Kallikrein-modified mesenchymal stem cell implantation provides enhanced protection against acute ischemic kidney injury by inhibiting apoptosis and inflammation

Abstract

Mesenchymal stem cells (MSCs) migrate to sites of tissue injury and serve as an ideal vehicle for cellular gene transfer. As tissue kallikrein has pleiotropic effects in protection against oxidative organ damage, we investigated the potential of kallikrein-modified MSCs (TK-MSCs) in healing injured kidney after acute ischemia/reperfusion (I/R). TK-MSCs secreted recombinant human kallikrein with elevated vascular endothelial growth factor levels in culture medium, and were more resistant to oxidative stress-induced apoptosis than control MSCs. Expression of human kallikrein was identified in rat glomeruli after I/R injury and systemic TK-MSC injection. Engrafted TK-MSCs exhibited advanced protection against renal injury by reducing blood urea nitrogen, serum creatinine levels, and tubular injury. Six hours after I/R, TK-MSC implantation significantly reduced renal cell apoptosis in association with decreased inducible nitric oxide synthase expression and nitric oxide levels. Forty-eight hours after I/R, TK-MSCs inhibited interstitial neutrophil and monocyte/macrophage infiltration and decreased myeloperoxidase activity, superoxide formation, p38 mitogen-activated protein kinase phosphorylation, and expression of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1. In addition, tissue kallikrein and kinin significantly inhibited H2O2-induced apoptosis and increased Akt phosphorylation and cell viability in cultured proximal tubular cells. These results indicate that implantation of kallikrein-modified MSCs in the kidney provides advanced benefits in protection against ischemia-induced kidney injury by suppression of apoptosis and inflammation.

FIG. 1.

Immunocytochemical characterization of MSCs and ROS-induced apoptosis. (A) TK-MSCs were identified by positive immunostaining to human tissue kallikrein, α-SMA, and vimentin. A transduction efficiency of 70–80% was confirmed by GFP expression. (B) Expression of human tissue kallikrein in TK-MSC culture medium was determined by ELISA (n = 6–8). (C) Expression of VEGF in TK-MSCs culture medium 4 days after Ad.CMV-TK transduction (n = 4, *p < 0.05 vs. GFP-MSCs). (D) Representative Hoechst staining images of MSCs treated with H₂O₂ for 6 hr. (E) Quantitative analysis of apoptotic MSCs expressed as a percentage of total cell number. (F) Caspase-3 activity in cell lysates of MSCs treated with H₂O₂ for 6 hr. Caspase-3 activity is expressed as a percentage of control MSCs (n = 6, *p < 0.05 vs. MSCs and GFP-MSCs). Values are expressed as means ± SEM.

FIG. 2.

Expression of human tissue kallikrein in the kidney and the effect of TK-MSCs on renal I/R injury. (A) Representative photomicrographs of immunohistochemical staining of human tissue kallikrein in kidney 6 and 48 hr after TK-MSC implantation. Original magnification, ×400. (B and C) BUN and creatinine levels 6 and 48 hr after I/R. Values are expressed as means ± SEM, n = 6–8 (*p < 0.05 vs. I/R; ^#p < 0.05 vs. I/R + GFP-MSCs).

FIG. 3.

TK-MSCs ameliorate tubular injury 48 hr after I/R. (A) Representative images of histological staining with PAS in kidney cortex and medulla. Original magnification, ×200. (B) Semiquantitative analysis of tubular damage. Values are expressed as means ± SEM, n = 6–8 (*p < 0.05 vs. I/R; ^#p < 0.05 vs. I/R + GFP-MSCs).

FIG. 4.

TK-MSCs reduce renal cell apoptosis, NO, and iNOS mRNA expression 6 hr after I/R. (A) Representative images of TUNEL staining. Propidium iodide stains red and TUNEL stains green; yellow is overlay. Original magnification, ×200. (B) Apoptotic tubular cells in the outer stripe of the medulla. (C) Nitrate/nitrite (NOx) content and (D) mRNA levels of iNOS in kidney tissue. Values are expressed as means ± SEM, n = 6–8 (*p < 0.05 vs. I/R; ^#p < 0.05 vs. I/R + GFP-MSCs).

FIG. 5.

TK-MSCs reduce monocyte/macrophage and neutrophil accumulation in the renal interstitium 48 hr after I/R. (A) Representative images of immunohistochemical staining for ED-1, a specific marker for monocytes/macrophages. Original magnification, ×200. Quantitative analysis of (B) ED-1-positive cells and (C) neutrophils. Values are expressed as means ± SEM, n = 6–8 (*p < 0.05 vs. I/R; ^#p < 0.05 vs. I/R + GFP-MSCs).

FIG. 6.

TK-MSCs reduce oxidative stress, p38^MAPK phosphorylation, and inflammatory mediator expression 48 hr after I/R. (A) MPO activity and (B) superoxide formation in kidney tissue. (C) Representative Western blot of phosphorylated and total p38^MAPK (top) and densitometric analysis (bottom). Relative mRNA levels of (D) TNF-α, (E) MCP-1, and (F) ICAM-1 determined by real-time PCR. Values are expressed as means ± SEM, n = 6–8 (*p < 0.05 vs. I/R; ^#p < 0.05 vs. I/R + GFP-MSCs).

FIG. 7.

Tissue kallikrein (TK) and bradykinin (BK) inhibit cell injury induced by H₂O₂. (A) Representative Hoechst staining of proximal tubular cells treated with H₂O₂ for 24 hr. (B) Quantitative analysis of apoptotic MSCs expressed as a percentage of total cell number. (C) Cell viability of IRPTCs assessed by MTS assay after incubation with H₂O₂ for 24 hr. (D) Representative Western blot of phosphorylated and total Akt (top) and densitometric analysis (bottom). Values are expressed as means ± SEM, n = 6 (*p < 0.05 vs. H₂O₂).