Administration of mesenchymal stem cells in diabetic kidney disease: a systematic review and meta-analysis

Background Mesenchymal stem cell (MSC) therapy shows great promise for diabetic kidney disease (DKD) patients. Research has been carried out on this topic in recent years. The main goals of this paper are to evaluate the therapeutic effects of MSCs on DKD through a meta-analysis and address the mechanism through a systematic review of the literature. Method An electronic search of the Embase, Cochrane Library, ISI Web of Science, PubMed, and US National Library of Medicine (NLM) databases was performed for all articles about MSC therapy for DKD, without species limitations, up to January 2020. Data were pooled for analysis with Stata SE 12. Result The MSC-treated group showed a large and statistically significant hypoglycemic effect at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, and 6 months. Total hypoglycemic effect was observed (SMD = − 1.954, 95%CI − 2.389 to − 1.519, p < 0.001; I2 = 85.1%). The overall effects on serum creatinine (SCr) and blood urea nitrogen (BUN) were analyzed, suggesting that MSC decreased SCr and BUN and mitigated the impairment of renal function (SCr: SMD = − 4.838, 95%CI − 6.789 to − 2.887, p < 0.001; I2 = 90.8%; BUN: SMD = − 4.912, 95%CI − 6.402 to − 3.422, p < 0.001; I2 = 89.3%). Furthermore, MSC therapy decreased the excretion of urinary albumin. Fibrosis indicators were assessed, and the results showed that transforming growth factor-β, collagen I, fibronectin, and α-smooth muscle actin were significantly decreased in the MSC-treated group compared to the control group. Conclusion MSCs might improve glycemic control and reduce SCr, BUN, and urinary protein. MSCs can also alleviate renal fibrosis. MSC therapy might be a potential treatment for DKD.


Introduction
Diabetes mellitus (DM) is a chronic metabolic disease with a rising incidence rate, and its microvascular and macrovascular complications are associated with a large global burden of morbidity and mortality [1]. Diabetic kidney disease (DKD) is a serious kidney-related complication that is present in approximately 40% of patients with DM [2], and patients with DKD have an increased risk of cardiovascular events and all-cause mortality [3]. Abnormal blood glucose status leads to oxidative stress and induces the release of inflammatory mediators, resulting in glomerular lesions in DM patients. The current evidence indicates that it is very difficult to prevent the progression of DKD. The creatinine clearance rate (CCr), serum creatinine (SCr), blood urea nitrogen (BUN), microalbuminuria, urinary albumin excretion, etc. are important indicators to assess the renal damage associated with DKD.
Mesenchymal stem cells (MSCs) are being used systemically or locally to treat many diseases, as they exhibit great self-renewal and differentiation potential [4,5]. Stem cells are self-renewing, self-replicating pluripotent cells and can be classified according to their origin: embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Among them, adult stem cells, the undifferentiated cells in differentiated tissues, can be isolated from the bone marrow, adipose tissue, umbilical cord blood, and deciduous teeth. MSCs have been used for tissue regeneration and repair [6], treatment of inflammatory disease [7], prevention of transplant rejection [8], and other clinical applications.
At present, there are some data indicating that MSCs might improve complications from DM [9][10][11]. We conducted this systematic review and meta-analysis to evaluate the effects of MSC therapy on DKD.

Search strategy
We searched the Embase, Cochrane Library, ISI Web of Science, PubMed, and US National Library of Medicine (NLM) databases through January 2020 for original papers that assessed the effects of MSC administration on DKD animal models or patients without language restrictions. Keywords in this research included the following: (mesenchymal stem cells OR MSC OR multipotent stromal cells OR mesenchymal stromal cells OR mesenchymal progenitor cells OR Wharton jelly cells OR adipose-derived mesenchymal stem cells OR bone marrow stromal stem cells) AND (diabetic nephropathy OR DN OR diabetic kidney disease OR DKD).
Randomized controlled trials, comparative studies, or controlled trials that assessed the efficacy or safety of MSC therapy for treatment as an intervention in DKD animal models (without species limitations) or patients with DKD were included. The included studies were required to contain biochemical data on renal function or adverse events and to report albuminuria and impaired renal function in patients or animals with DM. The precise distinction between DKD and diabetic nephropathy (DN) was outside the scope of this paper, and both were included. Reviews, case reports, metaanalyses, comments, and letters were excluded. Articles that studied embryonic stem cells, induced pluripotent stem cells, or MSC components (rather than actual MSCs) for the treatment of DKD were excluded. In addition, studies that lacked a control arm or did not provide essential data such as renal function and sample size were excluded. We also searched for additional relevant reports by browsing the references of the articles.

