soler MJ, et al
. Impact of recurrent acute kidney injury on patient outcomes. Kidney Blood Press Res 2018; 43
Shahrzad H, Mojtaba S, Gholam H, Keyvan S, Hamid R, Hojjat N, et al
. In vivo
effects of allogeneic mesenchymal stem cells in a rat model of acute ischemic kidney injury. Iran J Basic Med Sci 2018; 21
Gijs F, Susanne S, Aarnoudse A-JH, Robert Z, Michiel G. Long-term sequelae of severe acute kidney injury in the critically ill patient without comorbidity: a retrospective cohort study. PLoS One 2015; 10
Il Y, Joo H, Dong W, Soo B, Harin R, Eun Y, et al
. Fluid overload and survival in critically ill patients with acute kidney injury receiving continuous renal replacement therapy. PLoS One 2017; 12
Ballús J, Sabater J, Pérez X, Nin N, Ordonez-Llanos J, Betbesé AJ. Cell-cycle arrest biomarkers in urine to predict acute kidney injury in septic and non-septic critically ill patients. Ann Intensive Care 2017; 7
Albert Q, Joseph V. Regenerating the nephron with human pluripotent stem cells. Curr Opin Organ Transplant 2015; 20
Barnes CJ, Distaso CT, Spitz KM, Verdun VA, Haramati A. Comparison of stem cell therapies for acute kidney injury. Am J Stem Cells 2016;5:1-10.
Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 2009; 4
Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother 2010; 1
David W, James C, Robert H. Microfluidic high gradient magnetic cell separation. J Appl Phy 2006; 99
Singbartl K, Joannidis M. Short-term effects of acute kidney injury. Crit Care Clin 2015; 31
Ashwani K, Sachin H, Naresh K, Soniya N. Fetal kidney cells can ameliorate ischemic acute renal failure in rats through their anti-inflammatory, anti-apoptotic and anti-oxidative effects. PLoS One 2015; 10
Liu Y, Tang SCW. Recent progress in stem cell therapy for diabetic nephropathy. Kidney Dis 2016; 2
Patschan D, Buschmann I, Ritter O, Kribben A. Cell-based therapies in acute kidney injury (AKI). Kidney Blood Press Res 2018; 43
Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, et al
. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 2012;7:e33115.
Tsuda H, Yamahara K, Otani K, Okumi M, Yazawa K, Kaimori JY, et al
. Transplantation of allogenic fetal membrane-derived mesenchymal stem cells protects against ischemia/reperfusion-induced acute kidney injury. Cell Transplant 2014;23:889–99.