MicroRNA-140-5p Mediates Renal Fibrosis Through TGF-ß1/Smad Signaling Pathway by Directly Targeting TGFBR1.

Renal tubulointerstitial fibrosis is usually the final outcome of various end-stage renal diseases. Recent studies have reported that microRNAs (miRNAs) play an important role in renal fibrosis. However, the biological function of microRNAs in renal fibrosis is complicated and remains unclear. In this study, our results show that miR-140-5p expression is significantly down-regulated in mice with unilateral ureteral obstruction and human proximal tubule epithelial cells (HK2) treated with TGF-ß1. The knockdown of miR-140-5p upregulates the expression levels of collagen I, collagen IV, and α-SMA, decreases E-cadherin expression, and increases Smad-2/3 phosphorylation. In contrast, the overexpression of miR-140-5p decreases the expression levels of collagen I, collagen IV, and α-SMA, enhances E-cadherin expression, and inhibits the phosphorylation of Smad-2/3 in HK2 cells treated with TGF-ß1. The dual-luciferase reporter assay revealed that TGFBR1 is a direct target gene of miR-140-5p. The enforced expression of miR-140-5p significantly inhibited the expression of TGFBR1 in HK2 cells. Furthermore, the knockdown of TGFBR1 has a similar effect of miR-140-5p overexpression on blocking the TGF-ß1/smad signal pathway activation. In contrast, the overexpression of TGFBR1 reverses the effect of miR-140-5p inhibition on the activation of the TGF-ß1/smad signal pathway. This study demonstrates that miR-140-5p regulates the TGF-ß1/smad signaling pathway by suppressing the expression of TGFBR1. Therefore, miR-140-5p may have a therapeutic potential for preventing fibrotic kidney diseases through inhibiting the TGF-ß1/Smad signaling pathway by directly targeting TGFBR1.

HIF-1α-BNIP3-mediated mitophagy in tubular cells protects against renal ischemia/reperfusion injury.

In the present study, we hypothesized that hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy plays a protective role in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Mitophagy was evaluated by measuring the changes of mitophagy flux, mitochondria DNA copy number, and the changes of mitophagy-related proteins including translocase of outer mitochondrial membrane 20 (TOMM20), cytochrome c oxidase IV (COX IV), microtubule-associated protein 1 light chain 3B (LC3B), and mitochondria adaptor nucleoporin p62 in HK2 cells, a human tubular cell line. Results show that HIF-1α knockout significantly attenuated hypoxia/reoxygenation (H/R)-induced mitophagy, aggravated H/R-induced apoptosis, and increased the production of reactive oxygen species (ROS). Similarly, H/R induced significantly increase in Bcl-2 19-kDa interacting protein 3 (BNIP3), a downstream regulator of HIF-1α. Notably, BNIP3 overexpression reversed the inhibitory effect of HIF-1α knockout on H/R-induced mitophagy, and prevented the enhancing effect of HIF-1α knockout on H/R-induced apoptosis and ROS production. For in vivo study, we established HIF-1αflox/flox; cadherin-16-cre mice in which tubular HIF-1α was specifically knockout. It was found that tubular HIF-1α knockout significantly inhibited I/R-induced mitophagy, and aggravated I/R-induced tubular apoptosis and kidney damage. In contrast, adenovirus-mediated BNIP3 overexpression significantly reversed the decreased mitophagy, and prevented enhanced kidney damage in tubular HIF-1α knockout mice with I/R injury. In summary, our study demonstrated that HIF-1α-BNIP3-mediated mitophagy in tubular cells plays a protective role through inhibition of apoptosis and ROS production in acute kidney damage.

Astragalin induced selective kidney cancer cell death and these effects are mediated via mitochondrial mediated cell apoptosis, cell cycle arrest, and modulation of key tumor-suppressive miRNAs.

