<b><i>Background:</i></b> Chloroquine (CQ), a classic autophagy inhibitor, is used clinically for malaria prophylaxis and pulmonary hypertension treatment. The adverse effects of CQ on morphological and functional changes in the kidney were investigated in the current study due to CQ accumulation in the kidney. <b><i>Methods:</i></b> Twelve male Sprague-Dawley rats were randomly divided into 2 groups for 4 weeks: group 1, control (<i>n</i> = 6); and group 2, CQ administration group (50 mg<sup>–1</sup>·kg per day ip; <i>n</i> = 6). Serum aldosterone and vasopressin were measured by radioimmunoassay. Immunofluorescence was used to colocalize Tunel with aquaporin 1, aquaporin 2 (AQP2), and Tamm-Horsfall protein. Expression of AQP2 and mineralocorticoid (MR) was detected by western blot and immunohistochemistry. <b><i>Results:</i></b> In the present study, 4 weeks of CQ administration were shown to induce severe kidney injury, including glomerular sclerosis and tubular cells apoptosis, especially distal tubular cells. Decreased expression of LC3II/I and p-AKT was demonstrated in CQ-treated rats. Glomerular and proximal tubule injury were associated with impaired autophagy flux, and distal tubule injury may be associated with downregulated cyclic adenosine monophosphate (cAMP)/PKA/AKT signaling. Both MR and AQP2, which are mainly located in the distal tubule and collecting duct, were significantly reduced in CQ-treated rats, thus leading to increased exosomal secretion of AQP2 in urine. Additionally, chronic CQ administration increased aldosterone and vasopressin levels in serum, but lowered the blood pressure, glomerular filtration rate, and urine concentration. <b><i>Conclusions:</i></b> CQ administration damages glomerular, proximal tubule autophagy, and severe distal tubular cells apoptosis by inhibiting cAMP/PKA/AKT signaling.
Oxygen homeostasis disturbances play a critical role in the pathogenesis of acute kidney injury (AKI). The transcription factor hypoxia-inducible factor-1 (HIF-1) is a master regulator of adaptive responses to hypoxia. Aside from post-translational hydroxylation, mechanism of HIF-1 regulation in AKI remains largely unclear. In this study, the mechanism of HIF-α regulation in AKI was investigated. We found that tubular HIF-1α expression significantly increased at the transcriptional level in ischemia/reperfusion (I/R)-, unilateral ureteral obstruction (UUO)-, and sepsis-induced AKI models, which was closely associated with macrophage-dependent inflammation. Meanwhile, nuclear factor-κB (NF-κB), which plays a central role in inflammation response, was involved in the increasing expression of HIF-1α in AKI, as evidenced by pharmacological modulation (NF-κB inhibitor BAY11-7082). Mechanistically, NF-κB directly bound to the HIF-1α promoter and enhanced its transcription, which occurred not only in hypoxic condition, but also in normoxic condition. Moreover, the induced HIF-1α by inflammation protected against the tubular injury in AKI. Thus, our findings not only provide novel insight into HIF-1 regulation in AKI but also offer to understand the pathophysiology of kidney diseases.
The reduction of podocyte injury is a key strategy in controlling proteinuria, which is the main early clinical manifestation of diabetic nephropathy (DN). Impaired autophagic flux is the primary mechanism responsible for podocyte injury in DN. The aim of the present study was to elucidate the effect of connexin 43 (Cx43) on impaired autophagic flux in podo-cyte injury and to explore its molecular mechanism of action in DN. Sprague-Dawley rats were administered streptozocin (STZ) to construct a DN animal model. Podocytes were incubated in media containing either buffer or high glucose (HG; 30 mM) for variable time periods. The podocytes were then examined and the mechanism of injury was investigated using an Annexin V/PI assay, immunofluorescence staining, western blotting, and RNA interference. In vivo, STZ-induced DN rats with or without Cx43 knockdown were established to observe the role of Cx43 in autophagic flux and podocyte injury. We observed that HG induced podocyte injury, accompanied by increases in Cx43 expression and impaired autophagic flux, as evidenced by the accumulation of LC3II/LC3I and p62. Interestingly, the silencing of Cx43 expression ameliorated autophagic flux impairment and reduced podocyte injury via suppression of the mammalian target of rapamycin pathway. Furthermore, impaired autophagic flux also blocked the degradation of Cx43. In vitro studies indicated that higher numbers of Annexin V/PI-positive podocytes, impaired autophagic flux and increased Cx43 expression were observed in HG-induced podocyte injury relative to the control group. The pathogenic effect of Cx43 on impaired autophagic flux and podocyte injury was also confirmed by Cx43 knockdown. The present study provided preliminary evidence indicating that the interdependence of Cx43 and impaired autophagic flux represents a novel mechanism of podocyte injury in DN. Hence, the Cx43-autophagy loop is a potentially relevant therapeutic target for the treatment of DN.
