Autologous cell therapy for cisplatin-induced acute kidney injury by using non-expanded adipose tissue-derived cells

Abstract Background aims. Recent studies have demonstrated that cultured mesenchymal stromal cells derived from adipose tissue are useful for regenerative cell therapy. The stromal vascular fraction (SVF) can be obtained readily without culturing and may be clinically applicable. We investigated the therapeutic effects of SVF and used it in the treatment of acute kidney injury (AKI). Methods. Liposuction aspirates were obtained from healthy donors who had provided written informed consent. We harvested the SVF and determined the growth factor secretion and anti-apoptotic ability with conditioned medium. To investigate the effect of SVF on AKI, cisplatin was injected into rats and SVF was administrated into the subcupsula of the kidney. Results. Both human and rat SVF cells secreted vascular endothelial growth factor-A (VEGF) and hepatocyte growth factor (HGF). Human SVF-conditioned media had an anti-apoptotic effect, which was inhibited by anti-HGF antibody (Ab) but not by anti-VEGF Ab. In vivo, SVF significantly ameliorated renal function, attenuated tubular damage and increased the cortical blood flow speed. In the SVF-treated group, VEGF levels in the cortex and HGF levels in both the cortex and medulla, especially tubules in the medulla, were significantly higher. Immunostaining revealed that SVF cells expressing VEGF and HGF and remained in the subcapsule on day 14. Conclusions. The present study demonstrates that a subcapsular injection of non-expanded SVF cells ameliorates rat AKI, and that the mechanism probably involves secretion of renoprotective molecules. Administration of human SVF may be clinically applicable and useful as a novel autologous cell therapy against kidney diseases.     

LncRNA OIP5-AS1 facilitates ox-LDL-induced endothelial cell injury through the miR-98-5p/HMGB1 axis

Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.

     

Downregulation of XIST ameliorates acute kidney injury by sponging miR-142-5p and targeting PDCD4

Acute kidney injury (AKI) is a common kidney disease that markedly affects public health. To date, the roles of long noncoding RNA XIST in AKI are poorly understood. Here, we investigated the biological functions of XIST in AKI. We observed that XIST expression increased in patients with AKI and HK-2 cells stimulated by CoCl₂. In addition, a rat AKI model induced by ischemia–reperfusion was established. Tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2 messenger RNA expression were induced in vivo; moreover, XIST expression was upregulated. Knockdown of XIST significantly repressed CoCl₂-triggered injury in HK-2 cells. However, microRNA (miR)-142-5p, a downstream target of XIST, was downregulated in AKI. miR-142-5p was repressed by XIST and miR-142-5p could inhibit CoCl₂-induced injury in HK-2 cells. Moreover, PDCD4 expression was significantly increased in AKI. PDCD4 was predicted to be the target of miR-142-5p. Subsequently, loss of PDCD4 was able to retard injury in HK-2 cells exposed to CoCl_2. Thus, we suggest that XIST regulates miR-142-5p and PDCD4, and it has the potential to function as a biomarker in therapeutic strategies for AKI.     

Silencing circ-USP1 protects the renal tubular from kidney injury induced by hypoxia via modulating miR-194-5p/DNMT3A axis in acute renal allografts

Recent studies indicate that circular RNAs are involved in dysregulation of kidney injury. Nevertheless, the underlying mechanisms remain largely unclear. Therefore, this study sought to investigate the role of circ-USP1 in the pathogenesis of early renal allografts. Thirty-two male C57BL/6J mice aged between 6 and 8 weeks were divided into the sham and allograft groups. Thereafter, the association between miR-194-5p, circ-USP1 and DNMT3A was confirmed using a combination of bioinformatics and the luciferase reporter gene assay. Additionally, the expression of circ-USP1, miR-194-5p and DNMT3A mRNA was detected through qPCR. Afterwards, the Western blot assay was performed to examine the expression of DNMT3A protein. Finally, the TUNEL assay was conducted to determine the rate of apoptosis in DNMT3A cells. The expression of circ-USP1 increased, while that of miR-194-5p decreased in renal allografts. Additionally, silencing circ-USP1 reduced kidney injuries caused by renal allografts in mice. Moreover, miR-194-5p was a target for circ-USP1, and DNMT3A was a target of miR-194-5p. Finally, it was shown that silencing circ-USP1 reduced DNMT3A expression in the kidney of mice that received renal allografts. Circ-USP1 functions as a competing endogenous RNA for miR-194-5p. This occurs in order to regulate DNMT3A expression in kidney injury induced by hypoxia in acute renal allografts.     

