ABS 054: HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy

Man Chen ¹ , Madhav C. Menon ², Wenlin Wang ¹ , Jia Fu ¹ , Zhengzi Yi ¹ , Zeguo Sun ¹ , Jessica Liu ¹ , John Cijiang He ¹ , Chengguo Wei ¹

¹ Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
² Division of Nephrology, Yale School of Medicine, New Haven, CT, USA.

Van Wickle (2025) Volume 1, ABS054

Introduction: Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.

Methods: HCK-knockout of bone marrow-derived macrophages were used in mice models. Immunohistochemistry staining was used to confirm the presence of HCK in macrophages. qPCR was used to quantify mRNA concentrations of M1 and M2 macrophage markers in HCK KO cells. Western blot assays were used to quantify the amount of autophagy-related protein present as well as M1 and M2 markers in HCK KO cells and the unilateral ureteral obstruction mice model (UUO).

Results: Immunohistochemistry staining demonstrates high levels of phosphorylated-HCK and HCK in close proximity to CD68 in high chronic allograft damage index (CADI) and low CADI allograft kidneys. qPCR demonstrates decreased levels of pro-inflammatory and autophagy-inhibiting M1 markers as well as increased levels of anti-inflammatory and autophagy-activating M2 markers in HCK KO cells. Western blot assay demonstrates increased LC3II/LC3I ratio in BMDM cells as well as decreased M1 (iNOS) and increased M2 (CD206) levels adjusted by macrophage number in UUO model kidneys. Boron-containing inhibitor BT424 demonstrates decreased M1 and increased M2 in UUO model kidneys.

Discussion: This study confirms that HCK positively stained cells are co-localized with macrophage marker CD68 in the interstitial area of the kidney. HCK KO decreases macrophage polarization to M1 markers and increases polarization to M2 phenotype, as seen with markers iNOS and CD206. These effects are likely through direct interaction with autophagy proteins (ATG2A and CBL) which culminate in suppression of autophagy in macrophages. KO of HCK increases autophagy activity as reflected by increased LC3II/ LC3I ratio. HCK-selective inhibitor BT424, derived from dasatinib, inhibits M1-like polarization and restores autophagy activity in BMDM and kidney-derived macrophages in UUO model kidneys.