Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155.

BACKGROUND: Chronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence. METHODS: Among the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155-/- mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. RESULTS: Compared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated ß-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs. CONCLUSIONS: Our work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

Satellite cell-derived exosome-mediated delivery of microRNA-23a/27a/26a cluster ameliorates the renal tubulointerstitial fibrosis in mouse diabetic nephropathy.

Renal tubulointerstitial fibrosis (TIF) is considered as the final convergent pathway of diabetic nephropathy (DN) without effective therapies currently. MiRNAs play a key role in fibrotic diseases and become promising therapeutic targets for kidney diseases, while miRNA clusters, formed by the cluster arrangement of miRNAs on chromosomes, can regulate diverse biological functions alone or synergistically. In this study, we developed clustered miR-23a/27a/26a-loaded skeletal muscle satellite cells-derived exosomes (Exos) engineered with RVG peptide, and investigated their therapeutic efficacy in a murine model of DN. Firstly, we showed that miR-23a-3p, miR-26a-5p and miR-27a-3p were markedly decreased in serum samples of DN patients using miRNA sequencing. Meanwhile, we confirmed that miR-23a-3p, miR-26a-5p and miR-27a-3p were primarily located in proximal renal tubules and highly negatively correlated with TIF in db/db mice at 20 weeks of age. We then engineered RVG-miR-23a/27a/26a cluster loaded Exos derived from muscle satellite cells, which not only enhanced the stability of miR-23a/27a/26a cluster, but also efficiently delivered more miR-23a/27a/26a cluster homing to the injured kidney. More importantly, administration of RVG-miR-23a/27a/26a-Exos (100 µg, i.v., once a week for 8 weeks) significantly ameliorated tubular injury and TIF in db/db mice at 20 weeks of age. We revealed that miR-23a/27a/26a-Exos enhanced antifibrotic effects by repressing miRNA cluster-targeting Lpp simultaneously, as well as miR-27a-3p-targeting Zbtb20 and miR-26a-5p-targeting Klhl42, respectively. Knockdown of Lpp by injection of AAV-Lpp-RNAi effectively ameliorated the progression of TIF in DN mice. Taken together, we established a novel kidney-targeting Exo-based delivery system by manipulating the miRNA-23a/27a/26a cluster to ameliorate TIF in DN, thus providing a promising therapeutic strategy for DN.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

HIF-1α and adaptor protein LIM and senescent cell antigen-like domains protein 1 axis promotes tubulointerstitial fibrosis by interacting with vimentin in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Activation of the renin-angiotensin system, as a hallmark of hypertension and chronic kidney diseases (CKD) is the key pathophysiological factor contributing to the progression of tubulointerstitial fibrosis. LIM and senescent cell antigen-like domains protein 1 (LIMS1) plays an essential role in controlling of cell behaviour through the formation of complexes with other proteins. Here, the function and regulation of LIMS1 in angiotensin II (Ang II)-induced hypertension and tubulointerstitial fibrosis was investigated. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with Ang II to induce tubulointerstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) renal tubular-specific knockout mice or LIMS1 knockdown AAV was used to investigate their effects on Ang II-induced renal interstitial fibrosis. In vitro, HIF-1α or LIMS1 was knocked down or overexpressed in HK2 cells after exposure to Ang II. KEY RESULTS: Increased expression of tubular LIMS1 was observed in human kidney with hypertensive nephropathy and in murine kidney from Ang II-induced hypertension model. Tubular-specific knockdown of LIMS1 ameliorated Ang II-induced tubulointerstitial fibrosis in mice. Furthermore, we demonstrated that LIMS1 was transcriptionally regulated by HIF-1α in tubular cells and that tubular HIF-1α knockout ameliorates LIMS1-mediated tubulointerstitial fibrosis. In addition, LIMS1 promotes Ang II-induced tubulointerstitial fibrosis by interacting with vimentin. CONCLUSION AND IMPLICATIONS: We conclude that HIF-1α transcriptionally regulated LIMS1 plays a central role in Ang II-induced tubulointerstitial fibrosis through interacting with vimentin. Our finding represents a new insight into the mechanism of Ang II-induced tubulointerstitial fibrosis and provides a novel therapeutic target for progression of CKD.

