Repeated administration of adipose-derived mesenchymal stem cells added on beneficial effects of empagliflozin on protecting renal function in diabetic kidney disease rat

Abstract

Background: Diabetic kidney disease (DKD) is one of the most significant public health burdens worldwide. This study explored the renal protections of combined adipose-derived mesenchymal stem cells (ADMSCs) and empagliflozin (EMPA) in DKD rats.

Methods: Adult-male-SD rats were equally allocated into group 1 (sham-operated-control), group 2 (DKD), group 3 (DKD + EMPA/20 mg/kg/day since day-14 after CKD-induction), group 4 [DKD + ADMSCs (6.0 × 10⁵/intrarenal-arterial-injection/post-day-28, followed by 1.2 × 10⁶/intravenous injection post-days 35 and 42 after CKD-induction, i.e., defined as repeated administration)] and group 5 (DKD + ADMSCs + EMPA) and kidney was harvested post-day-60 CKD-induction.

Results: The result showed that the blood sugar and circulatory levels of BUN/creatinine and the ratio of urine protein/creatinine at day 60 were greatly increased in group 2 as compared the SC (i.e., group 1), significantly increased in groups 3 and 4 than in groups 5, but these parameters showed the similar manner in groups 3 and 4, except for blood sugar that was significantly lower in group 3 than in group 4 (all p < 0.0001). The protein levels of inflammation (NF-κB/FNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized protein/p22-phox)/apoptosis (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax)/fibrosis (TGF-β/Smad 3)/mitochondrial/DNA-damaged (p-DRP1/γ-H2AX) biomarkers revealed a similar manner of creatinine level among the groups (all p < 0.0001). Kidney injury score/fibrotic area/oxidative-stress score (8-OHdG) and cellular levels of kidney-damaged biomarkers (KIM-1/γ-H2AX) showed a unanimous manner. In contrast, the cellular expressions of podocyte components (ZO-1/synaptopodin) revealed an antithetical manner of creatinine among the groups (all p < 0.0001).

Conclusion: Combined ADMSCs-EMPA was superior to just one therapy for protecting kidney function and ultra-structural integrity in DKD rodents.

Keywords: Diabetic kidney disease; Inflammation; Oxidative stress; Renal function.

Fig. 1

Time courses of the circulating levels of blood sugar, blood urine nitrogen, and creatinine, and the ratio of urine protein to urine creatinine (A) Circulating blood sugar level at day 0, p > 0.5. (B) Circulating level of blood sugar at day 7 after streptozotocin treatment, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Circulating level of blood sugar at day 60 after CKD induction, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Circulatory level of BUN at day 0, p > 0.5. (E) Circulatory level of BUN at day 14 after CKD induction, ∗ vs. †, p < 0.0001. (F) Circulatory level of BUN at day 28 after CKD induction, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (G) Circulatory level of BUN at day 60 after CKD induction, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (H) Circulatory level of creatinine at day 0, p > 0.5. (I) Circulatory level of creatinine at day 14 after CKD induction, ∗ vs. †, p < 0.0001. (J) Circulatory level of creatinine at day 28 after CKD induction, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (K) Circulatory level of creatinine ay day 60 after CKD induction, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (L) Ratio of urine protein to urine creatinine (R^UPr/UCr) at day 0, p > 0.5. (M) R^UPr/UCr at day 14 after CKD induction, ∗ vs. †, p < 0.0001. (N) R^UPr/UCr at day 28 after CKD induction, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (O) R^UPr/UCr at day 60 after CKD induction, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 7 to 11 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; BUN : blood urea nitrogen; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 2

Serial changes of renal artery resistive index (RARI) (A) The RARI at day 0, p > 0.5. (B) The RARI at day 14 after CKD induction, ∗ vs. †, p < 0.0001. (C) The RARI at day 28 after CKD induction, ∗ vs. other groups with different symbols (†, ‡), p < 0.0001. (D) The RARI at day 60 after CKD induction, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 7 to 11 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 3

