The Role And Mechanism Of Macrophage-specific Adenosine Kinase Gene Knockout In Chronic Kidney Injury

Part I the role and mechanism of macrophage with genetic depletion of adenosine kinase in renal fibrosisObjective:To investigate the role and mechanism of macrophage with genetic depletion of adenosine kinase in renal fibrosisMethods:We chose 12 wild-type dult male mice and 12 ADK-deficient dult male mice weighing 22 to 28 g at the age of 6 and 8 weeks.The mice were divided into four groups:Group of WT control,Group of WT UUO,Group of ADK^-/-Control and Group of ADK^-/-UUO.WT control and ADK^-/-Control undergo a sham surgery.Groups of WT UUO and ADK^-/-UUO undergo a unilateral ureteral occlusion.All the mice were sacrificed 14 days after surgery and kidney cortex was preserved.Renal pathological changes were observed by Masson staining.The expression ofα-SMA and E-cadherin was detected by Western Blot.F4/80 immunofluorescence staining was used to detect the degree of macrophage infiltration and immunohistochemical staining was used to detect M1 and M2 macrophages respectively.Results:（1）Masson staining showed that the group UUO 14 days after UUO had a disordered renal structure,tubular vacuolar degeneration,tubular basement membrane nudity,renal fibrosis and infiltration of interstitial cells.Interstitial fibrosis in ADK^-/-UUO group was heavier than that in WT UUO group（P<0.05）.（2）The results of Western Blot showed that WT UUO group had a significantly increasedα-SMA expression and a reducted E-cadherin compared with that in controls（P<0.05）,and the ADK^-/-UUO group worsen the results ompared with that in WT UUO group（P<0.05）.（3）Infiltrated F4/80-positive macrophages in the WT UUO group were significantly increased than controls（P<0.05）,as detected by immunofluorescence staining,and the number of macrophages in the ADK^-/-UUO group was higher than the WT UUO group（P<0.05）.Imunohistochemical staining of i NOS,MR and Arg-1 shows M2 macrophages in the ADK^-/-UUO group ware significantly increased compared with WT UUO group（P<0.05）,and the M1 macropages were reducted（P<0.05）.Conclusion:Genetic depletion of adenosine kinase of macrophage exacerbates UUO-induced fibrotic lesions in vivo by mediating Macrophage migration and promoting M2 macrophages transformation.Part II Effect of adenosine kinase inhibition on macrophages and its mechanismObjective: To investigate the effect of adenosine kinase inhibition on macrophages and its mechanismMethods: RAW264.7 macrophages cultured in vitro were divided into control group,ADKI group（1u M）,LPS group（5ug/L）,LPS（5ug/L）+ ADKI（1u M）group,and cell protein and RNA were collected after 24 hours of intervention.The expression of i NOS,MR were detected by Western Blot and the expression of i NOS,IL-1β,TNF-α,IL-10 and MCP-1 genes were detected by PCR.Transwell Assay were divided into Control group and ADKI（1u M）group,and macrophage migration was observed after 8 hours of migration.Seahorse Energy Detector measures oxygen consumption（OCR）and ECAR in cells of the Control and ADKI（1u M）groups.Results:（1）LPS-stimulated macrophages increased the expression of i NOS,a marker of M1 macrophage,in a concentration-dependent manner.（2）ADKI promoted the migration of RAW264.7 macrophages（P<0.05）.（3）ADKI inhibited the gene level of i NOS and some pro-inflammatory cytokines,such as IL-1 β,TNF-α but upregulated MCP-1induced and IL-10 by LPS（P<0.05）.ADKI inhibited the upregulation of i NOS protein induced by LPS（P<0.05）.（4）The mitochondrial energy metabolism test showed that the basic oxygen demand（OCR）and maximal oxygen consumption of macrophagestreated with ADKI were higher than Contol group（P<0.05）,but the extracellular acidification rate（ECAR）was lower than that of Contol group（P<0.05）.Conclusion: ADKI can promote the migration of macrophages,and may promote the conversion of macrophages to M2 by inhibiting the glycolytic pathway.Part III The Expression of M2 macrophages in the Kidney of patients with diabetic kidney diseaseObjective:To investigate the expression of M2 macrophages in the patients with DKDMethods: Renal autopsy samples were obtained from 52 patients with histologically proven diabetic kidney disease in Department of Nephrology,first affiliated Hospital of Nanjing Medical University from January 2015 to June 2017,and stained for CD68 and MR for the expression of M2 macrophages by Immunofluorescence.The clinical and pathological data of the patients were collected and the correlation between the infiltration of M 2 macrophages and the clinical parameters was analyzed,and the infiltration of M 2 macrophages in different pathological stages was compared.Renal tissue specimens were scored by histopathologist to compared the infiltration of M2 macrophages in patients with different grade IF/TA scores according to the degree of interstitial fibrosis and tubular atrophy.Results:（1）The patients’ mean age was 51.0 ±12.71 years and 69.2% were male.The average level of e GFR was 61.2 ±35.02ml/min/1.73 m.2.17 of these patients developed into a nephrotic syndrome.9 of these patients had Class IIa DKD and 13 patients had Class IIb DKD,21 patients had Class III DKD,Finally,the remaining nine patients had Class IV DKD.（2）M2 macrophages wre noted in the kidney of all the patients with DKD.（3）The numbers of M2 macrophages were negatively correlated with hemoglobin（P<0.01,r=0.386）,e GFR（P<0.01,r=0.381）,serum total protein（P<0.01,r=0.365）and serum albumin（P<0.05,r=0.326）.However,It was positively correlated with 24 hour urine protein（P<0.01,r=0.412）,serum creatinine（P<0.05,r=0.342）,urea nitrogen（P<0.01,r=0.405）and fasting urine p H（P<0.05,r=0.384）.The number of M2 macrophages in nephrotic syndrome group was more than that without nephrotic syndrome（P<0.05）.（4）The number of macrophages infiltrated in patients of Class III and IV DN was significantly higher than Class IIa or IIb stage,but there is no significant difference between Class III and IV DN.Macrophage infiltration in group of score 2 was higher than that in group of score 1by IF/TA（P < 0.05）,while the degree of macrophage infiltration in group of score 3was was higher than that in group of score 2.Conclusion: The presence of M2 macrophages in DKD is common and the infiltration can occur in the class IIa.The infiltration of macrophages was correlated with higher histopathological class.