To investigate the effects of exosomes (Exo) derived from human induced pluripotent stem cells (hiPSCs) on renal ischemia-reperfusion injury (IRI).

HiPSCs were cultured, and Exo were isolated from the supernatant by ultracentrifugation. The isolated Exo were characterized by detecting the specific markers CD9, CD63, CD81 and the negative marker Calnexin via Western blot (WB), observing their morphology by transmission electron microscopy, and analyzing particle size via nanoparticle tracking analysis. SD rats were randomly assigned to four groups (n=6 per group): sham-operated group, IRI group, IhiPSCs group, and IhiPSCs-Exo group. At 48 hours post-intervention, serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured, and renal pathological sections were stained with hematoxylin and eosin for evaluation using a modified Paller tubular injury score. The mRNA expression levels of IL-1β, TNF-α, SOD-2, and Nrf-2 were determined by quantitative real-time polymerase chain reaction. The protein expression of CD163, CD68, α-SMA, COL-1, and FN-1 was assessed by immunohistochemistry. WB was performed to determine the protein expression levels of p-MEK1/2, p-ERK1/2, Caspase-3, Bcl-2, and Bax. Using human renal tubular epithelial cells (HK-2 cells), a hypoxia/reoxygenation (H/R) model was established, including H/R group and H/R + hiPSCs-Exo group. Cell proliferation was measured by CCK-8 assay, apoptosis was detected by Annexin V-FITC/PI staining, and the expression of Caspase-3, Bcl-2, and Bax was analyzed by WB. One-way analysis of variance was used for comparisons between groups, and further pairwise comparisons were conducted using the Tukey HSD test, with a P value <0.05 considered statistically significant.

The isolated Exo exhibited a typical cup-shaped morphology with a double-membrane structure, an average particle size of (88.0±0.6) nm, and expressed the specific marker proteins CD9, CD63, and CD81. In vivo experiments showed that compared with the IRI group, the IhiPSCs group and IhiPSCs-Exo group exhibited significantly lower levels of Scr, BUN, and modified Paller tubular injury scores (all P<0.05). Furthermore, these groups showed decreased mRNA levels of IL-1β and TNF-α, increased mRNA levels of SOD-2 and Nrf-2, reduced protein expression of CD68, α-SMA, COL-1, and FN-1 by immunohistochemistry, and elevated protein expression of CD163 (all P<0.05). WB analysis revealed that compared with the IRI group, the relative protein expression levels of p-MEK1/2, p-ERK1/2, Caspase-3, and Bax were decreased, while the expression of Bcl-2 was increased in the IhiPSCs group and IhiPSCs-Exo group (all P<0.05). In vitro experiments demonstrated that compared with the H/R group, the hiPSCs-Exo group showed enhanced HK-2 cell proliferation, reduced numbers of apoptotic and necrotic cells, decreased protein expression of Caspase-3 and Bax, and increased expression of Bcl-2.

Exo derived from hiPSCs exert a protective effect against renal IRI through multiple pathways, including the suppression of oxidative stress, inflammatory factor expression, and apoptosis. This protective effect is associated with the inhibition of the MEK/ERK signaling pathway.