*Purpose: Interstitial fibrosis and tubular atrophy (IF/TA) is common histological abnormality in biopsy of kidney transplants. Our previous study from a prospective human kidney transplant cohort identified hematopoietic cell kinase (HCK) was upregulated in chronic allograft injury patients and associated with renal fibrosis. We recently find HCK could regulate autophagy by interaction with autophagy key proteins ATG2A and CBL. But how HCK regulates kidney fibrosis in renal allograft injury through macrophage autophagy and activation is unclear.

*Methods: Bone marrow-derived macrophage (BMDM) and RAW264.7 macrophage cell line were used to measure HCK regulation on autophagy and macrophage polarization. HCK exon3 loxp flanked KO mice were developed at EuMMCR in Germany. We isolated BMDM from HCK KO and WT mice to test HCK effects on macrophage autophagy and polarization. Next, we performed UUO in global (CMV-cre), macrophage (LYZ2-cre) and RTEC (Pax8-cre) homozygous HCK KO and WT mice to assess role of HCK in kidney fibrosis. In the UUO mice, we performed CD45 leukocytes selection with immunomagnetic bead from StemcellTM and flow cytometry to measure macrophage subpopulation in WT and HCK KO UUO kidneys.

*Results: Single cell sequencing data confirmed that HCK is mainly expressed in macrophages (Fig. 1A). We found endogenous HCK interacts with autophagy proteins ATG2A and CBL in macrophage cell line RAW264.7 by IP/WB analysis (Fig. 1B). We isolated BMDM from HCK KO and WT mice and found HCK KO increased autophagy activity as reflected by increased LC3II/LC3I ratio and decreased P62 (Fig. 1C). We also found HCK KO decreased macrophage M1 polarization and increased M2 polarization when BMDM were stimulated with LPS/INF γ or IL4, as assessed by mRNA and protein levels of M1 and M2 markers (Fig. 1D, 1E). We found M1 macrophages CD86+F4/80+CD11B+ subtypes decreased and CD206+F4/80+CD11B+ M2 macrophage subpopulation increased in HCK KO UUO kidneys (Fig. 1F). HCK exon3 loxp flanked KO mice were developed at EuMMCR in Germany (Fig. 2A). HCK KO was confirmed by WB in isolated BMDM from CMV-Cre and LysM-Cre HCK-KO mice (Fig. 2B). In the UUO model, we found that both global and macrophage HCK KO mice had significantly attenuated tubulointerstitial fibrosis, as shown by decreased mRNA levels of profibrotic markers (Fig. 2C) and Masson’s trichrome stain (Fig. 2D, 2E). HCK KO in RTEC did not significantly reduce renal fibrosis in the UUO mice (data not shown), indicating HCK regulates kidney fibrosis in macrophages.

*Conclusions: HCK regulates kidney fibrosis through macrophage autophagy and polarization in chronic allograft nephropathy.

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