The Role of HNF4a in CKD - PROJECT SUMMARY Progressive loss of kidney function in chronic kidney disease (CKD) is associated with adverse clinical outcomes including inflammation, anemia, and bone and mineral disorders that contribute to cardiovascular events and increased mortality. Limited knowledge of molecular mechanisms that contribute to CKD progression impedes development of desperately needed therapeutics aimed at reducing morbidity and mortality in patients with CKD. Expression of hepatocyte nuclear factor 4 alpha (Hnf4α) is dramatically reduced in bone and kidney from patients and animals with CKD, but the role of HNF4α in CKD progression and CKD-associated outcomes is unknown. In preliminary data for this project, we establish that deletion of Hnf4α in osteoblasts results in bone loss and we demonstrate that inflammation is a strong suppressor of Hnf4α expression in bone. We show that HNF4A2 regulates the expression of Pth1r in osteoblasts and that HNF4A deficiency contributes to PTH hyporesponsiveness in CKD. In additional preliminary data, we show that osteoblast HNF4α provides a major niche for erythropoiesis by inducing the secretion of EPO from osteoblasts. Consequently, osteoblast deletion of Hnf4α aggravates iron deficiency anemia. We demonstrate that similar reductions in bone HNF4α in patients and animals with CKD result in impaired osteogenesis and that overexpression of Hnf4α2 in osteoblasts of mice with CKD corrects PTH response, skeletal and erythropoietic abnormalities. We further show that Hnf4α is reduced in the kidney of patients and mice with CKD, and that inhibition or genetic deletion of kidney HNF4α induces fibrosis and accelerates CKD progression. We also show that hyperphosphatemia and resulting inflammation suppresses Hnf4α via a STAT3 mediated mechanism. In final preliminary data, we demonstrate that rescue of HNF4α signaling in the kidney delays CKD progression and propose the reconciling hypothesis that multiorgan HNF4α deficiency contributes to ROD, anemia and CKD progression. In Aim 1, we will establish that inflammation suppresses Hnf4α in bone leading to a feed-forward loop, whereby reduced Hnf4α amplifies the inflammatory signaling, and induces PTH hyporesponsiveness. We will demonstrate the protective role of HNF4α in bone by genetically inducing and suppressing Hnf4α2 expression in pre- osteoblasts in mice receiving anabolic PTH challenges and in mice with moderate or advanced CKD. In Aim 2, we will investigate HNF4α as a regulator of osteoblast-induced bone marrow erythropoiesis via EPO, using deletion and overexpression of Hnf4α in osteoblasts, in co-cultures with hematopoietic precursors in presence and absence of EPO, in vivo in two established models of anemia, i.e. iron deficiency anemia and anemia of inflammation, and in CKD. Finally, in Aim 3 we will investigate the critical role of kidney HNF4a in CKD progression and STAT3 as an upstream regulator, using mice fed a high phosphate diet and the Col4a3KO mouse model of CKD. Finally, we will demonstrate the protective role of HNF4A in the kidney by genetically overexpressing Hnf4α in the proximal tubules in mice with early, moderate and advanced CKD.
