Effect of extracellular vesicles derived from induced pluripotent stem cells on mesangial cells underwent a model of fibrosis in vitro.

The fibrogenic process plays a significant pathophysiological role in the progression of chronic kidney disease. Inhibition of the renin-angiotensin system (RAS) is one strategy to delay disease progression but does not reverse established fibrosis. In this context, induced pluripotent stem cells (iPSCs) have been considered an alternative due to their regenerative potential. iPSCs exert their effects through paracrine signaling, which releases specific biomolecules into the extracellular environment, either directly or within extracellular vesicle (EVs), that can reach target cells. This study aims to evaluate the potential beneficial effects of iPSC-derived EVs (EV-iPSCs) in an in vitro model of fibrosis using mouse mesangial cells (MMCs) stimulated with TGF-ß. EV-iPSCs were obtained by differentially ultracentrifuging iPSCs culture medium. MMCs were stimulated with 5 ng/mL of TGF-ß and simultaneously treated with or without EV-iPSCs for 24 h. Markers of inflammation, fibrosis, and RAS components were assessed using RT-PCR, western blotting, and immunofluorescence. Under TGF-ß stimulus, MMCs exhibited increased expression of inflammation markers, RAS components, and fibrosis. However, these changes were mitigated in the presence of EV-iPSCs. EV-iPSCs effectively reduced inflammation, RAS activation, and fibrogenesis in this fibrosis model involving mesangial cells, suggesting their potential as a strategy to reduce glomerular sclerosis.

Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-ß.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-ß. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-ß.

Comparison of the Effects of Mesenchymal Stem Cells with Their Extracellular Vesicles on the Treatment of Kidney Damage Induced by Chronic Renal Artery Stenosis.

BACKGROUND: Chronic renal artery stenosis is considered one of the most common causes of renovascular hypertension (RH). Chronic hypoxia can lead to irreversible damage to renal tissue and to a progressive deterioration of renal function. We have previously shown that bone marrow-derived mesenchymal stem cells (BMSCs) improved renal parenchyma and function in a model of RH (2 kidneys, 1 clip model (2K-1C) in rats. Microvesicles (MVs) and exosomes (EXs) released by MSCs have been shown to induce effects similar to those induced by whole cells but with fewer side effects. In this study, we compared the effects of adipose-derived MSCs (ASCs) with those of the MVs and EXs released by ASCs on tissue inflammation and renal function in 2 K-1C rats. RESULTS: Flow cytometry analysis showed that even after 15 days, ASCs were still detected in both kidneys. The expression of a stem cell homing marker (SDF1-α) was increased in ASC-treated animals in both the stenotic and contralateral kidneys. Interestingly, SDF1-α expression was also increased in MV- and EX-treated animals. A hypoxia marker (HIF1-α) was upregulated in the stenotic kidney, and treatments with ASCs, MVs, and EXs were effective in reducing the expression of this marker. Stenotic animals showed a progressive increase in systolic blood pressure (SBP), while animals treated with ASCs, MVs, and EXs showed a stabilization of SBP, and this stabilization was similar among the different treatments. Stenotic animals developed significant proteinuria, which was reduced by ASCs and MVs but not by EXs. The increased expression of Col I and TGFß in both kidneys was reduced by all the treatments, and these treatments also effectively increased the expression of the anti-inflammatory cytokine IL-10 in both kidneys; however, only ASCs were able to reduce the overexpression of the proinflammatory cytokine IL-1ß in both kidneys of 2K-1C animals. CONCLUSION: The results of this study demonstrated that the EVs released by ASCs produced beneficial results but with lower efficacy than whole cells. ASCs produced stronger effects in this model of renal chronic hypoxia, and the use of EVs instead of whole cells should be evaluated depending on the parameter to be corrected.

Author Correction: Influence of high glucose on mesangial cell-derived exosome composition, secretion and cell communication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influence of high glucose on mesangial cell-derived exosome composition, secretion and cell communication.

Mesangial cells stimulated with high glucose (HG) exhibit increased intracellular angiotensin II (AngII) synthesis that is correlated with the upregulation of AngII target genes, such as profibrotic cytokines. The intracrine effects of AngII can be mediated by several molecules transferred to other cells via exosomes (Exos), which play a key role in cellular communication under many physiological and pathological conditions. The aim of this study was to investigate the effects of exosomes derived from HG-stimulated human mesangial cells (HG-HMCs) on normal unstimulated HMCs. Exosomes from HMCs (C-Exos) and HG-HMCs (HG-Exos) were obtained from cell culture supernatants. HMCs were incubated with C-Exos or HG-Exos. HG stimulus induced a change in the amount but not the size of Exos. Both C-Exos and HG-Exos contained angiotensinogen and renin, but no angiotensin converting enzyme was detected. Compared with HMCs treated with C-Exos, HMCs treated with HG-Exos presented higher levels of fibronectin, angiotensinogen, renin, AT1 and AT2 receptors, indicating that HG-Exos modified the function of normal HMCs. These results suggest that the intercellular communication through Exos may have pathophysiological implications in the diabetic kidney.

Klotho and PPAR Gamma Activation Mediate the Renoprotective Effect of Losartan in the 5/6 Nephrectomy Model.

Renin angiotensin system (RAS) blockade reduces the progression of chronic kidney disease (CKD) independently of its antihypertensive effect. Ang II-induced fibrosis can be mediated by molecules such as klotho, peroxisome proliferator-activate receptor γ (PPAR-γ), and the Wnt/ß-catenin pathway; however, the interaction among these molecules and RAS activation is not completely known. The aim of this study was to investigate a possible link between RAS, PPAR-γ, and Klotho in the 5/6 nephrectomy (NX) animals. NX rats presented hypertension that was blunted by both losartan and propranolol, however, only losartan was able to reduce the expression levels of fibronectin FSP1 and TGF-ß in the remnant kidney. The anti-fibrotic Klotho and PPAR-γ were reduced in the remnant kidney, and losartan, but not propranolol, restored their levels. In contrast, the profibrotic Wnt 7a and Wnt 3 were upregulated and losartan prevented the increase in Wnts. In vitro, Ang II induced a decrease in both klotho and in PPAR-γ in Madin-Darby canine kidney (MDCK) cells, and this effect was blunted by losartan. However, klotho expression was increased by pioglitazone, an agonist of PPAR-γ, and suppressed by BADGE, an antagonist of PPAR-γ, suggesting that the effect of Ang II downregulating klotho is mediated by PPAR-γ. These data suggest that activation of the Wnt pathway together with downregulation of PPAR-γ that in turn suppresses klotho contribute to potentiating the profibrotic effect of Ang II.

miR-26a modulates HGF and STAT3 effects on the kidney repair process in a glycerol-induced AKI model in rats.

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2.

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT1 and AT2), defined as intracrine response. The aim of this study was to examine the presence of AT1 and AT2 receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT1 through an intracrine mechanism. Subcellular distribution of AT1 and AT2 was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.