Upregulation of lncRNA NONRATG019935.2 suppresses the p53-mediated apoptosis of renal tubular epithelial cells in septic acute kidney injury

Although increasing evidence has confirmed that the apoptosis of renal tubular epithelial cells (RTECs) is a crucial contributor to the onset and development of septic acute kidney injury (AKI), the pathological mechanism by which RTEC apoptosis is upregulated during septic AKI is not entirely clear. In this study, a rat model of septic AKI was induced by a cecal ligation puncture procedure or lipopolysaccharide (LPS) injection. Four differentially expressed long noncoding RNAs (DE-Lncs) in the rat model of septic AKI were determined using RNA-sequencing and verified by qRT-PCR. Among the four DE-Lncs, the expression level of lncRNA NONRATG019935.2 (9935) exhibited the most significant reduction in both septic AKI rats and LPS-treated NRK-52E cells (a rat RTEC line). The overexpression of 9935 suppressed cell apoptosis and p53 protein level in LPS-treated NRK-52E cells, and retarded septic AKI development in the rat model of septic AKI. Mechanistically, 9935 decreased the human antigen R (HuR)-mediated Tp53 mRNA stability by limiting the combination of HuR and the 3′UTR region of Tp53 mRNA in RTECs. The overexpression of HuR abrogated the inhibitory effect of pcDNA-9935 on the LPS-induced apoptosis of NRK-52E and rat primary RTECs. In conclusion, 9935 exerts its role in septic AKI by suppressing the p53-mediated apoptosis of RTECs, and this essential role of 9935 relies on its destructive effect on HuR-mediated Tp53 mRNA stability.Subject terms: Cell biology, Molecular biology     

PSTPIP2 inhibits cisplatin-induced acute kidney injury by suppressing apoptosis of renal tubular epithelial cells

Cisplatin (CP) is an effective chemotherapeutic agent widely used in the treatment of various solid tumours. However, CP nephrotoxicity is an important limitation for CP use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Recently, we identified a specific role of proline–serine–threonine phosphatase-interacting protein 2 (PSTPIP2) in cisplatin-induced AKI. PSTPIP2 was reported to play an important role in a variety of diseases. However, the functions of PSTPIP2 in experimental models of cisplatin-induced AKI have not been extensively studied. The present study demonstrated that cisplatin downregulated the expression of PSTPIP2 in the kidney tissue. Administration of AAV-PSTPIP2 or epithelial cell-specific overexpression of PSTPIP2 reduced cisplatin-induced kidney dysfunction and inhibited apoptosis of renal tubular epithelial cells. Small interfering RNA-based knockdown of PSTPIP2 expression abolished PSTPIP2 regulation of epithelial cell apoptosis in vitro. Histone acetylation may impact gene expression at the epigenetic level, and histone deacetylase (HDAC) inhibitors were reported to prevent cisplatin-induced nephrotoxicity. The UCSC database was used to predict that acetylation of histone H3 at lysine 27 (H3K27ac) induces binding to the PSTPIP2 promoter, and this prediction was validated by a ChIP assay. Interestingly, an HDAC-specific inhibitor (TSA) was sufficient to potently upregulate PSTPIP2 in epithelial cells. Histone acetylation-mediated silencing of PSTPIP2 may contribute to cisplatin nephrotoxicity. PSTPIP2 may serve as a potential therapeutic target in the prevention of cisplatin nephrotoxicity.Subject terms: Cancer, Cancer prevention     

Exosomal lncRNA-p21 derived from mesenchymal stem cells protects epithelial cells during LPS-induced acute lung injury by sponging miR-181

Long noncoding RNAs(IncRNAs)act as essential regulators of various diseases.However,the func-tions of IncRNAs in sepsis-induced acute lung injury(SALI)remain un...     

