Increased Myocardial Ischemia/Reperfusion Injury In Renal Failure Involves Cardiac Adiponectin Signal Deficiency

BackgroundChronic kidney disease (CKD) is a major public health problem worldwide, which hasbeen repeatedly documented to be associated with increased risk for cardiovascularmorbidity and mortality. It has been increasingly apparent that accelerated cardiovasculardisease (CVD) is prevalent in individuals with CKD and has become the leadingcomplication of renal insufficiency. In particular, the incidence of acute myocardialinfarction among patients with CKD is more than twice that of patients without CKD.Therefore, the prevention and treatment of cardiac insult are critical considerations in themanagement of individuals with chronic kidney disease.Adiponectin(APN), a30-kDa adipocyte-derived vasoactive peptide involved in theregulation of inflammation and energy metabolism, has shown beneficial effects oncardiovascular disorders. It is highly abundant in the circulation and exists either as trimers of truncated (globular) or full-length isoforms, or as high molecular weightcomplexes. Hypoadiponenemia has been observed in individuals with increased body fatmass and various obesity related pathologic disorders such as insulin resistance, type IIdiabetes mellitus, hypertension and cardiovascular disease. Previous studies by us as wellas others have reported that plasma levels of APN decline following myocardialischemia/reperfusion (MI/R) injury, and that exogenous administration of APN protectesheart via inhibition of oxidative/nitrative stress. However, in patients with chronic kidneydisease, plasma levels of APN are significantly increased and are inversely related to therisk of cardiovascular morbidity and mortality. The mechanism underlying the paradoxicalrelationship between high adiponectin and poor cardiac outcome remains unclear. Moreimportantly, there are no publications to date that evaluate the manipulation of APN levelon cardiovascular outcome in the presence of CKD.Objectives�'�The purposes of the present study were to determine the changes in plasma and urinaryAPN in a mouse model of renal failure and the cardiac responses to I/R injury.�'�The purposes of the present study were to delineate the role of APN in I/R in the contextof renal failure and the underlying mechanisms using APN knockout (APN-KO) mice andadministration of human recombinant globular domain of adiponectin (gAd).Methods�'�Renal failure in mice was induced by subtotal nephrectomy (SN):Animals wereanesthetized, the left kidney was decapsulated and approximately two thirds resected. Theanimals were allowed14days to recover before undergoing the second stage of theprocedure, right total nephrectomy, which followed a similar procedure as above.�'�Induction of myocardial ischemia/reperfusion:Mice were anesthetized andmyocardial infarction (MI) was produced by temporarily exteriorizing the heart via a leftthoracic incision and placing a6-0silk suture slipknot around the left anterior descendingcoronary artery. After30minutes of MI, the slipknot was released, and the myocardiumwas reperfused for3hours (for myocardial apoptosis evaluation) or24hours (for cardiacfunction and infarct size determination). At the end of reperfusion, the suture around the coronary artery was retied, and2%Evans Blue dye was injected into the left ventricularcavity.�'�The method of gAd treatment after SN:All SN mice were randomized to receiveeither vehicle (PBS,pH7.5) or human recombinant gAd (2μg/g body weight) viaintraperitoneal injection once a day for7consecutive days.�'�Biochemical assays:Plasma creatinine, urinary creatinine, urea nitrogen, urinaryalbumin and cystatin C levels were measured using commercial assay kits. Plasma orurinary concentrations of totalAPN were determined using mouse totalAPN ELISAkit.�'�Determination of cardiac function, myocardial infarct size and apoptosis: Cardiacfunction was determined by noninvasive echocardiography. Upon completion of thefunctional determination, the ligature around the coronary artery was retied, and MI sizewas determined by the Evans blue/TTC double staining method. Myocardial apoptosiswas determined within the entire I/R region via TUNEL staining and caspase-3activityassay.�'�Determination of total nitric oxide,nitrotyrosine content and superoxideproduction:The tissue nitric oxide (NO) was measured with nitrate reductase kits.Nitrotyrosine content was determined by ELISA and myocardial superoxide content wasdetermined by lucigenin-enhanced luminescence. In situ superoxide detection wasperformed with dihydroethidium staining.�'�Western blot analysis:At4weeks after SN, proteins from cardiac tissue homogenateswere separated for immunoblotting analysis. Primary antibodies included antibodiesagainst endothelial NO synthase (eNOS), phosphorylated eNOS (peNOS), inducible NOS(iNOS) and AdipoR1, AdipoR2, and pAMPK and totalAMPK.�'�Immunohistochemistry:Paraformaldehyde-fixed tissues were stained with antibodiesagainst nitrotyrosine and APN.Results�'�Renal failure is induced successfully by SN:SN mice exhibited significantly higherplasma urea nitrogen, creatinine and cystatin C, as well as urinary albumin level thannormal mice after24h of SN, whereas creatinine clearance was significantly lowersuggesting severe renal impairment resulted from SN. �'�APN concentration increased in plasma and urine but decreased in kidneyfollowing SN:Plasma total APN levels slightly declined at24h after SN, and achievedsignificant reduction at72h. This trend was reversed at1wk and gradually increasedthrough the remainder of the study (4wk). Urinary total APN concentration presented acontinuous increase after SN (4wk). In addition, a strong staining for APN was detectedon the endothelial surface of both intrarenal arteries/arterioles and glomerular andperitubular capillaries in normal WT mice, whereas the SN mice shows markedlydecreased APN staining.�'�MI/R injury is increased in SN mice:Compared with nomal mice, SN mice displayedsignificantly depressed cardiac function and enlarged infarct size. Moreover, TUNELstaining and caspase-3activity assay demonstrated markedly increased cardiomyocyteapoptosis in SN mice follwoing MI/R mice.�'�MI/R injury is markedly increased in APN-KO mice with SN and is rescued byadministration of gAd:Compared with WT mice with SN, cardiac function was furtherdepressed and MI size was enlarged in KO mice with SN. However,7days of gAdtreatment after SN significantly enhanced cardiac function and reduced infarct size inboth WT and KO mice with SN mice. gAd treatment also reduced cardiomyocyteapoptosis in both WT mice and KO mice with SN.�'�SN increases myocardial NO production via upregulation of iNOS expression: SNreduced eNOS phosphorylation and upregulated iNOS expression, both contributing toincreased NO production in cardiac tissue, and gAd treatment produced the oppositeeffects.�'�SN increases myocardial O-2and peroxynitrite production, which can be reversedby administration of gAd：Superoxide and peroxynitrite induced by SN were furtherintensified in cardiac tissue obtained from WT and KO animals, and gAd treatmentattenuated both superoxide and peroxynitrite production.�'�Change in cardiac APN receptor expression after SN：AdipoR1expression wasreduced4-wk after SN whereas AdipoR2expression showed no significant change. Moreimportantly, AMPK activation was also inhibited after SN and exogenous gAdsupplementation reversed this change. Conclusion�'�The present study demonstrates that renal dysfunction increases cardiac susceptibilityto ischemic/reperfusion injury, which is associated with downregulated APN/AdipoR1/AMPK signaling and increased oxidative/nitrative stress in local myocardium.�'�The present study provides the first evidence for the benefits of exogenous supplementofAPN on cardiac outcomes in renal failure.