Tubular epithelial cells (TECs) exposed to hypoxia incite tubu-lointerstitial 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-1a 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 amelio-rated when KIM-1 or Rab27a, a master regulator of sEV secre-tion, 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. Correspond-ingly, 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 tu-bules 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 devel-opment of TII in ischemic acute kidney injury.

Abstract BACKGROUND AND AIMS Tubular epithelial cells (TECs) exposed to hypoxia during kidney injury communicate with interstitial inflammatory cells and contribute to tubulointerstitial inflammation. However, whether injured TECs communicate under hypoxia in an autocrine or paracrine manner and contribute to tubulointerstitial inflammation has yet to be identified moieties. The purpose of this study is to explore the role of small extracellular vesicles(sEVs) secreted by hypoxic TECs in the development of renal tubulointerstitial inflammation and the mechanism of sEV-mediated TEC communication. METHOD Ischemia reperfusion induced kidney injury (IRI) models were established to explore the conditions of hypoxia induced Inflammation as well as tubular kidney injury molecule-1(KIM-1) expression and sEV production. In vitro, hypoxic TEC-derived sEVs (Hypo-sEVs) were applied to culture TECs to explore sEV-mediated tubule communication. Besides, inflammatory cytokine generation was analyzed in hypoxic TECs with silence of KIM-1 and Rab27a. To investigate the mechanism of sEV internalization, uptake efficiency of the recipient TECs was evaluated in conditions of KIM-1 overexpression. Phosphatidylserine (PS) on the surface of Hypo-sEVs was further assayed by nanoflow cytometry (NanoFCM). sEVs isolated from hypoxic TECs with or without labeling were transferred to IRI mice to detect its localization and the consequences on tubulointerstitial inflammation. RESULTS In IRI mice, hypoxia induced tubule injury as evidenced by tubule HIF-1α and KIM-1 expression as well as tubulointerstitial inflammation were observed. Impressively, we noticed the colocalization of sEV marker (CD63) with KIM-1, which suggested increased sEV production in injured tubules or uptake of sEVs by damaged tubules through KIM-1. Intriguingly, KIM-1 expression also correlated with CD3 + and CD68 + inflammatory cells infiltration. In vitro, stable HIF-1α and KIM-1 expression was detected in hypoxic TECs with significant inflammatory cytokine expression. Notably, increased sEV release was noticed in hypoxic TECs, while hypoxia induced inflammation could be ameliorated when KIM-1 and Rab27a were silenced. Importantly, the inflammatory response of hypoxic TECs induced by exogenous Hypo-sEVs could be partly relieved when KIM-1 was knock down by siRNA transfection. Besides, PS could readily be detected in Hypo-sEVs by NanoFCM suggesting that sEVs may accelerate TEC inflammation via recognizing PS by KIM-1. Correspondingly, Hypo-sEVs localized to tubules with KIM-1 expression and amplified tubulointerstitial inflammation in IRI mice. CONCLUSION Our studies demonstrate that KIM-1 expressed by injured tubules mediates sEVs uptake via recognizing PS, which participates in TEC communication and augment of tubulointerstitial inflammation induced by hypoxia.

Tubules injury and immune cell activation are the common pathogenic mechanisms in acute kidney injury (AKI). However, the exact modes of immune cell activation following tubule damage are not fully understood. Here we uncovered that the release of cytoplasmic spliceosome associated protein 130 (SAP130) from the damaged tubular cells mediated necroinflammation by triggering macrophage activation via miRNA-219c(miR-219c)/Mincle-dependent mechanism in unilateral ureteral obstruction (UUO) and cisplatin-induced AKI mouse models, and in patients with acute tubule necrosis (ATN). In the AKI kidneys, we found that Mincle expression was tightly correlated to the necrotic tubular epithelial cells (TECs) with higher expression of SAP130, a damaged associated molecule pattern (DAMP), suggesting that SAP130 released from damaged tubular cells may trigger macrophage activation and necroinflammation. This was confirmed in vivo in which administration of SAP130-rich supernatant from dead TECs or recombinant SAP130 promoted Mincle expression and macrophage accumulation which became worsen with profound tubulointerstitial inflammation in LPS-primed Mincle WT mice but not in Mincle deficient mice. Further studies identified that Mincle was negatively regulated via miR-219c-3p in macrophages as miR-219c-3p bound Mincle 3'-UTR to inhibit Mincle translation. Besides, lentivirus-mediated renal miR-219c-3p overexpression blunted Mincle and proinflammatory cytokine expression as well as macrophage infiltration in the inflamed kidney of UUO mice. In conclusion, SAP130 is released by damaged tubules which elicit Mincle activation on macrophages and renal necroinflammation via the miR-219c-3p-dependent mechanism. Results from this study suggest that targeting miR-219c-3p/Mincle signaling may represent a novel therapy for AKI.

