G Protein-coupled Receptor Kinase 4 Aggravates Acute Renal Ischemia-reperfusion Injury Via Necroptosis

Background:Acute renal ischemia/reperfusion(I/R)injury,a frequent postoperative complication of cardiac or renal surgeries,is a leading cause of acute renal failure and allograft damage.Despite the availability of supportive treatments,renal I/R injury is still associated with substantial morbidity and mortality.Therefore,developing new therapeutic methods is an urgent issue for renal I/R injury.G protein-coupled receptor kinases(GRKs),which play a critical role in G protein-coupled receptor(GPCR)desensitization,are involved in various ischemia-related organ injuries by excessive GPCR phosphorylation or non-canonical activities.GRK2 and GRK5 have been reported to participate in ischemic cardiomyocyte death and heart failure development.For example,the upregulation of GRK2 expression hyperphosphorylatesβ-adrenergic receptor,consequently leading to cardiac remodeling and heart failure after myocardial infarction.GRK2 inhibition is partially beneficial in the attenuation of cardiomyocyte death.GRK5 accumulates in the nuclei of cardiomyocytes and aggravates cardiac dysfunction in post-ischemic heart failure.GRK4,abundantly expressed in kidneys,plays an essential role in the development of essential hypertension by regulating renal dopamine and angiotensin II receptors.More recently,we reported that myocardial infarction markedly upregulates GRK4 expression in the nuclei of cardiomyocytes and impairs cardiac function by promoting autophagy and apoptosis.However,whether GRK4 plays a role in pathological processes during acute renal I/R injury is still unknown.The current project was performed to determine whether and how GRK4 influences acute renal I/R injury.Methods and Results:Two-month-old C57BL/6J mice were subjected to acute renal I/R injury.Among the GRK subtypes(GRK2-6)expressed in kidneys,the increase of GRK4 expression was much more apparent than the expressions of the other four GRKs 24 hours after renal I/R injury.I/R injury increased renal GRK4 protein expression in a time-dependent manner,markedly elevated at 3-6 hours,peaked at 24-48 hours,and lasted for 5-7 days.GRK4 was mainly expressed in the cytoplasm of renal tubules under normal state,while acute renal I/R injury induced GRK4 translocation from cytosol to nucleus,determined by confocal immunofluorescence with intensity profiles and nucleoplasm separation assays.In an observational study,patients with severe I/R-induced renal dysfunction had significant high levels of GRK4 expression and nuclear translocation in the injured renal tubule cells,which is of clinical significance.Moreover,gain-and loss-of-function experiments revealed that GRK4 overexpression exacerbated acute renal I/R injury,while tubule-specific knockout of GRK4 decreased I/R-induced renal dysfunction.Mechanistically,necroptosis is the major type of renal tubule cell death mediated by GRK4,because GRK4 significantly increased RIPK1 expression and phosphorylation,subsequently leading to RIPK3 and MLKL phosphorylation after renal I/R injury,which was reversed by RIPK1 inhibitor Nec-1 pretreatment.Compared with the marked increase in p-MLKL-positive renal tubules,the increase of GRK4-induced apoptosis was marginal.Using co-immunoprecipitation,mass spectrometry,and si RNA screening studies,we identified STAT1 as a GRK4 binding protein,which colocalized with GRK4 in the nuclei of renal tubule cells.Rather than Tyr701 site,GRK4 phosphorylated STAT1 at Ser727 site,whose inactive mutation effectively reversed GRK4-mediated RIPK1 activation and renal tubule cell death.Additionally,we identified the presence of a putative lysine-rich repeat NLS motif(residues 218-228)in kinase catalytic domain of GRK4.Using co-immunoprecipitation and mass spectrometry screening studies,KPNA3 was identified to mediate GRK4 translocation into the nucleus.The interaction between GRK4 and KPNA3 was markedly increased under H/R challenge,while KPNA3 knockdown significantly repressed GRK4 nuclear transportation validated by immunofluorescence,co-immunoprecipitation and nucleoplasm separation assays.In order to explore the clinical translation potential of GRK4 as a target for renal I/R treatment,kidney-targeted drug delivery of PLAG-PEI-PEG nanoparticles(NPs)encapsulating GRK4 si RNA with kidney-targeting peptide(KTP)was constructed.Both prophylactic and therapeutic injection of KTP-NPs loaded with GRK4 si RNA structurally and functionally attenuated acute renal I/R injury via suppressing GRK4 expression and RIPK1 activation.In summary,our findings showed that GRK4 inhibition was a novel intervention to mitigate I/R-induced AKI,which may have significant potential into clinical translation.Conclusion:1.GRK4 is the most prominently changed subtype of GRKs during acute renal I/R injury,whose expression and nuclear translocation is significantly elevated in the injured renal tubule cells.2.GRK4 overexpression aggravates whereas GRK4 knockout reduces acute renal I/R injury.3.Necroptosis is the major type of GRK4-mediated tubule cell death by increasing RIPK1 expression and phosphorylation.4.GRK4 phosphorylates STAT1 at Ser727 site to induce necroptosis under H/R injury.5.KPNA3 is responsible for H/R-induced GRK4 translocation into the nucleus.6.Kidney-targeted GRK4 silencing with nanoparticle delivery considerably ameliorates acute renal I/R injury.