SUN-038 HOMOCYSTEINE EXACERBATES IRI-INDUCED ACUTE KIDNEY INJURY VIA PROMOTING MEGAKARYOCYTE MATURATION AND PROPLATELET FORMATION

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Platelets are crucial regulatory factors of acute kidney injury (AKI), as they are present in high numbers in the circulation and could release massive bioactive factors, which participate in intra-renal inflammation and haemostasis. After ischaemia-reperfusion injury (IRI), unbalanced macrocirculation and microcirculation lead to hypoxaemic injury and inflammatory response and haemostatic processes (platelet sequestration and thrombin). Homocysteine (Hcy), the important intermediate product of methionine cycle, is elevated as related genetic enzymatic deficiencies or nutritional defects which interfere metabolism of methionine. Moderate hyperhomocysteinemia (HHcy) is defined as total plasma Hcy in the range of 16-30uM, which is emerged as prothrombotic factor, influencing coagulative status and endothelial function and modulating platelet aggregation. We therefore aimed to evaluate the effects of hcy levels on platelet in IRI-induced AKI. Experiments were performed using 6 to 8 weeks old male C57BL/6 mice. To generate HHcy mice, mice were fed with high methionine diet containing 19.56g/kg methionine for 3 weeks. IRI induced AKI were performed by bilateral renal pedicles clamped for 30min in 37℃ with or without oral clopidogrel. After 48h, mice were euthanized and blood and kidney tissues were collected. For FeCl3 carotid artery injury, filter paper soaked in FeCl3 solution (3.5%) was placed on the right carotid artery for 90 seconds. Flow was recorded by Doppler Image scanner. As to deep venous thrombosis, the inferior vena cava was ligated for 48h and the length and weight of thrombus were recorded. A preparation of MKs was obtained from femora and tibiae BM of C57BL/6. Fetal liver-derived MKs was from the liver of mouse fetuses aged 13-15 days. The MKs was incubated with Hcy or not. In the present research, we found hyperhomocysteinemia mice developed more severe renal injury after IRI shown as more severe renal tubular damage and higher serum creatinine. Furthermore, the excess injury would be counteracted by oral clopidogrel compared with normal plasma Hcy level mice. Intriguingly, we observed that platelet counts rise by 30% in methionine-induced HHcy. The time required to form an occlusive thrombus in mice subjected to FeCl3 carotid artery injury was sharply shorter and the length and weight of deep venous thrombosis was significantly increased in HHcy model. We hypothesized that Hcy could regulate platelet production by affecting megakaryocytes. MKs cultured with Hcy or obtained from HHcy model increased proplatelet production and had higher ploidy. However, the amount of MKs and precursor cells were not change differently. In conclusion, the present study demonstrates that HHcy increases platelet counts by promoting megakaryocyte maturation and proplatelet formation. The results further suggest that mechanism of HHcy exacerbates IRI-induced acute Kidney Injury.