plasmid and polyethylenimine (PEI).Sepsis was induced via cecal ligation and puncture (CLP) 7 days after transfection, .RESULTS: In vitro, suppressed the production of pro-inflammatory cytokines in RAW264.7 macrophage cells after lipopolysaccharide (LPS) stimulation.Of these, miR-146a displayed the highest suppressive effect, wherein the transcriptional activity of nuclear factor kappa B (NF-jB) was decreased via targeting of interleukin 1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis receptor-associated factor 6 (TRAF6).In vivo, Treatment with miR-146a-expressing plasmid/PEI complex significantly decreased the level of serum inflammatory cytokines, attenuated kidney injury, and led to increased survival from CLP induced sepsis.miR-146a-expressing plasmid was abundantly distributed in splenic macrophages, but not in renal parenchymal cells.CLP mice treated with miR-146a displayed significantly decreased NF-jB activation and splenocyte apoptosis.Splenectomy diminished the anti-inflammatory effects of miR-146a. CONCLUSIONS:We demonstrated that the splenic macrophages governs the hierarchy of organs in the pathogenesis of sepsis.The induction of miR-146a in splenic macrophages prevents excessive inflammation and sepsis-induced kidney injury.This study establishes a novel and critical pathophysiological role for splenic macrophage interference in sepsis-related AKI.

Tubulointerstitial inflammation is a common characteristic of acute and chronic kidney injury. However, the mechanism by which the initial injury of tubular epithelial cells (TECs) drives interstitial inflammation remains unclear. This paper aims to explore the role of exosomal miRNAs derived from TECs in the development of tubulointerstitial inflammation. Global microRNA(miRNA) expression profiling of renal exosomes was examined in a LPS induced acute kidney injury (AKI) mouse model and miR-19b-3p was identified as the miRNA that was most notably increased in TEC-derived exosomes compared to controls. Similar results were also found in an adriamycin (ADR) induced chronic proteinuric kidney disease model in which exosomal miR-19b-3p was markedly released. Interestingly, once released, TEC-derived exosomal miR-19b-3p was internalized by macrophages, leading to M1 phenotype polarization through targeting NF-κB/SOCS-1. A dual-luciferase reporter assay confirmed that SOCS-1 was the direct target of miR-19b-3p. Importantly, the pathogenic role of exosomal miR-19b-3p in initiating renal inflammation was revealed by the ability of adoptively transferred of purified TEC-derived exosomes to cause tubulointerstitial inflammation in mice, which was reversed by inhibition of miR-19b-3p. Clinically, high levels of miR-19b-3p were found in urinary exosomes and were correlated with the severity of tubulointerstitial inflammation in patients with diabetic nephropathy. Thus, our studies demonstrated that exosomal miR-19b-3p mediated the communication between injured TECs and macrophages, leading to M1 macrophage activation. The exosome/miR-19b-3p/SOCS1 axis played a critical pathologic role in tubulointerstitial inflammation, representing a new therapeutic target for kidney disease.

IgA nephropathy (IgAN) features variable renal pathology and a heterogeneous clinical course. Our aim was to search noninvasive biomarkers from urinary exosomes for IgAN patients; membrane nephropathy and minimal change disease were included as other glomerulopathy controls. Transmission electron microscopy and nanoparticle tracking analysis confirmed the size and morphology characteristic of urinary exosomes. Exosome markers (Alix and CD63) as well as renal cell. markers [aquaporin 2 (AQP2) and nephrin] were detected, which indicate the renal origin of urinary exosomes. Exosome excretion was increased markedly in IgAN patients compared with controls and correlated with levels of proteinuria and tubular injury. More important, urinary exosome excretion correlated with greater histologic activity (mesangial hypercellularity, crescents, and endocapillary hypercellularity). Profiling of the inflammation-related mRNA revealed that exosomal chemokine (C-C motif) ligand 2 (CCL2) was up regulated in IgAN patients. In a validation study, CCL2 was exclusively highly expressed in IgAN patients compared with healthy controls as well as minimal change disease and membrane nephropathy patients. Also, a correlation between exosomal CCL2 and estimated glomerular filtration rate levels was found in IgAN. Exosomal CCL2 was correlated with tubulointerstitial inflammation and C3 deposition. High CCL2 levels at the time of renal biopsy were associated with subsequent deterioration in renal function. Thus, urinary exosomes and exosomal CCL2 mRNA are promising biomarkers reflecting active renal histologic injury and renal function deterioration in IgAN.