PURPOSE: Kidney cancer is responsible for a significant number of deaths worldwide. This cancer is often diagnosed at advanced stages and there are frequent relapses following chemotherapy. Target therapies are used now for kidney cancer, while the use of chemotherapy declines. The currently used chemotherapeutic drugs have a number of adverse effects. Herein, we examined the anticancer effects of Astragalin against a panel of kidney cancer cells. METHODS: CellTiter-Glo Luminescent Cell Viability Assay Kit was used to examine the anti-proliferative effects of Astragalin. Acridine orange (AO)/ethidium bromide (EB), DAPI and annexin V/promidium iodide (PI) staining assays were used to examine the apoptotic cell death. Cell cycle analysis was performed by flow cytometry. The mRNA expression was checked by qRT-PCR and protein expression was examined by western blotting. RESULTS: Astragalin inhibited the growth of the all kidney cancer cell lines with IC50 ranging between 20 to 50 µM. Of note, Astragalin had low cytotoxic effects on the normal kidney cells with an IC50 of 110 µM. The experiments have shown that Astragalin exerts antiproliferative effects on the A498 kidney cancer cells by apoptotic cell death. This effect was concomitant with upregulation of apoptotic proteins such as caspase 3 and 9 and Bax. Astragalin also induced arrest of the A498 cells at the G2/M checkpoint of the cell cycle. Also, Astragalin could upregulate the expression of tumor-suppressive microRNAs. CONCLUSIONS: These results suggest that Astragalin exerts potent anticancer effects on kidney cancer cells and could pave the way in the management of kidney cancer provided clinical studies are carried out.

p62/SQSTM1 protects against cisplatin-induced oxidative stress in kidneys by mediating the cross talk between autophagy and the Keap1-Nrf2 signalling pathway.

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.

Inhibition of neddylation plays protective role in lipopolysaccharide-induced kidney damage through CRL-mediated NF-κB pathways.

It has been shown that NF-κB signaling path is very effective pharmacological target for the treatment of various inflammatory diseases, including bacterial infection-associated acute kidney injury (AKI), which remains a main cause of disability and death in patients. Notably, IκB, the upstream molecular of NF-κB, plays an important role by inhibiting NF-κB activity, and IκB is regulated by cullin-RING E3 ligases (CRLs)-mediated proteasomal degradation. Therefore inhibition of CRLs-mediated neddylation and degradation of IκB would prevent NF-κB-mediated inflammation. MLN4924, a potent neddylation-inhibiting pharmacological agent, has been shown to have significant protective effects against lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production through restriction of the CRL-mediated NF-κB pathway. However, it is still unclear whether MLN4924 plays a protective role through its anti-inflammatory properties in sepsis-induced AKI. In the current research, we explored whether MLN4924 have anti-inflammatory action in LPS-induced AKI mice. Our results show that MLN4924 dramatically decreased the cytotoxicity of LPS and inhibited LPS-induced synthesis and release of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß, in HK2 cells, a renal tubular cell line. In addition, MLN4924 inhibited Nedd8-activating enzymes, which broke the process of cullin proteins neddylation and subsequent CRL target proteins degradation. The MLN4924-induced degradation of CRL attenuated the phosphorylation modification of IκB and IKK-α/ß and blocked the nuclear translocation of P50-NF-κB and P65-NF-κB in HK2 cells under LPS stimulation. Finally, our in vivo results show that MLN4924 protected against LPS-induced AKI at relatively low doses. Collectively, these results suggest that pharmacologically blocking neddylation by MLN4924 results in the suppression of pro-inflammatory cytokines generation through the CRL/NF-κB pathway in LPS-stimulated HK2 cells, and attenuated renal inflammation in LPS-induced AKI.

Blood concentrations of lead, cadmium, mercury and their association with biomarkers of DNA oxidative damage in preschool children living in an e-waste recycling area.

Reactive oxygen species (ROS)-induced DNA damage occurs in heavy metal exposure, but the simultaneous effect on DNA repair is unknown. We investigated the influence of co-exposure of lead (Pb), cadmium (Cd), and mercury (Hg) on 8-hydroxydeoxyguanosine (8-OHdG) and human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) mRNA levels in exposed children to evaluate the imbalance of DNA damage and repair. Children within the age range of 3-6 years from a primitive electronic waste (e-waste) recycling town were chosen as participants to represent a heavy metal-exposed population. 8-OHdG in the children's urine was assessed for heavy metal-induced oxidative effects, and the hOGG1 mRNA level in their blood represented the DNA repair ability of the children. Among the children surveyed, 88.14% (104/118) had a blood Pb level >5 µg/dL, 22.03% (26/118) had a blood Cd level >1 µg/dL, and 62.11% (59/95) had a blood Hg level >10 µg/dL. Having an e-waste workshop near the house was a risk factor contributing to high blood Pb (r s  = 0.273, p < 0.01), while Cd and Hg exposure could have come from other contaminant sources. Preschool children of fathers who had a college or university education had significantly lower 8-OHdG levels (median 242.76 ng/g creatinine, range 154.62-407.79 ng/g creatinine) than did children of fathers who had less education (p = 0.035). However, we did not observe a significant difference in the mRNA expression levels of hOGG1 between the different variables. Compared with children having low lead exposure (quartile 1), the children with high Pb exposure (quartiles 2, 3, and 4) had significantly higher 8-OHdG levels (ß Q2 = 0.362, 95% CI 0.111-0.542; ß Q3 = 0.347, 95% CI 0.103-0.531; ß Q4 = 0.314, 95% CI 0.087-0.557). Associations between blood Hg levels and 8-OHdG were less apparent. Compared with low levels of blood Hg (quartile 1), elevated blood Hg levels (quartile 2) were associated with higher 8-OHdG levels (ß Q2 = 0.236, 95% CI 0.039-0.406). Compared with children having low lead exposure (quartile 1), the children with high Pb exposure (quartiles 2, 3, and 4) had significantly higher 8-OHdG levels.

