It has been established that the covalent modification of proteins occurs in vivo as a consequence of reaction with reactive lipids such as 4-hydroxy-2-nonenal (HNE). The fact that HNE occurs in blood under physiological as well as pathophysiological conditions suggests that erythrocytes undergo modification by HNE. Here we describe the immunochemical characterization of HNE-treated erythrocytes by demonstrating the Michael-type HNE addition to both membrane and cytosolic proteins. Exposure of erythrocytes to HNE (0-0.5 mM) for 2 h resulted in HNE labeling of multiple membrane proteins. Pretreatment of erythrocytes with a sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant decrease of HNE attached to the proteins, suggesting that HNE primarily reacts with the sulfhydryl groups of erythrocyte membrane proteins, whereas enhanced HNE labeling of the membrane proteins was observed when the erythrocytes were pretreated with H2O2 (0.1-5 mM) for 15 min. On the other hand, highly selective modification of a 30 kDa protein was observed in the hemolysates of erythrocytes treated with HNE. The protein, which represents a major intracellular target of HNE in erythrocytes, was identified as carbonic anhydrase, based on the observations that (i) a reverse-phase HPLC analysis of the chloroform/ethanol extract of HNE-treated erythrocytes detected two major proteins, which cross-reacted with anti-carbonic anhydrase antibody as well as with the anti-HNE adducts antibody, (ii) the chloroform-ethanol extraction of authentic carbonic anhydrase gave a similar HPLC pattern, and (iii) the HNE treatment of erythrocytes resulted in the partial inhibition of the carbonic anhydrase activity.
Heart failure often presents with prognosis-relevant impaired renal function. To investigate whether the chronic activation of guanylate cyclase-A (GC-A) protects both heart and kidney, we examined the effects of TDT, a neprilysin (NEP)-resistant natriuretic peptide (NP) derivative, on cardiac and renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. Pretreatment with NEP or NEP inhibitor did not influence GC-A activation by TDT both in vitro and in vivo, resulting in a long-acting profile of TDT compared with native human atrial NP (hANP). The repeated administration of TDT to DS rats suppressed the progress of cardiac hypertrophy, systolic/diastolic dysfunction, and proteinuria in a dose-dependent manner. Compared with vehicle and hANP, salt diet-induced podocyte injury was reduced by TDT, as analyzed by urinary podocalyxin concentration, renal expression of nephrin mRNA, and glomerular expression of desmin protein. Since glomerular TRPC6 plays detrimental roles in podocyte homeostasis, we examined the renal expression of TRPC6 in DS rats and found that salt diet upregulated the expression of TRPC6. Importantly, TRPC6 induction was significantly decreased in TDT-treated rats, compared with vehicle and hANP. Consistently, in primary-culture podocytes from DS rats, TDT inhibited ATP-induced calcium influx, similar to TRPC inhibitor SKF96365. Finally, TDT-mediated protection of podocytes was abolished by protein kinase G inhibitor KT5823. In conclusion, TDT treatment attenuated heart and kidney dysfunction, accompanied by podocyte protection through inhibition of TRPC6. Thus, long-acting NPs could be a new avenue for treatment of heart failure.
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