In total, 33 studies in 29 publications were included, among which 28 publications were based on animal studies [13-40] and 1 was based on a clinical trial . 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 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 1A.
The only clinical trial was a multicenter, randomized, double-blind, dose-escalating, sequential, placebo-controlled study, finished in 2016. Two doses of allogeneic mesenchymal precursor cells were separately infused into 10 patients with type 2 diabetes and advanced DN, and the efficacy and adverse events were observed. The main features of the clinical trial are shown in Table 1B.
None of the animal experiments reported the occurrence of graft-rejection after administration, but 2 MSC-treated 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 et al.  reported the deaths of 6 model rats during the construction of the diabetes model (21.4%, 6/28), and Wang et al.  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. , 1 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) . Similarly, Xian et al.  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.  found no marked change in the immune system of rhesus macaques DN models in response to hUCB-MSC treatment.
Quality assessments of animal experiments and clinical trials were performed (Table 2A and Table 2B). Table 2A 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 2B, a total score of 7 suggested the high methodological quality of the included clinical trial.
Assessment of glucose
Glucose was detected after MSC treatment in all but 2 studies [32, 37]. Sixteen studies measured glucose once at the end of the experiment [18-21, 23-29, 31, 33-36]. Seven studies conducted blood glucose monitoring at several points in time [14-17, 22, 30, 40]. Five studies, 7 studies, 5 studies, 12 studies, 17 studies, 7 studies and 2 studies were included to assess the effect of treatment on blood glucose levels at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months and 6 months, respectively, all of which showed a highly significant hypoglycemic effect in the MSC-treated group (1-week: SMD=-1.484, 95%CI: -2.586 to -0.381, p＜0.001; 2-week: SMD=-2.312, 95%CI: -3.743 to -0.882, p=0.002; 3-week: SMD=-4.007, 95%CI: -6.472 to -1.541, p=0.001; 1-month: SMD=-1.740, 95%CI: -2.660 to -0.821, p＜0.001; 2-month: SMD=-1.830, 95%CI: -2.633 to -1.028, p＜0.001; 3-month: SMD=-1.649, 95%CI: -2.838 to -0.461, p=0.007; 6-month: SMD=-3.045, 95%CI: -5.895 to -0.195, p=0.036). The total hypoglycemic effect was also analyzed (SMD=-1.954, 95%CI: -2.389 to -1.519, p＜0.001). (Figure 1)
Assessment of serum creatinine (SCr)
There were 4 studies, 2 studies and 5 studies that assessed SCr at 1 month, 2 months and 3 months, respectively. All of them showed significantly reduced creatinine values in the MSC-treated group (1-month: SMD=-4.126, 95%CI: -7.936 to -0.315, p=0.034; 2-month: SMD=-3.506, 95%CI: -4.735 to -2.278, p＜0.001; 3-month: SMD=-6.736, 95%CI: -10.311 to -3.162, p＜0.001). The total effect on SCr was also analyzed, suggesting that MSCs decreased SCr and improved renal function (SMD= -4.838, 95%CI: -6.789 to -2.887, p＜0.001). (Figure 2)
Assessment of blood urea nitrogen (BUN)
BUN was evaluated at 5 different time points, each of which was used by relatively few studies. At 2 weeks (2 studies included), 3 weeks (2 studies included), 1 month (2 studies included), 2 months (3 studies included), and 3 months (4 studies included), BUN decreased in the MSC-treated group, although no statistical significance was seen at 3 weeks or 1 month (2-week: SMD=-2.463, 95%CI: -3.292 to -1.634, p＜0.001; 3-week: SMD=-4.432, 95%CI: -9.220 to -0.356, p=0.070; 1-month: SMD=-10.392, 95%CI: -21.247 to 0.464, p=0.061; 2-month: SMD=-3.389, 95%CI: -6.679 to -0.099, p=0.044; 3-month: SMD=-5.902, 95%CI: -8.988 to -2.815, p＜0.001). The total effect on BUN was also analyzed, suggesting that MSCs decreased BUN (SMD= -4.912, 95%CI: -6.402 to -3.422, p＜0.001). (Figure 3)
Assessment of creatinine clearance rate (CCr)
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-month: SMD=-1.881, 95%CI: -2.842 to -0.921, p＜0.001). (Figure 4)
Assessment of blood insulin level
Two studies assessed insulinemia. The insulin level increased at 3 months after MSC treatment, although the significance was not notable (3-month: SMD=3.051, 95%CI: -0.091 to 6.193, p=0.057).
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; 2-month: SMD=-4.386, 95%CI: -5.891 to -2.881, p＜0.001). The total effect on urinary albumin excretion was also analyzed, suggesting that MSCs decreased urinary albumin excretion (SMD= -4.830, 95%CI: -6.602 to -3.058, p＜0.001).
