Tubule-derived CCN1 Drives Renal Repair via αvβ5-STAT6-ARG1-dependent Reprogramming of Macrophages

Acute kidney injury (AKI) affects approximately 10-15% of all hospitalized patients, with ischemia-reperfusion (I/R) injury being one of the most common causes. Macrophages play a critical role in injury and repair following AKI, with specific subsets such as arginase1 (Arg1)⁺ populations promoting renal tubular epithelial cell (RTEC) proliferation, but their regulatory mechanisms remain incompletely understood. Cellular communication network factor 1 (CCN1), a secreted matricellular protein and early biomarker of AKI, may regulate macrophage function during kidney injury. In this study, we investigated CCN1's interaction with macrophages in a murine model of I/R-induced AKI.

The role of CCN1 was investigated using multiple experimental approaches including recombinant CCN1 protein administration, RTEC-specific CCN1 knockdown mouse model via adeno-associated virus, and in vitro studies with bone marrow-derived macrophages (BMDMs). Transcriptomic profiling of kidney tissues and BMDM RNA-seq were performed, along with BayesPrism deconvolution analysis. Co-immunoprecipitation coupled with mass spectrometry was used to identify CCN1-binding partners. Tissue analysis included H&E staining and immunostaining for KIM-1 and F4/80. Functional assays examining RTEC proliferation were conducted both in vitro and in vivo using transwell co-culture systems and EdU incorporation assays.

In response to I/R injury, RTECs upregulated and secreted CCN1, which colocalized with infiltrating F4/80⁺ macrophages. CCN1 expression peaked at 24 hours post-I/R with approximately 6-fold increase and remained elevated through 72 hours. Exogenous CCN1 administration significantly ameliorated renal dysfunction and increased Ly6C⁻ macrophage infiltration. Conversely, RTEC-specific CCN1 knockdown exacerbated renal injury, reduced macrophage infiltration, and decreased tubular epithelial proliferation, as confirmed by H&E, KIM-1, F4/80, and Ki67 staining. Transcriptomic profiling revealed that CCN1 expression was strongly associated with immune cell infiltration, particularly macrophages. BMDM RNA-seq demonstrated that CCN1 promoted pro-repair Arg1⁺ macrophage differentiation, upregulated tissue repair-associated genes (Pdgfb, Igf1, Hbegf, Vegfa), and activated the STAT6 signaling pathway. BayesPrism deconvolution further confirmed the enrichment of Arg1+ macrophages following CCN1 treatment under both basal and inflammatory conditions. Flow cytometry showed CCN1 treatment increased ARG1⁺ macrophages from 8.8% to 15.6% under basal conditions and from 22.5% to 35.3% under LPS/IFN-γ stimulation. Co-immunoprecipitation coupled with mass spectrometry identified integrin αvβ5 as a direct CCN1-binding partner mediating STAT6/ARG1 activation. Functionally, CCN1-treated macrophages enhanced growth factor secretion and promoted RTEC proliferation both in vitro and in vivo. These pro-proliferative and protective effects were abolished by ARG1 inhibition, αvβ5 integrin blockade, or macrophage depletion.

CCN1 regulates macrophages via the αvβ5-STAT6-ARG1 axis to promote tubular epithelial proliferation and improve kidney function in I/R-AKI. This study highlights a novel tubular-immune communication pathway and identifies potential therapeutic targets for ischemic AKI.