Data extraction
The main features of the included studies were summarized, and the data were extracted independently by two authors using a standardized datasheet. Adverse events and biochemical indicator data were extracted from the articles, such as blood glucose, CCr, SCr, BUN, Ualbumin/U-creatinine ratio (U-ACR), microalbuminuria, urinary albumin excretion, urine protein/Cr, kidney weight, body weight, and kidney weight/body weight ratio. If a paper contained no specific information, data were obtained by measuring the chart in the paper or by contacting the primary authors. Any disagreements in the extracted data were resolved by the third author.

Validity and quality assessment
For clinical trials, quality assessment was performed using 4 items based on the Jadad scale [12]: randomization, concealment of allocation, blinding method, and description of withdrawals and dropouts. A total score of ≥ 3 was considered high quality.
For animal studies, the methodological quality assessment was carried out using a risk of bias (RoB) tool by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE), which is based on the Cochrane RoB tool and adjusted for animal experiments. The following ten items were assessed. (1) Sequence generation: Were the subjects randomly assigned to the case or control groups with an adequately generated allocation sequence? (2) Baseline characteristics: Were the baseline characteristics of the two groups comparable? (3) Allocation concealment: Was the allocation of all the subjects adequately concealed? (4) Random housing: Were all the subjects randomly housed in the same environment during the experiment? (5) Researcher blinding: Were the researchers blinded to which subjects had received treatment (in this case, MSC treatment)? (6) Random outcome assessment: Were the animals selected in random order for outcome assessment? (7) Blinding of outcome assessors: Were the outcome assessors blinded to the group information? (8) Incomplete outcome data: Were incomplete outcome data or dropouts adequately addressed? (9) Selective outcome reporting: Was the study free of selective outcome reporting for significant results? (10) Other sources of bias: Was the study apparently free of other problems that could result in a high risk of bias, such as contamination of MSCs, inappropriate influence of the funder, errors in units of analysis, design-specific risk of bias, and additional animals to replace dropouts? An answer of "yes" means a low risk of bias, while "no" means a high risk of bias, and "unclear" means the risk of bias cannot be assessed for the lack of sufficient information. Disagreements were resolved by consensus-oriented discussion.

Statistical analysis
Stata SE 12 was used for statistical analysis. For continuous variables, standard mean differences (SMDs) were obtained by pooling the mean values, standard deviations, and sample sizes. For binary data, the odds ratio (OR) was calculated. Moreover, 95% confidence intervals (95%CIs) were calculated between the MSC-treated groups and the control groups. If there were multiple MSC-treated groups in an article, the data in the control group were reused. Heterogeneity across studies was quantified using I 2 and was considered significant at a p value of < 0.1. The data were pooled using a fixed-effect model without heterogeneity or a random-effect model. A p value of < 0.05 was regarded as statistically significant for all analyses. Potential publication bias was assessed with Begg's test, Egger's test, and the trim-andfill method.

