The Effects And Mechanisms Of HIF-1? Activation In Tubulointerstitial Inflammation And Fibrosis

Objective: Tubulointerstitial inflammation(TI)is a critical pathological feature of kidney diseases and triggers the development of interstitial fibrosis(TIF).Renal hypoxia,which is one of the most common causes of kidney injury,is a key instigator of TI.Hypoxic TECs can facilitate TI by promoting macrophage.However,the molecular signals through which hypoxic TECs activate macrophages remain obscure.Hypoxia inducible factor-1(HIF-1)is a key transcription factor mediating adaptive responses to hypoxia.In response to hypoxia in the kidney,HIF-1? is expressed predominantly in TECs and works as a master regulator of hypoxic stress.However,whether HIF-1? expressed in TECs mediates hypoxia-induced renal TI through TECs-macrophage interactions is a fundamental question for further study.Exosomes are small extracellular vesicles secreted by various cell types,with size ranging from 30 to 150 nm.Exosomes could transfer micro RNA(mi RNA)and thus shuttle information to target cells in the immediate vicinity of,or at a distance from,the parent cell.The exosomal transfer of mi RNAs could be a novel mechanism for intercellular communication by exerting their regulatory effects on recipient cells.Therefore,whether HIF-1? in TECs promotes TI through exosomal mi RNAs during hypoxia-related TI need further to be explored.The identification of oxygen-dependent prolyl hydroxylase domain(HIF-PHD)enzymes as regulators of HIF has led to the development of novel therapeutic agents for renal anemia.HIF-PH inhibitor(HIF-PHI)primarily functions by mimicking the hypoxia-driven expression of HIF.Convincing evidence suggests that HIF-? could be activated by HIF-PHI in a dose-dependent manner.Although HIF-PHIs were well-tolerated in clinical trials to treat CKD patients with anemia,there are several safety concerns related to the potential non-erythropoietic effects due to repeated or persistent HIF activation.However,the effects of HIF activation induced by HIF-PHI on TIF in CKD remain unclear.MK-8617(MK),a recently identified,selective,orally bioavailable HIF-PHI,actively stimulates erythropoiesis.However,the effects and mechanisms of HIF-1? activation,which is induced by MK administration with long time,on TIF in CKD mice need to be elucidated.The present study aimed to investigate whether HIF-1? expressed in TECs promotes TI through exosomal mi RNAs during hypoxia-related TI.Furthermore,the effects and mechanisms of HIF-1? activation,which is induced by MK administration with long time,on TIF in CKD mice were explored.The study includes four parts:Part one: The study on relationship between TI and hypoxia-induced renal tubular epithelial cells HIF-1? and exosomal micro RNA-23aMethod: Bilateral ischemia/reperfusion(I/R)injury and unilateral ureteral obstruction(UUO)mice models were estabilshed.Mice were killed on days 1,3,or 7 after I/R injury or UUO under general anesthesia,and their urine,serum and kidneys were harvested for renal function analysis,pathologic and immunohistochemistry staining and molecular biological analysis,respectively.Exosomes from hypoxic kidney and hypoxic tubules were isolated using differential centrifugation.The mi RNA-23 a inhibitor was transfected into I/R injury kidneys using the in vivo-jet PEI.Mice were killed 24 hours after injected,and their urine,serum and kidneys were harvested for renal function analysis,pathologic and immunohistochemistry staining and molecular biological analysis,respectively.Results: In I/R injury and UUO models,renal TI was observed on days 1,3,and 7 associated with an increase in F4/80+ macrophages infiltration.Concomitantly,there were significant increases in m RNA expression of renal inflammatory cytokines(MCP-1,TNF-?,and IL-1?)and phosphorylation of NF-?B p65(p-p65)at days 1 and 3,preceding a