Impact of ADMSCs-EMPA therapy on attenuating the kidney injury score by day 60 after CKD induction (A1 to E1) Light microscopic findings (200x; H&E stain) illustrating the remarkably increased loss of brush border in renal tubules (yellow arrows), tubular necrosis (green arrows), tubular dilatation (red asterisk), protein cast formation (black asterisk), and dilatation of Bowman's capsule (blue arrows) in DKD group than in other groups. All scale bars in the lower right corner represent 50 μm. (A2 to E2) Showing the magnified blue square box for observing the more clear kidney ultrastructure. (F) Analytical result of kidney injury score, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; BUN : blood urea nitrogen; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC: adipose-derived mesenchymal stem cell.

Fig. 4

Impact of ADMSCs-EMPA therapy on alleviating fibrosis in renal tissue by day 60 after CKD induction (A1 to E1) Demonstrating the microscopic finding of Masson's trichrome stain (200x) for verification of fibrosis in kidney parenchyma (blue color). All scale bars in the lower right corner represent 50 μm. (A2 to E2) Exhibiting the magnified blue square box for inspecting kidney ultrastructure more clearly. (F) Analytical result of fibrotic area, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 5

Impact of ADMSCs-EMPA therapy on ameliorating oxidative stress in renal tissue by day 60 after CKD induction (A1 to E1) Displaying the microscopic finding (200x) of 8-OHdG stain for recognizing the intensity of oxidative stress expression. All scale bars in the lower right corner represent 50 μm. (A2 to E2) Exhibiting the magnified blue square box for inspecting more clearly oxidative-stress expression in renal tubules. (F) Analytical result of expression of the 8-OHdG score, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 6

Impact of ADMSCs-EMPA therapy on preserving the podocyte components in kidney parenchyma by day 60 after CKD induction (A to E) Demonstrating the immunofluorescent (IF) microscopic finding (200x) for identification of the expression of ZO-1 (green color). (F) Analytical result of the expression of ZO-1, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (G to K) Demonstrating the IF microscopic finding (200x) for identification of the expression of synaptopodin (green color). (L) Analytical result of the expression of synaptopodin, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All scale bars in the lower right corner represent 50 μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 7

Impact of ADMSCs-EMPA therapy on attenuating the protein levels of apoptosis, fibrosis, damaged and autophagic biomarkers in renal tissue by day 60 after CKD induction (A) Protein level of cleaved caspase 3 (c-Casp 3), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (B) Protein level of cleaved Poly (ADP-ribose) polymerase (PARP), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (C) Protein level of mitochondrial Bax (mit-Bax), ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (D) Protein expression of transforming growth factor (TGF-β), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (E) Protein level of phosphorylated (p)-Smad 3, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (F) Protein level of p-Smad 1/5, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (G) Protein level of bone morphogenetic protein 2 (BMP-2), ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (H) Protein levels of p22 phox. (I) Protein level of phosphorylation of dynamin-related protein 1 (p-DRP1), ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (J) Protein level of γ-H2AX, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (K) Protein level of Atg 5, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §, ¶) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.

Fig. 8

Impact of ADMSCs-EMPA therapy on downregulating the protein levels of oxidative stress and inflammation in renal tissue by day 60 after CKD induction (A) Protein level of NOX-1, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (B) Protein level of NOX-2, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (C) The oxidized protein level, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. Left and right lanes expressed on the upper panel indicated the molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1–3 dinitrophenylhydrazone. (D) Protein level of phosphorylated nuclear factor (p–NF)–κB, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (E) Protein level of tumor necrosis factor (TNF)-α, ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. (F) Protein level of matrix metalloproteinase (MMP-9), ∗ vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §, ¶) indicate significance (at 0.05 level). Abbreviations: CKD : chronic kidney disease; SC : sham-operated control; DKD : diabetic kidney disease; EMPA : empagliflozin; ADMSC : adipose-derived mesenchymal stem cell.