Search results
In total, 33 studies in 29 publications were included, among which 28 publications were based on animal studies  and 1 was based on a clinical trial [41]. In addition, there are 4 ongoing clinical trials registered with the NLM.
Among 32 animal studies, 24 studies used rat models, 7 used mouse models, and 1 used a rhesus macaque model. A single method or a combination of multiple methods was used to induce DM, including streptozotocin (STZ) injection, high-fat diet dietary induction, nephrectomy, and natural development of models. However, the dosage and frequency of STZ injection and the time when the animals were tested for the establishment of DN were different. Although MSCs were used in all the included studies, the details of the source, dosage, frequency, administration, and point in time varied. The sources of MSCs were bone marrow mesenchymal stem cells (BM-MSCs) in 22 studies, adipose-derived stem cells (ADSCs) in 4 studies, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in 5 studies, and stem cells from exfoliated deciduous teeth in 1 study. Allogeneic administration was used in 23 studies, xenoplastic administration was used in 8 studies, and autologous administration was used in 1 study. The characteristics of the included animal studies are summarized in Table 1.
The only clinical trial was a multicenter, randomized, double-blind, dose-escalating, sequential, placebocontrolled study, finished in 2016. Thirty patients were randomized to receive one of two doses of mesenchymal precursor cells or placebo, and the efficacy and adverse events were observed. The main features of the clinical trial are shown in Table 2.
None of the animal experiments reported the occurrence of graft rejection after administration, but 2 MSCtreated human patients developed antibodies specific to the donor HLA in the clinical trial, one of these cases occurred transiently, whereas the other presented at baseline and persisted throughout the observation period without the appearance of adverse events. Strangely, however, antibodies specific to the donor HLA were also found in one placebo-treated patient. Six animal experiments specified the deaths or dropouts. Lang and Dai [27] reported the deaths of 6 model rats during the construction of the diabetes model (21.4%, 6/28), and Wang et al. [21] reported 1 death each in the MSC-treated group (8.3%, 1/14) and the DN group (10%, 1/10) as well as 2 deaths because of anesthesia. In the study of Li et al. [32], no rat died in the DN group (0.0%, 0/14), and 2 died in the MSC-treated group (18.2%, 2/11). During a 12-week observation, the MSC-treated group (25%, 3/ 12) had lower mortality than the DN-treated group (66.7%, 8/12) [33]. Similarly, Xian et al. [34] found 2 deaths in the hUCB-MSC group (16.7%, 2/12), making for a markedly lower mortality rate than the T1DM group (40%, 6/15) at the end of the study. An et al. [39] found no marked change in the immune system of rhesus macaque DN models in response to hUCB-MSC treatment.

Quality assessment
Quality assessments of animal experiments and clinical trials were performed (Tables 3 and 4). Table 3 shows a number of "unclear" judgments in the quality assessment of animal experiments; in particular, outcome assessment in a random order, concealment of allocation and blinding of outcome assessors in all included experiments were rated "unclear," largely due to a lack of awareness of randomization and blinding methods in animal experiments. As shown in Table 4, a total score of 7 suggested the high methodological quality of the included clinical trial.  (Fig. 1).

Assessment of creatinine clearance rate
The data of six studies were pooled to evaluate CCr at 2 months after MSC treatment; CCr was significantly decreased in the MSC-treated group compared to the DKD group (2 months: SMD = − 1.881, 95%CI − 2.842 to − 0.921, p < 0.001; I 2 = 79.7%) (Fig. 4).

Assessment of urine protein
The measurement of urine protein varied in the included studies. Microalbuminuria, urinary albumin excretion, the urinary albumin/urinary creatinine ratio, and the urinary protein/creatinine ratio were used to assess urine protein excretion in the DKD animals.
Urinary albumin excretion levels at 1 month (2 studies included) and at 2 months (7 studies included) were observed to be lower in the MSC-treated group than in the DKD group, although no significance at 1 month was observed (1 month: SMD = − 6.507, 95%CI − 17.935 to 4.921, p = 0.264; I 2 = 98.3%; 2 months: SMD = − 4.386, 95%CI − 5.891 to − 2.881, p < 0.001; I 2 = 85.5%). The total effect on urinary albumin excretion was also analyzed, suggesting that MSCs decreased urinary albumin All infusions were prepared by an unblinded pharmacist at the phase 1 unit who provided to the blinded clinical staff visually identical infusion products comprising rexlemestrocel-L or saline suspended in 100 mL normal saline.
Patients, investigators, and the sponsor were blinded to the treatment allocation through the entire 60-week study.

Assessment of kidney weight
Kidney weight and the kidney weight/body weight ratio were used to assess kidney hypertrophy. No significant intergroup difference in kidney weight was found between the MSC and untreated DKD groups at 1 month (2 studies included; SMD = − 0.674, 95%CI − 2.052 to 0.704, p = 0.337; I 2 = 67.0%).

Assessment of body weight
There were 3 studies and 5 studies that assessed body weight at the 1-month and 2-month time points, respectively. No significant difference in 1-month body weight was found between the two groups (SMD = 2.634, 95%CI − 0.730 to 5.999, p = 0.125; I 2 = 95.5%). At 2 months, the body weight of the MSC-treated groups significantly increased compared to that of the DKD groups (SMD = 0.903, 95%CI 0.346 to 1.459, p = 0.001; I 2 = 40.2%). An overall effect of MSC treatment on body weight was also found (SMD = 1.499, 95%CI 0.461 to 2.536, p = 0.005; I 2 = 87.3%).

