Transplantation of Human Amniotic Mesenchymal Stem Cells Up-Regulates Angiogenic Factor Expression to Attenuate Diabetic Kidney Disease in Rats

Figures & data

Figure 1 Morphological, phenotypic characteristics, and various inducing differentiation capabilities of hAMSCs. (A) hAMSCs were fusiform, polygonal, and star-shaped. Immunocytochemical staining revealed that hAMSCs expressed vimentin, a mesenchymal cell marker. (B) The staining results; 21 days after differentiation of hAMSCs into osteoblasts, chondroblasts, and adipocytes.

Notes: (a): osteocytes on Day 21 after Alizarin Red staining (100×); (b): chondrocytes on Day 21 after Toluidine Blue staining (100×); (c): lipocytes on Day 21 after Oil Red O staining (200×). (C) The FCM analysis has shown that third-generation hAMSCs highly expressed CD44, CD73, CD90, and CD105 but did not express CD34, CD11b, CD19, CD45, and HLA-DR.

Figure 2 Image of hAMSCs homing in kidney tissue (×40 Green: MAB1281, blue: nucleus).

Table 1 Comparison of Blood Glucose and 24-h Urine Protein Levels Among Three Rat Groups

Group	Blood Glucose (mmol/L)			24-Hour Urine Protein (mg/24 h)
	6W	8W	12W	6W	8W	12W
NC	7.66 ± 0.23	7.39 ± 0.39	10.56 ± 0.23	10.14 ± 3.24	5.24 ± 0.61	6.20 ± 1.43
DKD	47.89 ± 4.46^Δ	43.39 ± 3.45^Δ	35.79 ± 0.90^Δ	67.60 ± 17.90^Δ	32.69 ± 6.32^Δ	59.73 ± 7.96^Δ
MSC	42.75 ± 5.96^Δ	38.81 ± 3.70^Δ	28.64 ± 0.49^Δ○	53.85 ± 14.60^Δ	28.47 ± 4.00^Δ	33.23 ± 2.48^Δ○

Note: (± s, n = 6); Compared to NC group, ^ΔP <0.05; Compared to DKD group, ^○P <0.05.

Table 2 Comparison of SCr and Urea Levels Among Three Rat Groups

Group	SCr (μmol/L)			Urea (mmol/L)
	6W	8W	12W	6W	8W	12W
NC	24.00 ± 2.00	19.33 ± 5.50	23.67 ± 3.79	8.00 ± 1.30	6.26 ± 0.24	6.50 ± 0.97
DKD	31.20 ± 3.96^Δ	21.75 ± 2.06	31.75 ± 4.35	14.47 ± 4.91^Δ	13.84 ± 2.85^Δ	37.29 ± 3.79^Δ
MSC	28.60 ± 5.32	28.50 ± 4.43^Δ	25.75 ± 6.45	9.62 ± 1.05^○	11.13 ± 1.98^Δ	12.11 ± 1.07^Δ○

Note: ( ± s, n = 6); Compared to NC group, ^ΔP <0.05; Compared to DKD group, ^○P <0.05.

Table 3 Comparison of Serum Cys-c and Urine KIM-1 Levels Among Three Rat Groups

Group	Cys-c (mg/L)			KIM-1 (ng/L)
	6W	8W	12W	6W	8W	12W
NC	1.06 ± 0.49	1.84 ± 0.96	1.25 ± 0.18	71.03 ± 2.35	76.96 ± 4.64	98.07 ± 1.73
DKD	2.54 ± 0.45^Δ	1.97 ± 0.52	1.96 ± 0.41^Δ	97.49 ± 5.72^Δ	114.79 ± 7.16^Δ	107.24 ± 6.59
MSC	1.87 ± 0.60^Δ	2.18 ± 0.42	1.46 ± 0.47	85.46 ± 6.63^Δ○	86.02 ± 8.72^○	122.98 ± 6.80^Δ○

Note: ( ± s, n = 6); Compared to NC group, ^ΔP <0.05; Compared to DKD group, ^○P <0.05.

Figure 3 Timetable and flowchart of rat treatment, as well as changes in blood and urine biochemical indicators after hAMSCs transplantation. (A) The flowchart of rat treatment from Day 0 to Week 12. (B–G) The histogram of blood glucose and 24-hour urine protein levels, and urea levels, Cys-c, and urine KIM-1 levels among three rat groups.

Note: Compared to NC group, ^ΔP<0.05; Compared to DKD group, ^○P<0.05.

Table 4 Comparison of Kidney/Body Weight Among Three Rat Groups

Group	6W	8W	12W
NC	3.32 ± 0.45	3.60 ± 0.44	2.88 ± 0.30
DKD	5.14 ± 0.19^Δ	5.16 ± 0.78^Δ	6.88 ± 0.72^Δ
MSC	4.67 ± 0.28^Δ○	4.85 ± 0.16^Δ	5.92 ± 0.37^Δ○

Note: (mg/g, ± s, n = 6); Compared to NC group, ^ΔP <0.05; Compared to DKD group, ^○P <0.05.

Figure 4 The effect of hAMSCs transplantation intervention on STZ-induced kidney pathological changes in rats. (A) Kidney/body weight: the DKD and MSC groups had a higher kidney/body weight ratio than the NC group at each time point (P <0.05). The MSC group had a lower kidney/body weight ratio than the DKD group. However, statistical significance was observed at 6 and 12 weeks (P <0.05). (B) Renal tissue stained with H&E among three rat groups (×400): light blue arrow: inflammatory cell infiltration; dark blue arrow: renal tubule hypertrophy. (C) Renal tissue stained with PAS among three rat groups (×400): black arrow: increased mesangial matrix; red arrow: thickened tubular basement membrane.

Note: Compared to group NC, ^ΔP<0.05; Compared to group DKD, ^○P<0.05.

Figure 5 hAMSCs transplantation improved mRNA expressions of angiogenic factors in rat kidneys. TSP-1, VEGF, Tie-2, Ang-1, and Ang-2 mRNA expression levels in the NC, DKD, and MSC groups at each time point.

Note: Compared to group NC, ^ΔP<0.05; Compared to group DKD, ^○P<0.05.

Figure 6 hAMSCs transplantation improved the protein expressions of angiogenic factors in rat kidneys. (A–C) The TSP-1, VEGF, Tie-2, and Ang-1 protein expression levels in the NC, DKD, and MSC groups at each time point.

Note: Compared to group NC, ^ΔP<0.05; Compared to group DKD, ^○P<0.05.

Figure 7 hAMSCs transplantation improved protein expressions of VEGF, Ang-1, and TSP-1 in T1DRM at 8 weeks (as determined by WB analysis) (A–D). Note: Compared with the NC group, ^ΔP < 0.05; Compared to group DKD, *P < 0.01.

Figure 8 Correlation analysis results of various indicators. (A) Linear chart of correlation analysis between 24-hour urine protein and TSP-1, VEGF, Tie-2, and Ang-1. (B) Linear chart of correlation analysis between KIM-1 and 24-hour urine protein, TSP-1, Urea, and Tie-2. (C and D) Linear correlation analysis between TSP-1, Tie-2, and VEGF. (E and F) Linear correlation analysis between Ang-1, TSP-1, and VEGF. (G and H) Linear correlation analysis between Tie-2, Ang-1, and VEGF.