SUN-100 Exosome-packaged sonic hedgehog in cardiomyocyte plays a central role in type 2 cardiorenal syndrome

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Cardiorenal syndrome type 2 (CRS2) is characterized by chronic abnormalities in cardiac function causing progressive chronic kidney disease (CKD). However, the mediators connecting heart and kidney remains largely unknown. In this study, we provide evidence supporting that exosomes containing Shh generated by injured cardiomyocyte plays a vital role in mediating cardiorenal connection in mouse model of transverse aortic constriction (TAC). Four weeks after TAC, mice were randomized into three groups: 1) sham control; 2) TAC mice; 3) TAC mice injected daily with cyclopamine (CPN), a specific inhibitor of hedgehog signaling. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Another set of mice were randomized into three groups after 4 weeks of TAC: 1) sham control mice injected with pcDNA3 plasmid; 2) TAC mice injected with pcDNA3 plasmid; 3) TAC mice injected with shRab27a plasmid, a small GTPases implicated in regulating exosome biogenesis and secretion. These mice were injected with pcDNA3 or shRab27a plasmids once a week for three weeks beginning at 4 weeks after TAC. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Exosomes were isolated from TAC mice serum and CRS2 patients’ serum, and incubated with normal rat kidney interstitial fibroblast (NRK-49F) cells. At 8 weeks after TAC, cardiac hypertrophy and fibrosis were prominent, as evidenced by increased expression of β-myosin heavy chain, α-actin and fibronectin. Echocardiography also revealed an impaired cardiac function in TAC mice. These cardiac lesions were accompanied by an upregulation of Shh and increased production of exosomes in cardiac tissue. Electron microscopy confirmed abundant exosomes derived from the injured cardiomyocytes in the heart of TAC mice. Blockade of Shh signaling by CPN ameliorated cardiac injury and restored heart function. Moreover, TAC mice also developed kidney injury secondary to chronic heart failure, manifested by proteinuria, kidney fibrosis, podocyte injury, and renal upregulation of Shh. CPN mitigated all these lesions in the kidneys. Notably, serum derived from TAC mice or CRS patients was able to induce renal interstitial fibroblast activation in vitro, indicating the circulating factors as mediators of cardiorenal connection. Furthermore, exosomes isolated from TAC serum was sufficient to cause fibroblast activation and matrix production. Furthermore, blockade of exosome biogenesis and secretion by knockdown of Rab27a expression not only protected against cardiac hypertrophy and fibrosis but also ameliorated renal fibrotic lesions. These studies demonstrate that heart-derived, circulating exosomes are a unique and effective vehicle to deliver Shh into the kidney after TAC. Targeting exosome generation and secretion or inhibition of Shh signaling could be novel therapeutic strategies for protecting both heart and kidney in cardiorenal syndrome.