Background Emerging evidence of histopathological analyses suggests that endothelial progenitor cells (EPCs) play an important role in vascular diseases. Neointimal hyperplasia can be reduced by intravenous transfusion of EPCs after vascular injury in mice. Therefore, it would be advantageous to develop an in vivo technique that can explore the temporal and spatial migration of EPCs homing to the damaged endothelium noninvasively. Methodology/Principal Findings The left carotid common artery (LCCA) was injured by removal of endothelium with a flexible wire in Kunming mice. EPCs were collected by in vitro culture of spleen-derived mouse mononuclear cells (MNCs). EPCs labeling was carried out in vitro using Fe2O3-poly-L-lysine (Fe2O3-PLL). In vivo serial MR imaging was performed to follow-up the injured artery at different time points after intravenous transfusion of EPCs. Vessel wall areas of injured artery were computed on T2WI. Larger MR signal voids of vessel wall on T2WI was revealed in all 6 mice of the labeled EPC transfusion group 15 days after LCCA injury, and it was found only in 1 mouse in the unlabeled EPC transfusion group (p = 0.015). Quantitative analyses of vessel wall areas on T2WI showed that the vessel wall areas of labeled EPC transfusion group were less than those of unlabeled EPC transfusion group and control group fifteen days after artery injury (p<0.05). Histopathological analyses confirmed accumulation and distribution of transfused EPCs at the injury site of LCCA. Conclusions/Significance These data indicate that MR imaging might be used as an in vivo method for the tracking of EPCs homing to the endothelium injured artery.

PURPOSE:to study the feasibility of in vivo MR imaging in evaluation of endothelial progenitor cells (EPCs) on the progress of neointimal hyperplasia after carotid artery injury in mice. METHODS:fifteen Kunming mice were injured in left carotid artery by removal of endothelium with a flexible wire 7 days after splenectomy. EPCs were collected by in vitro culture of spleen-derived mouse mononuclear cells (MNCs) in endothelial basal medium. After artery injury, the mice received EPCs (n= 6), phosphate buffered solution (PBS) (n= 6), and DiI-Ac-LDL labeled EPCs (n= 3) intravenously. In vivo serial MR imaging were performed at different time points after artery injury, and vessel-wall thickness and vessel-wall area at injury site were measured on MR imaging. RESULTS:transfused Dil-Ac-LDL-labeled EPCs were found at the injury site by histopathological analyses. Vessel wall of injured artery was observed and quantitatively analyzed with MR imaging. Vessel-wall thickness was .487 ± .122 mm in the non-EPCs transfusion group and .294 ± .051 mm in the EPCs transfusion group 15 days after artery injury (P= .005). While vessel-wall area was .860 ± .182 mm(2) in the non-EPCs transfusion group and .468 ± .141 mm(2) in the EPCs transfusion group 15 days after artery injury (P= .002). Therefore, the neointimal hyperplasia of injured artery in the EPCs transfusion group was lesser than that in the non-EPCs transfusion group. CONCLUSION:neointimal hyperplasia can be reduced by intravenous transfusion of EPCs and analyzed on in vivo MR imaging after vascular injury.