Distal tubular epithelial cells of the kidney: Potential support for proximal tubular cell survival after renal injury

The tubular epithelium of the kidney is susceptible to injury from many causes, such as ischemia-reperfusion and the associated oxidative stress, nephrotoxins, inflammatory and immune disorders and many others. The outcome is often acute kidney injury, which may progress to chronic kidney disease and fibrosis. Acute kidney injury involves not only direct injury to the distal tubular (DT) and proximal tubular (PT) epithelium during and immediately following the injurious event, but the closely-associated and sometimes dysfunctional renal vascular endothelium also plays an important part in modulating the tubular epithelial injury. In comparison with the PT, the DT epithelium is less sensitive to cell death, especially after ischemic injury. It is more prone to apoptosis than necrosis when it dies, and has key paracrine and autocrine functions in secreting an array of inflammatory, reparative, and survival cytokines that include chemotactic cytokines, polypeptide growth factors, and vasoactive peptides. In a neighborly way, the cytokines and growth factors secreted by the DT epithelium may then act positively on the ischemia-sensitive PT that has receptors to many of these proteins, but may not be able to synthesize them. A more complete understanding of these cellular events will allow protection against nephron destruction, regeneration leading to re-epithelialization of the injured tubules, or prevention of progression to chronic kidney disease. This review looks at these functions in the DT epithelial cells, specifically the cells in the medullary thick ascending limb of the loop of Henle, in contrast with those of the straight segment of the PT.     

Bone mesenchymal stromal cell-derived small extracellular vesicles inhibit inflammation and ameliorate sepsis via delivery of microRNA-21a-5p

Background aimsSepsis is a potentially life-threatening disease that results from a severe systemic inflammatory response due to infection. Mesenchymal stromal cell-derived small extracellular vesicles (MSC sEVs) are able to transfer bioactive molecules and have been demonstrated to play an important role in the pathophysiological process of sepsis. Herein the authors aimed to investigate the potential role and downstream molecular mechanism of MSC sEVs in sepsis.MethodsMSC sEVs were acquired by ultracentrifugation and then injected into a cecal ligation and puncture mouse model. The efficacy of MSC sEVs in both in vitro and in vivo models of sepsis was evaluated.ResultsMSC sEV therapy improved survival, reduced sepsis-induced inflammation, attenuated pulmonary capillary permeability and improved liver and kidney function in septic mice. In addition, the authors found that microRNA-21a-5p (miR-21a-5p) was highly enriched in MSC sEVs, could be transferred to recipient cells, inhibited inflammation and increased survival in septic mice. Furthermore, the authors demonstrated that MSC sEV miR-21a-5p suppressed inflammation by targeting toll-like receptor 4 and programmed cell death 4. The therapeutic efficacy of MSC sEVs was partially abrogated by transfection with miR-21a-5p inhibitors.ConclusionsCollectively, the authors’ data suggest that miR-21a-5p-bearing MSC sEVs may be a prospective and effective sepsis therapeutic strategy.     

Impaired immunomodulatory capacity in adipose tissue-derived mesenchymal stem/stromal cells isolated from obese patients

Immune-modulatory properties of adipose tissue-derived mesenchymal stem/stromal cells (MSCs) might be susceptible to metabolic disturbances. We hypothesized that the immune-modulatory function of MSCs might be blunted in obese human subjects. MSCs were collected from abdominal subcutaneous fat of obese and lean subjects during bariatric or kidney donation surgeries, respectively. MSCs were co-cultured in vitro for 24 h with M1 macrophages, which were determined as M1or M2 phenotypes by flow cytometry, and cytokines measured in conditioned media. In vivo, lean or obese MSCs (5 × 10⁵), or PBS, were injected into mice two weeks after unilateral renal artery stenosis (RAS) or sham surgeries (n = 6 each). Fourteen days later, kidneys were harvested and stained with M1 or M2 markers. Lean MSCs decreased macrophages M1 marker intensity, which remained elevated in macrophages co-cultured with obese MSCs. TNF-α levels were four-fold higher in conditioned media collected from obese than from lean MSCs. RAS mouse kidneys were shrunk and showed increased M1 macrophage numbers and inflammatory cytokine expression compared with normal kidneys. Lean MSCs decreased M1 macrophages, M1/M2 ratio and inflammation in RAS kidneys, whereas obese MSCs did not. MSCs isolated from lean human subjects decrease inflammatory M1 macrophages both in vivo and in vitro, an immune-modulatory function which is blunted in MSCs isolated from obese subjects.     