[Bufalin inhibits PDGF-BB-induced mesangial cell proliferation via mediating gap junctional intercellular communication].

OBJECTIVE: To study the role and possible mechanisms of gap junctional intercellular communication (GJIC) involved in mesangial cell (MC) proliferation which could be inhibited by bufalin. METHODS: Rat mesangial cells were cultured in vitro. The effect of bufalin on platelet-derived growth factor-BB (PDGF-BB)-induced MC proliferation was evaluated by MTT assay. The function of GJIC was detected by Lucifer Yellow scrape loading and dye transfer (SLDT). mRNA levels of Cx43, Cx45 and Cx40 were measured by RT-PCR. Intracellular calcium concentrations ([Ca(2+)]i) were examined in laser scanning confocal microscopy after loading by Fura-3/AM. RESULTS: MTT indicated that bufalin could inhibited PDGF-BB-induced MC proliferation (P<0.01). Compared with the hormal control group, PDGF-BB inhibited GJIC function, increased the expression of Cx45 and Cx40 (P<0.01) without altering the Cx43 (P>0.05) in gene level and also increased [Ca(2+)]i. However, bufalin treatment enhanced GJIC function, decreased Cx45 mRNA and Cx40 mRNA expression (P<0.01), and reduced [Ca(2+)]i (P<0.01). CONCLUSIONS: Bufalin inhibits PDGF-BB-induced MC proliferation, and its possible mechanisms may be related to regulation of Cx45 and Cx40 expression in the gene level, reduction of [Ca(2+)]i and enhancement of GJIC function.

MiR-155 deficiency protects renal tubular epithelial cells from telomeric and genomic DNA damage in cisplatin-induced acute kidney injury.

Rationale: Cisplatin nephrotoxicity is an important cause of acute kidney injury (AKI), limiting cisplatin application in cancer therapy. Growing evidence has suggested that genome instability, telomeric dysfunction, and DNA damage were involved in the tubular epithelial cells (TECs) damage in cisplatin-induced AKI (cAKI). However, the exact mechanism is largely unknown. Methods: We subjected miR-155-/- mice and wild-type controls, as well as HK-2 cells, to cAKI models. We assessed kidney function and injury with standard techniques. The cell apoptosis and DNA damage of TECs were evaluated both in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. Results: The expression level of miR-155 was upregulated in cAKI. Inhibition of miR-155 expression protected cisplatin-induced AKI both in vivo and in vitro. Compared with wild-type mice, miR-155-/- mice had reduced mortality, improved renal function and pathological damage after cisplatin intervention. Moreover, inhibition of miR-155 expression attenuated TECs apoptosis and DNA damage. These protective effects were caused by increasing expression of telomeric repeat binding factor 1 (TRF1) and cyclin-dependent kinase 12 (CDK12), thereby limiting the telomeric dysfunction and the genomic DNA damage in cAKI. Conclusion: We demonstrated that miR-155 deficiency could significantly attenuate pathological damage and mortality in cAKI through inhibition of TECs apoptosis, genome instability, and telomeric dysfunction, which is possibly regulated by the increasing expression of TRF1 and CDK12. This study will provide a new molecular strategy for the prevention of cAKI.

[Effects of exogenous connective tissue growth factor on collagen III synthesis of human renal tubular epithelial cells].

OBJECTIVE: To explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro. METHODS: Cultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein. RESULTS: After 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group. CONCLUSIONS: CTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

[Transdifferentiation and collagen-synthesis effects of exogenous connective tissue growth factor on renal tubular epithelial cells in vitro].