Microalbuminuria was detected at 3 weeks and 3 months; each of these time points was addressed by 2 studies that satisfied the inclusion criteria. Microalbuminuria was found to be decreased in the MSC-treated group at 3 months (3-week: SMD=-9.112, 95%CI: -21.627 to 3.404, p=0.154; 3-month: SMD=-4.431, 95%CI: -5.771 to -3.091, p＜0.001). The total effect on microalbuminuria was analyzed, suggesting that microalbuminuria was significantly lower in the MSC-treated group than in the DKD group (SMD= -5.791, 95%CI: -8.681 to -2.901, p＜0.001).
The urinary albumin/urinary creatinine ratios at 1 month (6 studies included) and at 2 months (10 studies included) were observed to be significantly lower in the MSC-treated group than in the untreated DKD group (1-month: SMD=-2.419, 95%CI: -3.070 to -1.769, p＜0.001; 2-month: SMD=-2.648, 95%CI: -3.454 to -1.842, p＜0.001). The total effect on the urinary albumin/urinary creatinine ratio was analyzed, and the analysis suggested that the urinary albumin/urinary creatinine ratio was significantly lower in the MSC-treated group than in the DKD group (SMD= -2.539, 95%CI: -3.075 to -2.003, p＜0.001).
Urinary protein/creatinine ratios were not significantly different between groups at 2 weeks (2 studies included) after MSC treatment (SMD=-2.779, 95%CI: -7.617 to 2.059, p=0.260).
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).
The kidney weight/body weight ratio was found to be significantly decreased in the MSC-treated group at 2 months (8 studies included, SMD=-1.364, 95%CI: -2.164 to -0.565, p=0.001), while no significant difference was found between the two groups at 3 months (2 studies included, SMD=-10.012, 95%CI: -29.753 to 9.729, p=0.320). The total effect on the kidney weight/body weight ratio was analyzed, and the analysis suggested that a reduced kidney weight/body weight ratio was found in the MSC-treated group (SMD=-1.624, 95%CI: -2.594 to -0.655, p=0.001).
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). At 2 months, the body weight of the MSC-treated groups significantly increased compared to that of the DKD groups (SMD=0.869 95%CI: 0.442 to 1.296, p＜0.001). 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).
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).
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 (2 studies included), TGF-β was significantly decreased in the MSC-treated group (1-month PCR: SMD=-3.258, 95%CI: -4.133 to -2.384, p＜0.001; 2-month PCR: SMD=-7.594, 95%CI: -13.274 to -1.915, p=0.009; 2-month WB: SMD=-9.287, 95%CI: -11.322 to -7.252, p＜0.001). The same was true for total expression of TGF-β (SMD=-6.839, 95%CI: -9.367 to -4.312, p＜0.001).
Collagen I (Col-I) was detected by immunohistochemistry (IHC) and PCR. According to PCR at 2 months (3 studies included), Col-I was significantly decreased (SMD=-11.468, 95%CI: -13.685 to -9.252, p＜0.001) in the MSC-treated group, although no significance intergroup difference was found by IHC at 2 months (2 studies included; SMD=-4.714, 95%CI: -10.670 to 1.242, p=0.121). An overall effect on Col-I expression was also found (SMD= -9.081, 95%CI: -14.233 to -3.929, p=0.001).
Three included studies evaluated fibronectin (FN) by IHC at 2 months after MSC treatment, and a statistically significant decrease was found in the MSC-treated group (SMD=-7.781, 95%CI: -10.680 to -4.881, p＜0.001).
Two studies evaluated α-smooth muscle actin (α-SMA) by WB at 1 month after MSC treatment, and 3 studies quantified its expression by PCR at 2 months. Both measures of α expression were significantly decreased in the MSC-treated group (1-month WB: SMD=-2.514, 95%CI: -3.550 to -1.479, p＜0.001; 2-month PCR: SMD=-2.098, 95%CI: -3.721 to -0.476, p=0.011). An overall effect of MSC treatment on the expression of α-SMA was found (SMD= -2.249, 95%CI: -3.311 to -1.186, p＜0.001).
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 E-cadherin deposition (SMD=3.600, 95%CI: -2.338 to 4.861, p＜0.001).
Assessment of inflammatory mediators
Monocyte chemokine protein-1 (MCP-1) was detected by IHC at 2 months (2 studies included) after MSC treatment, and no significant difference was found between the two groups (SMD=-8.913, 95%CI: -20.994 to 3.167, p=0.148).
Tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA) at 2 weeks (3 studies included) and by PCR at 1 month (2 studies included) after MSC treatment, both of which showed statistically significant decreases in the MSC-treated group (2-week ELISA: SMD=-3.853, 95%CI: -7.207 to -0.499, p=0.024; 1-month PCR: SMD=-3.727, 95%CI: -4.684 to -2.769, p＜0.001). An overall effect of MSC treatment on the expression of TNF-α was found (SMD= -4.027, 95%CI: -5.955 to -2.098, p＜0.001).
Sensitivity analysis was tested by omitting each individual trial at a time, and we found that the results in this sensitivity analysis were similar to the overall results (data not shown).
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 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 (Figure 5).