Impact of Factor XII, Prekallikrein and C1 Inhibitor in Renal Fibrosis

Chronic kidney disease, CKD, is projected to be the 5^th highest cause of death by 2050. Renal fibrosis is a major determinant of progression of CKD. Tubule-interstitial fibrosis (TIF) is a major determinant of renal functional decline in CKD, regardless of etiology. The precise mechanism(s) driving renal fibrosis and TIF, however, are unknown. Factor XII (FXII) is a central component of several proteolytic systems. Zymogen FXII autoactivates on artificial or biologic surfaces to become activated FXII (FXIIa). FXIIa cleaves several substrates, including factor XI, prekallikrein, C1r and C1s, and plasminogen to initiate blood coagulation, liberate bradykinin, activate the classical complement pathway, and induce fibrinolysis, respectively. FXII, the zymogen, also has unique and important cell biology functions. It promotes smooth muscle and endothelial cell growth, proliferation and angiogenesis and neutrophil adhesion, migration, chemotaxis, and NETosis. A role for FXII in the development of fibrosis is unknown. We hypothesized that FXII and related proteins may influence the development of renal fibrosis.

Initial investigations examined two independent renal RNA-Seq datasets from patients with CKD. We found that the genes that code for factor XII (FXII) (F12), C1inhibitor (C1INH) (SERPING1), and prekallikrein (PK) (KLKB1) had increased expression and were associated with poor renal function, as measured by increased serum creatinine (SCr) and lower estimated glomerular filtration rate (eGFR). F12 also co-expressed with genes encoding proximal tubule injury markers KIM1, NGAL and MYC. Next, using a murine CKD model induced by a single folic acid (FA) injection in wild-type (WT), FXII (F12^-/-), PK (Klkb1^-/-) or C1INH (Serping1^-/-) deficient mice followed by analysis after 4 weeks, we observed that SCr and BUN levels were increased by 2-fold in FA-treated WT, Klkb1^-/- and Serping1^-/-, but not in F12^-/- mice. Unlike WT, Klkb1^-/- and Serping1^-/- mice, F12^-/- mice were protected from tubular injury, interstitial inflammation and renal fibrosis after FA treatment. No changes in plasma FXII, PK or C1INH levels were seen by immunoblotting when comparing FA-treated WT mice with controls. However, renal immunofluorescence showed that proximal tubules from FA-treated WT mice expressed higher levels of FXII antigen compared to untreated mice. Incubation of human proximal tubule cell line HK-2 cells with FA also induced FXII expression. Bulk RNA-seq studies showed major renal transcriptomic changes induced by FA. Gene set enrichment analysis showed that Cytokine-Cytokine Receptor, Chemokine, Fibrosis and Senescence pathways were increased in WT mice after FA treatment. The expression of these pathways were markedly increased in Serping1^-/- mice, mildly increased in Klkb1^-/- mice, and strikingly not increased in F12^-/- mice. Furthermore, biological pathways related to neutrophil migration, chemotaxis and extracellular trap formation also were enriched in FA-treated WT, Serping1^-/- and Klkb1^-/- mice but were non-significantly different between F12^-/- mice and untreated WT. Flow cytometry analysis showed FA-induced renal infiltration of CD45⁺/Ly6G⁺ neutrophils in WT, but not in F12^-/- mice. In conclusion, these data indicate that FXII is a profibrotic agent that induces renal fibrosis and CKD development. Regulation of FXII in the kidney has potential to blunt the development of renal fibrosis after injury states.