#4369 MYELOID CELL-DERIVED HMGB1 AGGRAVATES THE PROGRESSION OF CHRONIC KIDNEY DISEASE INDUCED BY CHRONIC OXALATE EXPOSURE
Authors: Kuang Meisi , Anders Hans-Joachim , Steiger Stefanie , Kusunoki Yoshihiro 
Published: June 2023

Abstract

Background and Aims
HMGB1, long form High Mobility Group Box 1, a highly conserved DNA chaperone stabilizes chromatin in the nucleus, regulates gene transcription, and supports DNA repair. In the cytosol, it regulates mitochondrial function, autophagy, and apoptosis. In addition, when secreted or passively released into the extracellular space during cell necrosis, it acts as danger signal (DAMP). As such HMGB1 amplifies necroinflammation in ischemic and toxic acute kidney injury. In contrast, little is known about the contribution of HMGB1 release from infiltrating immune cells in this context. We hypothesized that HMGB1 from resident and infiltrating myeloid cells would contribute to the progression of CKD by driving tubular atrophy and tissue remodeling.

Method
We generated myeloid deletion of HMGB1 by crossing Hmgb1 floxed mice (Hmgb1f/f) with endogenous Lyz2 promoter (Lyz2Cre/+) to investigate the role of myeloid cell HMGB1 in a model of chronic kidney disease induced by sodium oxalate-rich diet (50 μmol/g), i.e., calcium oxalate nephropathy. The Lyz2Cre/+ mice were on a C57BL/6J background and crossed with mice homozygous for the floxed Hmgb1 gene to generate mice with a constitutive deletion of the Hmgb1 gene in all myeloid cells. We used littermates of 6- or 8-week-old males for all experiments. Mice with no Lyz2Cre/+ but the homozygous of floxed Hmgb1 gene served as wildtype controls (Lyz2Cre/−Hmgb1f/f WT) and mice with Lyz2Cre/+ and the homozygous of floxed Hmgb1 gene were used as Lyz2Cre/+Hmgb1f/f KO. The primary endpoint for the comparison between Lyz2Cre/+Hmgb1f/f KO and WT mice was glomerular filtration rate at 21 days of oxalate feeding, assessed by FITC-sinistrin clearance. Plasma samples were collected only on day 21 before sacrifice by cervical dislocation. Urine samples were collected as well as GFR was measured from all experimental groups on day 0, day7, day14 and before sacrifice by cervical dislocation on day 21. Kidneys were harvested after sacrifice. The kidney was divided into two equal parts. One part was kept in RNA later solution at −80°C for RNA isolation and the second part was kept in 4% formalin to be embedded in paraffin for histology analysis.

Results
KO animals did not reveal any abnormalities within an observation phase of 6 months. Healthy mice showed normal kidney function parameters in urine and blood. Observation is continued and will be supplemented by histopathological analyzes at the age of 12 months. Primary endpoint: Deletion of HMGB1 from myeloid cells attenuated the decline of GFR as compared to WT control mice. Secondary endpoints: The difference in GFR was consistent with the respective levels of serum creatinine at day 21 upon oxalate-rich diet compared with controls. The ablation of HMGB1 in myeloid cells was associated with less tubulointerstitial fibrosis at day 21 after oxalate-rich diet

Conclusion
Our data suggest that HMGB1 release from myeloid cells promotes crystal-induced chronic kidney injury, which is consistent with the role of HMGB1 as an extracellular DAMP and persistent inflammation in CKD. However, this effect may also relate to the intracellular role of HMGB1 as a transcriptional regulator.