Human Liver Stem Cell-Derived Extracellular Vesicles Prevent Aristolochic Acid-Induced Kidney Fibrosis

Abstract

With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.

HLSC-Derived Extracellular Vesicles Attenuate Liver Fibrosis and Inflammation in a Murine Model of Non-alcoholic Steatohepatitis

“…Therefore, EV-associated proteins may implement the biological activity of regulatory RNAs present in the EVs. 10,11 In conclusion, the results of the present study indicated that EVs released from HLSCs exert anti-inflammatory and anti-fibrotic effects in a model of chronic liver disease by carrying molecules that may modulate genes involved in fibrosis. Fibrosis is an abnormal mechanism of tissue repair after repeated and sustained injuries that has common pathways in different organs.…”

“…We previously showed in two different models of chronic kidney diseases that the anti-fibrotic effect of EV-HLSCs is linked to the presence of microRNA (miRNA) patterns targeting the fibrotic genes. 10,11 In particular, we found that EV-HLSCs contain miRNA-29a, the let-7 family, miRNA-30a, miRNA-24, and miRNA-21, which are known to target Collagen I, Snail, and the FAS ligand. 11 In the present study, we performed proteomic analyses of EV-HLSCs showing the presence of several anti-inflammatory proteins that may contribute to the final anti-fibrotic effect by downregulating inflammation.…”

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model

“…69 However, the advantages in the use of EVs engineered with Klotho have not been reported yet. In the present study, Klotho was loaded onto fibroblast EVs, a Klotho negative EV source previously described as non-effective in renal repair, 70,71 and the inefficacy of naive EVs in AKI was reverted in parallel with the restoration of its endogenous levels. Moreover, the effect of recombinant Klotho administered alone at a dose comparable with that of uEVs (1 pg/mouse) did not exert any regenerative effect.…”

Aristolochic Acid-Induced Nephrotoxicity: Molecular Mechanisms and Potential Protective Approaches

“…Moreover, NLRP3 or Caspase-1 deficiency showed protective effects against renal injury in the mouse model of acute AAN, suggesting the involvement of NLRP3 signaling in the pathogenesis of AAN [74]. Inflammation in a rat model of AA was also reported by Kholia et al [75]. and in HK-2 cells by Wang et al [65].…”