Identification of In Vitro Responses of Resident Renal Cells to Sterile and Nonsterile Inflammatory Triggers

Background: The kidney can be the site of either sterile or infectious injury, with immune cell recruitment to the site of injury as a hallmark of the injury response. Macrophage and monocyte recruitment are key to both types of injury responses and several homing chemokines have been reported to be involved. However, neither the specificity of the implicated chemokines nor the specific monocyte/macrophage cells recruited have been defined in sterile injury versus kidney infection. Methods: The addition of freeze-fractured debris from mouse proximal tubule (mPT) cell line to primary-cultured renal cells (PCRC) in vitro was used to model sterile injury, while lipopolysaccharide (LPS) was used to model gram negative infectious injury. PCRC from wildtype C57BL/6 or from Tlr2-/-Tlr4-/- mice were exposed to either mPT debris or E. coli LPS for 24 hours and RNA was extracted, reverse transcribed and subjected to quantitative polymerase chain reaction (qPCR) analyses for selected chemokines. Results: Debris-treatment of PCRC for 24 hours resulted in a modest increase in canonical inflammatory genes including Csf2, Tnfa, Il1b and Il6 expression (2.1-fold, 2.8-fold, 2.1-fold and 1.9-fold, respectively), while LPS induced their expression to a much greater degree (11.9-fold, 11.3-fold, 69.5-fold, 56.8-fold, respectively). The induction of all proinflammatory cytokines was Tlr2/Tlr4 dependent. Macrophage recruiting chemokines Ccl2 and Cx3cl1 were robustly induced by LPS, with little induction by debris. In contrast, debris but not LPS, induced Cxcl12 (Sdf1) expression and analysis of PCRC single cell sequencing data revealed that Cxcl12 expression was specific to the fibroblast/myofibroblast subpopulation. Reference mouse kidney disassociated single cell sequencing confirmed the myofibroblast population as the predominant cell-type that expresses Cxcl12. Expression of the stromal cell-derived factor (SDF) receptor, Cxcr4, was localized to polymorphonuclear cells and monocyte subpopulations. Further sub clustering of the monocyte subpopulation identified distinct subsets with either Ccr2 or Cxcr4 expression, but not both. Conclusion: Resident kidney cells selectively respond to sterile and non-sterile inflammatory triggers to recruit distinct monocyte populations and induce distinct patterns of proinflammatory activation. Funding: NIDDK Support