Renal tubulointerstitial fibrosis (TIF) is a hallmark in the continuous progression of chronic kidney disease (CKD), in which excessive activation of the renin-angiotensinaldosterone system serves a crucial role. Currently, there are no targeted therapies for the progression of TIF. microRNA (miR)-26a may be an ideal anti-fibrosis candidate molecule; however, the effect of miR-26 on aldosterone (ALD)-induced TIF remains unclear. This study aimed to elucidate the role of miR-26a in ALD-induced TIF. In the present study, we hypothesized that delivery of miR-26a by exosomes could attenuate ALD-induced TIF. miR-26a expression was downregulated in the kidney of ALD-induced mice compared with the mice in the sham group. Exosome-encapsulated miR-26a (Exo-miR-26a) was manufactured and injected into ALD-treated mice through the tail vein. In vivo experiments showed that Exo-miR-26a alleviated the downregulated miR-26a expression in the kidney, tubular injury and ALD-induced TIF, which was determined using Masson's trichrome staining and assessment of lipocalin 2, α-smooth muscle actin, collagen I and fibronectin expression. Moreover, in vitro experiments revealed that Exo-miR-26a inhibited epithelial-mesenchymal transition and extracellular matrix deposition in mouse tubular epithelial cells. Mechanistically, overexpressing miR-26a led to decreased expression levels of connective tissue growth factor by directly binding to its 3'-UTR and inhibiting the activation of SMAD3. These findings demonstrated that the exosomal delivery of miR-26a may alleviate ALD-induced TIF, which may provide new insights into the treatment of CKD.
Transfer RNA-derived fragments (tRFs), a novel class of small non-coding RNA produced by the cleavage of pre-and mature tRNAs, are involved in various diseases. Renal tubulointerstitial fibrosis is a common final pathway in diabetic nephropathy (DN) in which hyperglycemia-induced tubular extracellular matrix (ECM) accumulation serves a vital role. The present study aimed to detect and investigate the role of tRFs in the accumulation of tubular ECM. Differentially expressed tRFs were analysed with high-throughput sequencing in primary mouse tubular epithelial cells treated with high glucose (HG). The Gene Ontology (GO) was used to analyze the potential molecular functions of these differentially expressed tRFs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the associated signaling pathways involved in these differentially expressed tRFs. tRF-1:30-Gln-CTG-4 was overexpressed using tRF-1:30-Gln-CTG-4 mimic, followed by HG treatment. A total of 554 distinct tRFs were detected and 64 differentially expressed tRFs (fold change >2; P<0.05) were identified in tubular epithelial cells following high glucose (HG) treatment, among which 27 were upregulated and 37 were downregulated. Ten selected tRFs with the greatest difference (fold change >2; P<0.05) were verified to be consistent with small RNA-sequencing data, of which tRF-1:30-Gln-CTG-4 showed the most pronounced difference in expression and was significantly decreased in response to HG. GO analysis indicated that the differentially expressed tRFs were associated with 'cellular process', 'biological regulation' and 'metabolic process'. An analysis of the KEGG database suggested that these differentially expressed tRFs were involved in 'autophagy' and signaling pathways for 'forkhead box O', 'the mammalian target of rapamycin' and 'mitogen-activated protein kinase'. Finally, the overexpression of tRF-1:30-Gln-CTG-4 ameliorated HG-induced ECM accumulation in tubular epithelial cells. Therefore, the present study demonstrated that there may be a significant association between tRFs and HG-induced ECM accumulation in tubular epithelial cells; these differentially expressed tRFs warrant further study to explore the pathogenesis of DN.
Background: Glomerulosclerosis is a characteristic pathologic feature in chronic kidney disease (CKD). Convincing evidence indicates that the mesangial cells (MCs) play critical role in this process. However, the exact mechanism remains unclear. Using RNA-seq analysis, we previously found that lncRNA uc.412 was involved in the MC proliferation. Here, the effect of uc.412 on glomerular fibrosis and the potential mechanism were explored. Methods: In vivo, CKD mice models were established by 5/6 nephrectomy. The expression of lncRNA uc.412 in CKD was detected by Real-Time PCR. In vitro, MCs were intervened with TGF-β1 (10ng/mL). The uc.412 expression in MCs was detected by in site hybridization. MCs were transfected with uc.412 siRNA or a lentivirus targeting uc.412 and then examined using western blot, Real-Time PCR, RNA pull down assay and immunofluorescence staining. Results: We found that the expression of uc.412 was significantly increased in CKD mice and is induced by TGF-β1 via Smad3- dependent signal pathway. Overexpressing uc.412 caused MCs fibrosis and knockdown of uc.412 alleviated TGF-β1-induced MCs fibrosis. Using RNA pull down analysis, we found that the ELAVL1 was the specific binding protein for uc.412. Moreover, ELAVL1 expression was increased in TGF-β1-treated MCs and silencing ELAVL1 expression attenuated MCs fibrosis. Conclusions: Thus, here, we demonstrated that uc.412, which is regulated in a Smad3-dependent mechanism, is significantly increased during progression of CKD via regulating ELAVL1 expression. Our findings provided the therapeutic strategy for treatment of CKD.
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