Assessment of renal fibrosis
Four included studies evaluated the percentage of glomerulosclerosis at 2 months after MSC treatment, and no significant difference was found (SMD = − 0.350, 95%CI − 4.173 to 3.473, p = 0.858; I 2 = 96.2%).
Transforming growth factor-β (TGF-β) was measured at different time points using different methods. According to polymerase chain reaction (PCR) assays at 1 month (2 studies included) and 2 months (3 studies included) as well as western blot (WB) assays at 2 months E-cadherin was quantified by WB at 1 month (2 studies included) after MSC treatment; the treatment was associated with a significant and notable decrease in Ecadherin deposition (SMD = 3.600, 95%CI 2.338 to 4.861, p < 0.001; I 2 = 0.0%).

Risk of bias
Given sufficient data to assess publication bias, 2-month blood glucose was used for measurement. There was some degree of bias, indicated by a moderate asymmetry of the funnel plot, and Egger's test showed p = 0.013. However, the trim-and-fill method did not identify any missing studies (Fig. 5).

Discussion
Meta-analysis of medication in clinical trials is essential for clinical decisions in evidence-based medicine. Before medications are put into clinical use, preclinical experiments to explore their efficacy and safety must be performed, and these studies can be costly. In addition, in the absence of compelling evidence, testing directly on humans is both highly risky and unethical. A metaanalysis based on animals may provide a good reference to predict the outcomes of clinical trials. To evaluate the therapeutic effects of MSCs on DKD and review the mechanisms involved, we carried out this study. In this study, we performed a literature search with no species restrictions, yielding 32 animal studies in 28 publications and 1 clinical trial; on this basis, we conducted a metaanalysis of the animal studies and a systematic review.
The concept of DKD was proposed to replace DN in the Kidney Disease Outcomes Quality Initiative (K/ DOQI) by the National Kidney Foundation (NKF) in 2007 and has been used to specify renal lesions caused by DM. DN is characterized by proteinuria ≥ 300 mg/day in a diabetic patient, with or without diabetic retinopathy and hypertension. However, with a new pathological classification of diabetic kidney lesions involving lesions of the tubules, interstitium, and/or vessels as determined by renal biopsy, the concept of DN has shifted to DKD in recent years focusing on clinical diagnosis. Because of the conceptual update, the precise distinction between DN and DKD was considered outside the scope of this study to avoid confusion, and both clinical entities were included. In this paper, we found that MSCs might improve diabetic status, islet function, and glucose levels, as well as provide reno-protection. MSCs appeared to be effective in the treatment of diabetes, mitigating diabetic symptoms such as weight gain and decreased urine output and enhancing pancreatic islet function to improve inclusion secretion and glycemic control. Regarding the therapeutic effect of DKD, reductions in SCr, BUN, CCr, urinary protein, and renal hypertrophy were found in the MSC-treated group. In addition, molecular detection showed that MSCs might reduce the expression of renal fibrosis-related indicators, such as TGF-β, Col-I, FN, α-SMA, and E-cadherin, and the expression of inflammatory mediators such as MCP-1 and TNF-α.
To the best of our knowledge, this study is the first attempt to systemically evaluate MSC administration in DKD without species limitations. El-Badawy and El-Badri [42] conducted a meta-analysis of the therapeutic effects of different sources of stem cells in T1DM and T2DM by evaluating C-peptide, HbA1c, insulin requirements, and adverse effects, showing improved outcomes with stem cell therapy, especially CD34+ hematopoietic stem cell therapy. According to the study, the incidence of adverse effects was 21.72%, and no deaths were reported. To assess and quantify stem cells in animal studies of chronic kidney disease (CKD). Papazova et al. [43] performed a systematic review and meta-analysis and reported notable improvements in plasma creatinine, plasma urea, urinary protein, glomerular filtration rate (GFR), and blood pressure. Wang et al. [44] screened and pooled the data from small animal models of acute kidney injury (AKI) and CKD treated with MSCs and confirmed that impaired renal function was improved.
For glucose at 2 months, the moderate funnel plot asymmetry suggested the presence of bias, and Egger's test showed p = 0.013; however, the trim-and-fill method did not show any missing studies. We detected significant heterogeneity, one of the inevitable drawbacks of animal meta-analyses; its causes may have included the following: different construction methods of animal models, different MSC treatment schemes, and different detection methods.
The therapeutic effects of MSC treatment seemed to be promising in animal studies, but the lone human investigation appeared to tell another story. That trial, a randomized, double-blind, placebo-controlled study of MSCs published in 2016, primarily assessed the safety over a 60-week follow-up and the efficacy over a 12week follow-up. Regarding safety and tolerance, neither adverse events associated with MSCs nor persistent donor-specific anti-HLA antibodies were observed in the trial. However, except for interleukin-6 values and GFR stabilization, no significant difference from placebo was found in any other treatment outcome: urinary protein, CCr, lipid profile, HbA1c, blood pressure, TNF-α, adiponectin, TGF-β, uric acid, and fibroblast growth factor 23. Nevertheless, the results are not convincing, as they come from a single trial with a small sample size (N = 30).
Previous studies have indicated that MSCs can improve some other renal diseases. Chang et al. [45] assessed the effects of MSCs in an anti-Thy1.1-induced rat model of glomerulonephritis and found that intrarenal transplantation of MSCs with hypoxic preconditioning could reduce glomerular apoptosis, autophagy, and inflammation. Barbado et al. [46] conducted a clinical study in patients with lupus nephritis and found that MSC treatment dramatically improved proteinuria levels at the end of the first month, and the ameliorations were sustained throughout the follow-up period. Song et al. [47] conducted a study in rats with nephropathy induced by adriamycin (ADR) and showed that MSCs attenuated ADR-induced nephropathy by inhibiting NF-kB to diminish oxidative stress and inflammation and improve glomerulosclerosis and interstitial fibrosis.
How do MSCs improve renal lesions? MSC therapy has been reported to exert beneficial effects on renal impairment in animal models and patients [48][49][50]. However, the exact mechanisms of nephroprotection of MSCs remain unclear at present. To date, several potential mechanisms have been proposed. Immunoregulation is one important aspect, encompassing antiinflammatory, antiapoptotic, and antioxidant action [51,52]. In addition, one cannot ignore the inhibition of extracellular matrix accumulation, which may be achieved by promoting the secretion of antifibrotic factors and reducing the expression of renal fibrosis-related indicators [27,53]. Protection of renal cells such as podocytes [51] and renal tubular epithelial cells [52] also deserves a place on the list. Proangiogenic potential is one of the functional characteristics of MSCs and may play a part in kidney repair [54]. Furthermore, attention should be paid to the homing of exogenously administered MSCs to specific parts or organs owing to the number of cells that come into play [15], and with the dedifferentiation of tubular cells into stem-like cells, there is a possibility of organ regeneration in AKI with MSC therapy [52].
In this study, a sensitivity analysis was performed, and we found that the results for sensitivity analysis were similar to those of non-sensitivity analyses. It might indicate that the results might be robust to some extent.

