Zhu, Xiang

Abstract Renal artery stenosis (RAS) is the leading cause of secondary hypertension worldwide. However, current medical and surgical treatment modalities provide minimal benefits for kidney injury. Recent preclinical RAS models have demonstrated promising potential of human mesenchymal stem cells (MSC) and their daughter extracellular vesicles (EV) in improving murine renal function and attenuating inflammation. However, the extent and mechanisms underlying immune rejection of xenogeneic MSCs or EVs are yet undetermined. Therefore, adipose tissue was harvested from adult healthy patients. Adipose-derived MSCs were extracted and cultured, and EVs were isolated from their supernatants via ultra-centrifugation. Then, mice randomly assigned to RAS or sham surgery were divided into 6 groups: sham surgery, RAS, sham + MSC, RAS + MSC, sham + EV, and RAS + EV. Two weeks after intra-aortic injection of MSCs (5 × 105) or EVs (20 µg protein), we compared the intrarenal T-cell and macrophage accumulation, splenic B-cell numbers, circulating cytokines and anti-human antibodies levels among the groups. MSCs and EVs did not influence intrarenal immune cell infiltrations. However, MSCs significantly increased circulating anti-human antibodies. In the spleen, RAS + EV mice showed higher memory IgM+ B-cells but reduced CD19+ B-cells compared to RAS + MSC. In vitro T-cell recall assay showed that both MSCs and EVs exhibited reduced IFN-γ release upon re-stimulation, indicating an immunosuppressive effect. Therefore, xenogeneic MSCs induced a greater humoral response in mice, while EVs triggered a splenic cellular response, but neither elicits discernible kidney rejection. Our results provide key insights into the immunomodulatory mechanisms of MSCs and EVs and immune mechanisms underlying xenograft rejection.

Mesenchymal stromal cells (MSCs) possess therapeutic properties, which can be blunted by obesity. Autophagy, a cellular recycling process, is essential for MSC function. We investigated the mechanisms by which obesity affects the properties of MSCs, with a focus on autophagy. Adipose tissue was obtained from kidney donors [body mass index (BMI) <30 kg/m 2 , non-obese] or individuals undergoing weight loss surgery (BMI ≥30 kg/m 2 , obese) for MSC harvesting ( n = 11 each); samples were randomized to sequencing (seq; n = 5 each) or functional studies ( n = 6 each). MSCs were sequenced to determine their epigenetic (5-hydroxymethylcytosine) and transcriptomic profiles across autophagy-related genes using hydroxymethylated DNA immunoprecipitation sequencing and mRNA-seq, respectively. Genes with shared trends in both datasets underwent Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) validation. During functional studies, 2-h starvation was used to induce autophagy in vitro , enabling detection of changes in the protein expression of microtubule-associated protein 1A/1B-light chain-3 and in autophagic flux. Obesity amplified a starvation-induced reduction in autophagic flux in MSCs while promoting earlier generation of new autophagosomes during autophagy initiation. Integrated analysis of the two sequencing datasets revealed 124 differentially hydroxymethylated genes and 30 differentially expressed mRNAs. Among six overlapping autophagy-related genes, three exhibited same-direction trends. Of these, STX12 and SLC25A4 may be implicated in the impact of obesity on autophagic changes in MSCs. Therefore, human obesity may alter autophagy in adipose tissue–derived MSC, and thereby their metabolism and function.