KIM-1 augments hypoxia-induced tubulointerstitial inflammation through uptake of small extracellular vesicles by tubular epithelial cells.

Tubular epithelial cells (TECs) exposed to hypoxia incite tubulointerstitial inflammation (TII), while the exact mechanism is unclear. In this study, we identified that hypoxia evoked tubule injury as evidenced by tubular hypoxia-inducible factor-1α and kidney injury molecule-1 (KIM-1) expression and that renal small extracellular vesicle (sEV) production was increased with the development of TII after ischemia-reperfusion injury (IRI). Intriguingly, KIM-1-positive tubules were surrounded by macrophages and co-localized with sEVs. In vitro, KIM-1 expression and sEV release were increased in hypoxic TECs and the hypoxia-induced inflammatory response was ameliorated when KIM-1 or Rab27a, a master regulator of sEV secretion, was silenced. Furthermore, KIM-1 was identified to mediate hypoxic TEC-derived sEV (Hypo-sEV) uptake by TECs. Phosphatidylserine (PS), a ligand of KIM-1, was present in Hypo-sEVs as detected by nanoflow cytometry. Correspondingly, the inflammatory response induced by exogenous Hypo-sEVs was attenuated when KIM-1 was knocked down. In vivo, exogenous-applied Hypo-sEVs localized to KIM-1-positive tubules and exacerbated TII in IRI mice. Our study demonstrated that KIM-1 expressed by injured tubules mediated sEV uptake via recognizing PS, which participated in the amplification of tubule inflammation induced by hypoxia, leading to the development of TII in ischemic acute kidney injury.

[New advances in oxygen sensing and adaptive mechanism].

Under the hypoxic condition, organs or cells trigger a series of reactions or responses to adapt to the physiological requirement. These responses involve a complex regulation at different levels from organs, in particular the kidney (producing erythropoietin), to cells throughout the body. Actually, the responses to hypoxia from adaption to injury largely depend on the degree and time of hypoxia. In the past two decades, with the discovery of hypoxia-inducible factor (HIF), the mechanisms of erythropoiesis regulation were elucidated gradually, which has provided a novel therapeutic strategy for hypoxia-related diseases, especially renal anemia. In this review we focus on the latest advances in oxygen sensing and adaptive mechanism.

Identification of stable reference genes for relative quantification of long RNA expression in urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA-seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA-seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1-2, RPL4, GAPDH, RPS27A as candidate reference genes. RT-qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.

Tubular epithelial cells-derived small extracellular vesicle-VEGF-A promotes peritubular capillary repair in ischemic kidney injury.

Peritubular capillaries (PTCs) are closely related to renal tubules in structure and function, and both are pivotal regulators in the development and progression of acute kidney injury (AKI). However, the mechanisms that underlie the interaction between PTCs and tubules during AKI remain unclear. Here we explored a new mode of tubulovascular crosstalk mediated by small extracellular vesicles (sEV) after AKI. In response to renal ischemia/reperfusion (I/R) injury, endothelial proliferation of PTCs and tubular expression of vascular endothelial growth factor-A (VEGF-A) were increased, accompanied by a remarkable redistribution of cytoplasmic VEGF-A to the basolateral side of tubular cells. Meanwhile, the secretion mode of VEGF-A was converted in the injured tubular cells, which showed a much greater tendency to secrete VEGF-A via sEV other than the free form. Interestingly, tubular cell-derived VEGF-A-enriched sEV (sEV-VEGF-A) turned out to promote endothelial proliferation which was regulated by VEGF receptors 1 and 2. Furthermore, inhibition of renal sEV secretion by Rab27a knockdown resulted in a significant decrease in the proliferation of peritubular endothelial cells in vivo. Importantly, taking advantage of the newly recognized endogenous repair response of PTCs, exogenous supplementation of VEGF-A + sEV efficiently recused PTC rarefaction, improved renal perfusion, and halted the AKI to CKD transition. Taken together, our study uncovered a novel intrinsic repair response after AKI through renal tubule-PTC crosstalk via sEV-VEGF-A, which could be exploited as a promising therapeutic angiogenesis strategy in diseases with ischemia.

Exosomal Vaccine Loading T Cell Epitope Peptides of SARS-CoV-2 Induces Robust CD8+ T Cell Response in HLA-A Transgenic Mice.

Purpose: Current vaccines for the SARS-CoV-2 virus mainly induce neutralizing antibodies but overlook the T cell responses. This study aims to generate an exosomal vaccine carrying T cell epitope peptides of SARS-CoV-2 for the induction of CD8+ T cell response. Methods: Thirty-one peptides presented by HLA-A0201 molecule were conjugated to the DMPE-PEG-NHS molecules, and mixed with DSPE-PEG to form the peptide-PEG-lipid micelles, then fused with exosomes to generate the exosomal vaccine, followed by purification using size-exclusion chromatography and validation by Western blotting, liquid nuclear magnetic resonance (NMR) test and transmission electron microscopy. Furthermore, the exosomal vaccine was mixed with Poly (I:C) adjuvant and subcutaneously administered for three times into the hybrid mice of HLA-A0201/DR1 transgenic mice with wild-type mice. Then, the epitope-specific T cell responses were detected by ex vivo ELISPOT assay and intracellular cytokine staining. Results: The exosomal vaccine was purified from the Peak 2 fraction of FPLC and injected into the hybrid mice for three times. The IFN-γ spot forming units and the frequencies of IFN-γ+/CD8+ T cells were 10-82-fold and 13-65-fold, respectively, higher in the exosomal vaccine group compared to the Poly (I:C) control group, without visible organ toxicity. In comparison with the peptides cocktail vaccine generated in our recent work, the exosomal vaccine induced significantly stronger T cell response. Conclusion: Exosomal vaccine loading T cell epitope peptides of SARS-CoV-2 virus was initially generated without pre-modification for both peptides and exosomes, and elicited robust CD8+ T cell response in HLA-A transgenic mice.

Integrin, Exosome and Kidney Disease.

Integrins are transmembrane receptors that function as noncovalent heterodimers that mediate cellular adhesion and migration, cell to cell communication, and intracellular signaling activation. In kidney, latency associated peptide-transforming growth factor ß (TGF-ß) and soluble urokinase plasminogen activator receptor (suPAR) were found as the novel ligands of integrins that contribute to renal interstitial fibrosis and focal segmental glomerular sclerosis glomerulosclerosis (FSGS). Interestingly, recent studies revealed that integrins are the compositional cargo of exosomes. Increasing evidence suggested that exosomal integrin played critical roles in diverse pathophysiologic conditions such as tumor metastasis, neurological disorders, immunology regulation, and other processes. This review will focus on the biology and function of exosomal integrin, emphasizing its potential role in kidney disease as well as its implications in developing novel therapeutic and diagnosis approaches for kidney disease.