OBJECTIVE: To investigate the effects of recombinal human connective tissue growth factor (rhCTGF) stimulation on epithelial-myofibroblast transdifferentiation (EMT) and collagen-synthesis in human renal tubular epithelial cell line (HK2) in vitro. METHODS: The cultured HK2 cells were stimulated with rhCTGF of 5 ng/mL. The morphological changes were observed under an inverted microscope. The cells were collected at 0, 3, 6, 12, 24 and 48 hrs after rhCTGF stimulation. The expression of E-cadherin,alpha-smooth muscle actin (alpha-SMA), collagen Ialpha1 (Col Ialpha1) and collagen IValpha1 (Col IValpha1) mRNAs were detected by RT-PCR. RESULTS: rhCTGF stimulation changed the HK2 cell appearance from oval to fusiformdown-regulated the E-cadherin mRNA expression and up-regulated alpha-SMA mRNA expression, but had no effects the Col Ialpha1 and Col IValpha1 mRNA expression. CONCLUSIONS: Exogenous CTGF can mediate the EMT but has no collagen-synthesis effects on HK2 cells.

[Effect of insulin-like growth factor I on the transdifferentiation and collagen synthesis of human renal tubular epithelial cells].

AIM: To investigate the role of insulin-like growth factor I(IGF-I) in transdifferentiation and collagen synthesis of human renal tubular epithelial cell line HK2 in vitro. METHODS: Cultured HK2 cells were divided into two groups: (1) control group; (2) IGF-I-treated group(25, 50, 100, 200 microg/L, respectively). The cell morphological changes were traced with inverted microscope, and the expression of alpha-smooth muscle actin (alpha-SMA), collagen I alpha and collagen III alpha mRNA was detected by RT-PCR. Concentration of collagen I secreted into the culture supernatant was determined by ELISA. RESULTS: In HK2 cells, IGF-I stimulated the morphological oval-to-fusiform transdifferentiation of the cells, and upregulated alpha-SMA, collagen I alpha and collagen III alpha mRNA expression(P<0.05). Secreted collagen I level was also up-regulate by IGF-I in the concentration of 100 and 200 microg/L, respectively. CONCLUSION: IGF-I can promote the transdifferentiation of human renal tubular epithelial cells and stimulate the synthesis of collagen.

[Enhance effect of Ox-LDL on NF-kappaB activity in glomerular mesangial cells from the Zucker rats].

AIM: To study the effect of oxidized low-density lipoprotein (Ox-LDL) on nuclear NF-kappaB activity in cultured glomerular mesangial cells (GMCs) from the Zucker rats and the correlation between the change of NF-kappaB activity and ras's age and genotype. METHODS: Four groups of GMCs (O(3m),O(10m), L(3m) and L(10m)) from the 3- and 10- month (fa/fa) and 3- and 10- month lean (Fa/Fa) Zucker rats were stimulated with Ox-LDL. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activity of NF-kappaB. Gel supershift assay(GSA) was used to detect the subunit of NF-kappaB dimer. RESULTS: (1) NF-kappaB activity after induction with Ox-LDL in the 3 groups (O(3m),O(10m) and L(3m)) of GMCs were significantly higher than that in the control group (P<0.01). (2) With the increased of concentration and stimulation time of Ox-LDL, the NF-kappaB activity was increased correspondingly and reached the highest when GMCs were stimulated with 50 mg/L Ox-LDL for 4 hours. (3) Supershift assay demonstrated that Ox-LDL mainly activated the two subunits p65 and p50 in GMCs. (4) As the activity of NF-kappaB, O(3m) group vs O(10m) group, O(3m) group vs L(3m) group, and O(10m) group vs L(3m) group had all markedly differences (P<0.01), while the differences between L(3m) group vs L(10m) group was inferior to 3 groups above (P<0.05). CONCLUSION: Ox-LDL could significantly activate NF-kappaB in GMCs from Zucker rats, in the time- and dose- dependence manner. There was a close relationship between the NF-kappaB activity in GMCs induced with Ox-LDL, the age and genotype of Zucker rats. NF-kappaB activity in GMCs of the Zucker rats induced with Ox-LDL plays an important role in the early renal lesion of diabetes mellitus.