OBJECTIVE:to explore the value of in vivo dynamic monitoring of abdominal aortic atherosclerosis (AS) by high field magnetic resonance (MR) imaging (MRI) in apoE-/- mice fed a high fat diet or infused with angiotensin.METHODS:high fat diet or angiotensin II infusion was applied to apoE-/- mice for establishment of abdominal aortic atherosclerosis model. Abdominal aorta MRI was performed at 3 time points (baseline, 3 and 6 months) in 13 high fat diet fed apoE-/- mice aged 10-12 months and 3 wild-type control mice; 10 apoE-/- mice aged 6 months were infused with angiotensin II (1000 or 500 ng × kg(-1)× min(-1), n = 5 each) or saline for 14 d through Osmotic minipump. The abdominal aortic artery MRI was performed at baseline and 14 d after infusion. Black blood sequences of FLASH T1 weighted images and Proton density weighted-T2 weighted dual echo images were obtained. At each observation time post MRI, mice (n = 3, 5 and 5 for high fat diet group and n = 5 and 5 for angiotensin II infusion group) were sacrificed for pathological examination of the abdominal artery.RESULTS:(1) the abdominal aorta atherosclerosis was identified in both high fat diet and angiotensin II treated apoE-/- mice but in WT controls. Lesion progression was documented in high fat diet fed apoE-/- mice characterized by significantly increased vessel wall (a marker of atherosclerotic burden, F = 29.94, P < 0.05) and gradually increased plaque signal in PDW and T2W images. Results derived from MRI corresponded histopathology findings in high fat diet fed apoE-/- mice (correlative coefficient = 0.84, 0.95, 0.90, P < 0.05, respectively). Both MRI and histology showed increased lipid composition and decreased fibrotic composition in these mice. (2) The vessel wall area increased significantly [(1.21 ± 0.21) mm(2) vs. (2.65 ± 0.48) mm(2), P < 0.05] and the abdominal aortic dissection aneurysms was identified in apoE-/- mice infused with high angiotensin II. The vessel wall area also increased [(0.85 ± 0.11) mm(2) vs. (1.01 ± 0.17) mm(2), P < 0.05] in low angiotensin II infused apoE-/- mice and the coefficient between MR and histopathology is 0.934.CONCLUSION:abdominal aortic unstable plaque model could be established by both high fat diet and angiotensin II infusion in apoE mice, angiotensin II infusion can transiently accelerate the progression of AS and can induce abdominal aortic dissection. Serial MR black blood sequences could demonstrate the development and progression of atherosclerosis in mouse abdominal aorta with excellent agreement to histopathology finding in terms of atherosclerotic burden and plaque composition. Thus, MRI appears to be a useful tool for in vivo AS plaque dynamic monitoring in mice.

Rationale and Objectives: To evaluate the efficacy of a self-expanding metal stent seeded with autologous endothelial progenitor cells (EPCs) for preventing in-stent stenoses in transjugular intrahepatic portosystemic shunt (TIPS) in a swine model.Materials and Methods: TIPS was performed in 18 young adult pigs, using a self-expanding nitinol stent (control, n = 8) and an autologous EPC-seeded stent (treatment, n = 10). All pigs were sacrificed at 2 weeks post-TIPS procedure. Portography was performed immediately before the euthanasia. Gross, microscopic, and immunohistochemistry of the TIPS tract specimens were examined. The proliferative response of the shunt was quantified histologically.Results: TIPS was performed successfully in 16 swine, 2 animals died during the procedure. Another pig died of unknown causes 2 days post-procedure. At day 14 follow-up, portography and necropsy of the 15 remaining swine demonstrated that five shunts occluded and one shunt was stenotic (80%) in the control group (n = 6). Five shunts remained patent, two shunts were stenosed (50%, 70%), and the remaining two shunts were occluded in the treatment group (n = 9). The patency rate was significantly lower in the control group than in the treatment group, 0% versus 55.6% (P = .03). Histological analyses showed a significantly greater pseudointimal hyperplasia in the TIPS track of the control group than that of the treatment group (P < .05). Intact endothelium was documented in the lumina of all the EPC-implanted stent group.Conclusions: The EPC-seeded metal stent is feasibly fabricated in vitro and improves the patency in TIPS in a porcine model.

To induce a rabbit model of atherosclerosis at carotid artery, to visualize the lesion evolution with magnetic resonance imaging (MRI), and to characterize the lesion types by histopathology. Atherosclerosis at the right common carotid artery (RCCA) was induced in 23 rabbits by high-lipid diet following balloon catheter injury to the endothelium. The rabbits were examined in vivo with a 1.5-T MRI and randomly divided into three groups of 6 weeks (n=6), 12 weeks (n=8) and 15 weeks (n=9) for postmortem histopathology. The lesions on both MRI and histology were categorized according to the American Heart Association (AHA) classifications of atherosclerosis. Type I and type II of atherosclerotic changes were detected at week 6, i.e., nearly normal signal intensity (SI) of the injured RCCA wall without stenosis on MRI, but with subendothelial inflammatory infiltration and proliferation of smooth muscle cells on histopathology. At week 12, 75.0% and 62.5% of type III changes were encountered on MRI and histopathology respectively with thicker injured RCCA wall of increased SI on T(1)-weighted and proton density (PD)-weighted MRI and microscopically a higher degree of plaque formation. At week 15, carotid atherosclerosis became more advanced, i.e., type IV and type V in 55.6% and 22.2% of the lesions with MRI and 55.6% and 33.3% of the lesions with histopathology, respectively. Statistical analysis revealed a significant agreement (p < 0.05) between the MRI and histological findings for lesion classification (r=0.96). A rabbit model of carotid artery atherosclerosis has been successfully induced and noninvasively visualized. The atherosclerotic plaque formation evolved from type I to type V with time, which could be monitored with 1.5-T MRI and confirmed with histomorphology. This experimental setting can be applied in preclinical research on atherosclerosis.