Artesunate relieves acute kidney injury through inhibiting macrophagic Mincle-mediated necroptosis and inflammation to tubular epithelial cell

Artesunate is a widely used derivative of artemisinin for malaria. Recent researches have shown that artesunate has a significant anti-inflammatory effect on many diseases. However, its effect on acute kidney injury with a significant inflammatory response is not clear. In this study, we established a cisplatin-induced AKI mouse model and a co-culture system of BMDM and tubular epithelial cells (mTEC) to verify the renoprotective and anti-inflammatory effects of artesunate on AKI, and explored the underlying mechanism. We found that artesunate strongly down-regulated the serum creatinine and BUN levels in AKI mice, reduced the necroptosis of tubular cells and down-regulated the expression of the tubular injury molecule Tim-1. On the other hand, artesunate strongly inhibited the mRNA expression of inflammatory cytokines (IL-1β, IL-6 and TNF-α), protein levels of inflammatory signals (iNOS and NF-κB) and necroptosis signals (RIPK1, RIPK3 and MLKL) in kidney of AKI mouse. Notably, the co-culture system proved that Mincle in macrophage can aggravate the inflammation and necroptosis of mTEC induced by LPS, and artesunate suppressed the expression of Mincle in macrophage of kidney in AKI mouse. Overexpression of Mincle in BMDM restored the damage and necroptosis inhibited by artesunate in mTEC, indicating Mincle in macrophage is the target of artesunate to protect tubule cells in AKI. Our findings demonstrated that artesunate can significantly improve renal function in AKI, which may be related to the inhibition of Mincle-mediated macrophage inflammation, thereby reducing the damage and necroptosis to tubular cells that provide new option for the treatment of AKI.     

Human adipose stromal cell therapy improves survival and reduces renal inflammation and capillary rarefaction in acute kidney injury

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short‐term recovery and long‐term functional preservation post‐AKI. Human adipose stromal cells (hASCs) possess pro‐angiogenic and anti‐inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC‐treated rats had significantly greater survival compared to vehicle‐treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post‐AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC‐treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle‐treated rats as well as reduced accumulation of interstitial alpha‐smooth muscle actin‐positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R‐induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.     

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210–3p, and miR-224–5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210–3p or miR-224–5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210–3p, and miR-224–5p in sEVs was compared. The amount of miR-33a-5p and miR-210–3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224–5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210–3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.     

microRNA-21-5p from M2 macrophage-derived extracellular vesicles promotes the differentiation and activity of pancreatic cancer stem cells by mediating KLF3

Cell Biology and Toxicology - Given the fact that tumor-associated macrophage-derived extracellular vesicles (EVs) are attributable to tumor aggressiveness, this research intends to decode the...     

Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of non-small cell lung cancer patients

Objective was to assess and compare the relative expressions of miR-205-5p, miR-205-3p, and miR-21-3p in tissues and serum among non-small cell lung carcinoma (NSCLC) patients, benign pulmonary conditions patients, and healthy volunteers. Serum samples were obtained between October 2011 and September 2012 from 20 NSCLC patients undergoing surgical treatment, 20 patients diagnosed with a benign lung disease (pulmonary tuberculosis, pneumonia, chronic obstructive pulmonary disease, or interstitial pneumonia) (lesion group), and 20 healthy volunteers (control group). NSCLC patients provided cancer tissues and cancer-adjacent normal tissues during surgery (paired specimens). Quantitative RT-PCR was used to assess miR-205-5p, miR-205-3p, and miR-21-3p expressions in serum and tissue samples. The relative expressions of miR-205-5p and miR-205-3p were significantly higher in NSCLC tissues compared with cancer-adjacent paired specimens (both P < 0.001). In the serum, significantly higher miR-205-5p, miR-205-3p, and miR-21-3p relative expressions were observed in the NSCLC group compared with the two other groups (all P < 0.001). The relative expressions of miR-205-5p and miR-21-3p in NSCLC tissues and serum were significantly correlated (r = 0.553, P = 0.011; and r = ?0.541, P = 0.014, respectively), while no significant correlation was observed for miR-205-3P (P = 0.120). Expressions of miR-205-5p and miR-205-3P in squamous cell carcinoma specimens were significantly higher than in lung adenocarcinoma specimens (both P = 0.001). Similarly, higher serum miR-205-5p and miR-205-3p levels were observed in squamous cell carcinoma patients (P = 0.033 and P = 0.002, respectively). In this preliminary and novel study, miR-205-5p was more useful as a marker for NSCLC than miR-205-3p or miR-21, indicating a potential for future applications in NSCLC diagnosis and prognosis.