Limitations
Only one clinical trial was included in this study, meaning that human data were seriously lacking. As for animal experiments, notable heterogeneity and bias left the conclusions uncertain. Because of the limited longevity of animals, the included animal experiments generally had short observation periods. Heterogeneity was observed in this meta-analysis due to the factors such as the experimental models of DM (e.g., animal species, method used to induce diabetes, and type of diabetes) and MSC treatment (e.g., source, dosage, frequency and route of administration, and timing of administration in relation to the onset of diabetic kidney disease). Sensitivity analysis should be performed by omitting each individual study. Concomitant effects of glycemia confound interpretations about direct therapeutic effects on renal injury. MSC-related adverse events were also limited. Overall poor quality of the experimental studies incorporated in the meta-analysis was found, and further attention should be paid to the design methodology as well as animal experiments with higher quality and larger samples in the future. If preclinical experiments yield sufficient evidence of efficacy and safety, it is expected that more human investigations will be conducted in the future.

Conclusions
In animal models of DKD, MSCs might improve body weight, glycemic control, and pancreatic islet function to secrete insulin and reduce the SCr, BUN, CCr, urinary protein, and renal hypertrophy. MSCs can reduce the expression of inflammatory mediators and alleviate renal fibrosis. MSC therapy might be a potential treatment for DKD.