significant reduction at day 7.The expression of HIF-1? was significantly increased in the injured kidney at days 1 and 3,preceding a significant decrease at day 7.The hypoxic kidney secreted exosomes that were highly enriched in mi RNA-23 a at days 1 and 3,receding at day 7.Interestingly,tubular exosomes were highly enriched in mi RNA-23 a in I/R injured kidneys and kidneys with UUO at day 1 and day 3,respectively.Meanwhile,mi RNA-23 a inhibition represses F4/80+ macrophages infiltration,renal inflammatory cytokines and p-p65 expression in hypoxia-induced kidney injury.Conclusion: There are close temporal correlation between the TI and hypoxia induced TECs HIF-1? and exosomal mi RNA-23 a expression level.mi RNA-23 a plays a critical role in hypoxia-induced TI.Part two: The mechanism of TI induced by HIF-1?-mediated exosomal mi RNA-23aMethod: Exosomes from TECs were isolated using differential centrifugation.HIF-1? si RNAs were used to regulate the HIF-1? levels.The role of HIF-1? on mi RNA-23 a expression was studied using Chromatin immunoprecipitation-PCR(Ch IP-PCR).Besides,exosomal mi RNA-23 a was inhibited or overexpressed using mi RNA-23 a inhibitor or mi RNA-23 a mimics.The luciferase reporter assay was performed to test the interaction between the mi RNA-23 a and 3'-UTR of A20.In vivo,TECs exosomes silenced mi RNA-23 a was transferred to mice via kidney parenchyma injection(10 ?g in 60 ?l of PBS).Their kidneys were harvested for pathologic and immunohistochemistry staining and molecular biological analysis.Results: Hypoxic TECs presented with higher HIF-1? and upregulation of p-p65 as well as exosomal mi RNA-23 a.Ch IP-PCR assay showed that the binding of HIF-1? was enriched in the mi RNA-23 a promoter in TECs by hypoxia stimulation.Exosome from hypoxic TECs could develop more severe macrophage activation compared to exosome released from normal TECs.Furthermore,mi RNA-23 a secreted from hypoxic TECs can be transferred to macrophages via exosomes.More interestingly,exo(Hypo)-mi RNA-23a-mimic increased the release of inflammatory factors from macrophages,an effect that was abrogated by exo(Hypo)-mi RNA-23a-inhibitor.Furthermore,we demonstrated that exosomal mi RNA-23 a directly suppressed its target A20,leading to macrophage activation.Inhibition of mi RNA-23 a reversed macrophage activation.In vivo,the uptake of exo(Hypo)by intrarenal CD68+ macrophages,and infiltration of these cells was significantly increased.And mi RNA-23 a predominantly located in the cells of CD68 staining macrophages.F4/80+ macrophages infiltration,inflammatory cytokines and p-p65 expression of kidney were significantly increased when mice transferred with mi RNA-23 a enriched exosomes,which was not shown by mi RNA-23 a silenced exosomes.Conclusion: Transcriptionally regulated mi RNA-23 a by TECs HIF-1?,which can be transferred to macrophages via exosomes,could promote NF-?B signaling through inhibiting A20,activating macrophages to induce TI.Part three: The effect of HIF-1? activation on TIF in CKD miceMethod: CKD mice models were estabilshed in mice by 5/6 nephrectomy(5/6Nx).After 8 weeks,the CKD mice were dosed with vehicle [DMSO/PEG400/water(5:40:55,v/v/v)] or MK(a novel HIF-PHI,1.5,5 or 12.5 mg/kg/d in vehicle)for 12 weeks.Mice were killed on week 20 after 5/6Nx under general anesthesia,and their urine,blood and kidneys were harvested for renal function analysis and molecular analysis,pathologic and immunohistochemistry staining and molecular biological analysis,respectively.Results: Western blotting showed that HIF-? could be stabilized by MK dose-dependently.HIF-1? activation has concentration-dependent biphasic effects on kidney function.Compared to vehicle-administered CKD mice,serum creatinine,blood urea nitrogen and albuminuria are significantly decreased in mice with HIF-1? mild activation(1.5mpk)or HIF-1? moderate activation(5mpk)and increased in those with HIF-1? excessive activation(12.5mpk).HIF-1? activation has concentration-dependent biphasic effects on TIF in vivo.Notably,compared to vehicle-administered CKD mice,?