MicroRNA-30c-5p ameliorates hypoxia-reoxygenation-induced tubular epithelial cell injury via HIF1α stabilization by targeting SOCS3.

The cellular hypoxia-reoxygenation (H/R) model is an ideal method to study ischemia-reperfusion injury, which is associated with high mortality. The role of microRNA-30c-5p (miR-30c-5p) in the H/R epithelial cell model remains unknown. In the current study, we observed a significant reduction in apoptosis when miR-30c-5p was up-regulated. We also found decreased levels of C-caspase-3 (C-CASP3) and Bcl-2-associated X (BAX) proteins and increased levels of B-cell lymphoma-2 (BCL2). Epidermal growth factor receptor (EGFR) showed similar results. Down-regulating miR-30c-5p increased the levels of apoptosis and C-CASP3 and BAX expression; additionally, cell proliferation was inhibited. Hypoxia-inducible factor 1α (HIF1α) protein expression levels were up-regulated in response to up-regulation of miR-30c-5p expression. The anti-apoptotic and proliferative effects of miR-30c-5p decreased significantly after the HIF1α protein levels were knocked down. Using a luciferase reporter assay, we confirmed that miR-30c-5p targets suppressor of cytokine signaling-3 (SOCS3). HIF1α levels increased when SOCS3 was blocked. Our data show that SOCS3 expression enhances apoptosis in the H/R model. In conclusion, up-regulating miR-30c-5p protects cells from H/R -induced apoptosis and induces cell proliferation; furthermore, HIF1α markedly contributes to this protective effect. MiR-30c-5p stabilizes HIF1α expression by targeting SOCS3 to achieve anti-apoptotic and proliferative effects.

MicroRNA-140-5p attenuated oxidative stress in Cisplatin induced acute kidney injury by activating Nrf2/ARE pathway through a Keap1-independent mechanism.

Oxidative stress was predominantly involved in the pathogenesis of acute kidney injury (AKI). Recent studies had reported the protective role of specific microRNAs (miRNAs) against oxidative stress. Hence, we investigated the levels of miR140-5p and its functional role in the pathogenesis of Cisplatin induced AKI. A mice Cisplatin induced-AKI model was established. We found that miR-140-5p expression was markedly increased in mice kidney. Bioinformatics analysis revealed nuclear factor erythroid 2-related factor (Nrf2) was a potential target of miR-140-5p, We demonstrated that miR-140-5p did not affect Kelch-like ECH-associated protein 1 (Keap1) level but directly targeted the 3'-UTR of Nrf2 mRNA and played a positive role in the regulation of Nrf2 expression which was confirmed by luciferase activity assay and western blot. What was more, consistent with miR140-5p expression, the mRNA and protein levels of Nrf2, as well as antioxidant response element (ARE)-driven genes Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1) were significantly increased in mice kidney tissues. In vitro study, Enforced expression of miR-140-5p in HK2 cells significantly attenuated oxidative stress by decreasing ROS level and increasing the expression of manganese superoxide dismutase (MnSOD). Simultaneously, miR-140-5p decreased lactate dehydrogenase (LDH) leakage and improved cell vitality in HK2 cells under Cisplatin-induced oxidative stress. However, HK2 cells transfected with a siRNA targeting Nrf2 abrogated the protective effects of miR-140-5p against oxidative stress. These results indicated that miR-140-5p might exert its anti-oxidative stress function via targeting Nrf2. Our findings showed the novel transcriptional role of miR140-5p in the expression of Nrf2 and miR-140-5p protected against Cisplatin induced oxidative stress by activating Nrf2-dependent antioxidant pathway, providing a potentially therapeutic target in acute kidney injury.