-SMA,collagen-I and fibronectin was significantly attenuated in mice with HIF-1? mild activation(1.5mpk)or HIF-1? moderate activation(5mpk)and increased in those with HIF-1? excessive activation(12.5mpk).Conclusion: HIF-1? activation has concentration-dependent biphasic effects on TIF.TI was significantly attenuated in mice with HIF-1? mild or moderate activation and markedly increased in those with HIF-1? excessive activation.Part four: The mechanism of TECs HIF-1? excessive activation promoting TIF in CKD miceMethod: RNA-sequencing was used to characterize the expression patterns of m RNA genes in HK-2 cells treated with MK.Self-organizing maps(SOMs)were intuitively used to analyze the structure and interrogate transcriptome data.The role of HIF-1? on KLF5 expression was studied using Ch IP-PCR.Besides,KLF5 was inhibited using si RNA to explore its functional role in TECs.Furthermore,the KLF5 knockdown mice were established by Lentiviruses expressing short-hairpin RNAs(sh RNAs)targeting KLF5.These mice were administrated with MK(12.5mg/kg/d)for 10 weeks.Mice were killed under general anesthesia,and their urine,blood and kidneys were harvested for renal function analysis,pathologic and immunohistochemistry staining and molecular biological analysis,respectively.Results: As indicated by the levels of ?-SMA,collagen-I and fibronectin,fibrogenesis was promoted in HK-2 cells with HIF-1? excessive activation(500 and 1000 n M).Intriguingly,Krüppel-Like Factor 5(KLF5)expression appeared to be markedly upregulated in HK-2 cells with HIF-1? excessive activation by genome-wide sequencing analysis.Ch IP assay showed that the binding of HIF-1? was enriched in the KLF5 promoter in TECs with HIF-1? excessive activation.The role of KLF5 in fibrogenesis was validated in in vivo and in vitro.Furthermore,TGF-?1 is significantly increased in mice with HIF-1? excessive activation(12.5mpk).KLF5 stimulates ?-SMA,collagen-I and fibronectin expression by upregulating TGF-?1.Knockdown of KLF5 markedly diminished the increase of ?-SMA,collagen-I and fibronectin expression in TECs.And KLF5 knockdown also diminished the increase of ?-SMA,collagen-I and fibronectin expression induced by HIF-1? excessive activation in CKD mice.Conclusion: Excessive activation of HIF-1? in TECs could promote TIF in CKD mice by activating HIF-1?-KLF5-TGF-?1 signalling.Conclusions for the full text:1.Hypoxia is a key instigator of TI.There are close temporal correlation between the TI and hypoxia induced TECs HIF-1? and exosomal mi RNA-23 a expression level.mi RNA-23 a plays a critical role in hypoxia-induced TI.Interrupting this broad,vertically integrated mi RNA-23 a signaling cascade represents a promising therapeutic target for hypoxia-induced TI.2.We have discovered an important role for tubular HIF-1? in inciting TI in the hypoxic kidney via exosomal mi RNA-23 a mediated intercellular communication between TECs and macrophages.3.HIF-1? activation has concentration-dependent biphasic effects on TIF.4.Excessive activation of HIF-1? could promote TIF by activating HIF-1?-KLF5-TGF-?1 signaling.Innovations of this study:1.We find that there are close temporal correlation between the hypoxia-induced TI and TECs HIF-1? and exosomal mi RNA-23 a expression.Transcriptionally regulated mi RNA-23 a by TECs HIF-1?,which can be transferred to macrophages via exosomes,could promote NF-?B signaling activation in macrophages to induce TI.2.We firstly demonstrate that TECs HIF-1? activation,which is induced by HIF-PHI MK-8617,has concentration-dependent biphasic effects on TIF in CKD mice.The pro-fibrotic effect of HIF-1? excessive activation on TIF is mediated by activating HIF-1?-KLF5-TGF-?1 signaling.