A negative feedback loop involving NF-κB/TIR8 regulates IL-1β-induced epithelial- myofibroblast transdifferentiation in human tubular cells

Renal tubular epithelial-myofibroblast transdifferentiation (EMT) plays a central role in the development of renal interstitial fibrosis (RIF). The profibrotic cytokine interleukin (IL)-1 and the IL-1 receptor (IL-1R) also participate in RIF development, and Toll/IL-1R 8 (TIR8), a member of the Toll-like receptor superfamily, has been identified as a negative regulator of IL-1R signaling. However, the functions of TIR8 in IL-1-induced RIF remain unknown. Here, human embryonic kidney epithelial cells (HKC) and unilateral ureteric obstruction (UUO)-induced RIF models on SD rats were used to investigate the functions of TIR8 involving IL-1β-induced EMT. We showed that IL-1β primarily triggers TIR8 expression by activating nuclear factor-κB (NF-κB) in HKC cells. Conversely, high levels of TIR8 in HKC cells repress IL-1β-induced NF-κB activation and inhibit IL-1β-induced EMT. Moreover, in vitro and in vivo findings revealed that TIR8 downregulation facilitated IL-1β-induced NF-κB activation and contributed to TGF-β1-mediated EMT in renal tubular epithelial cells. These results suggested that TIR8 exerts a protective role in IL-1β-mediated EMT and potentially represents a new target for RIF treatment.

MO075KLF11 DEFICIENCY ENHANCES CHEMOKINE GENERATION AND INJURY IN MURINE UNILATERAL URETERIC OBSTRUCTION

Background and Aims Kruppel-like factors (KLFs) comprise a family of zinc-finger transcription factors that play a critical role in development, proliferation, and regeneration following injury. There are over 17 members of this family; recent studies have shown that KLF family members regulate podocyte differentiation, preservation of the glomerular filtration barrier, and regulation of mitochondrial function. However, a role for KLF11 in renal pathophysiology has not been previously established. Method Wild-type (WT) and KLF11 knockout (KO) mice were subjected to unilateral ureteric obstruction (UUO), a well-established model of renal inflammation and fibrosis; controls included mice subjected to manipulation of the ureter without ligation. Kidneys were harvested after 9 days (n=8 animals per group). Semiquantitative histopathologic analysis of renal atrophy, fibrosis, and inflammation was performed in a blinded fashion. Gene expression analysis was performed on renal cortex employing the Pathway Detect RNA array and RNASeq. Results In UUO, renal atrophy was more severe in KLF11 KO mice than WT mice (p<0.001). Deposition of collagen, as assessed by quantitative analysis of Sirus Red stained sections, was greater in KLF11 KO mice, compared to WT mice subjected to UUO; COL3A1 expression was also increased (p<0.05). Atrophy was associated with an increase in F4/80+ (p<0.01) and CD206+ macrophages (p<0.05), but not CD3+ T cells in KLF11 KO vs. WT mice. Induction of CC chemokines, including CCL2, CCL5, CCL7, CCL12, and CCL2 as well as CCR2 was significantly higher in KLF11 KO versus WT mice subjected to UUO (all p<0.001). Expression of NF-kB (p<0.01) and TNF alpha (p<0.01), but not IL-1 beta, IL-6, or IL-10 were significantly higher in KLF11 KO than WT mice with UUO. Expression of TGF-beta 1, Smad2, and Smad3 were also higher in KLF11 KO mice than WT mice with UUO (p<0.05). Conclusion Renal injury in UUO is exacerbated in KLF11 KO mice, compared to WT mice. Injury is associated with increased macrophage influx and production of pro-inflammatory chemokines. Future studies will determine how KLF11 deficiency directs transcription of pro-inflammatory and pro-fibrotic genes.

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

The kidney has a limited capacity to repair following injury, however, the endogenous reparative pathways are not well understood. Here we employ integrated droplet- and plate-based scRNA-seq in the murine reversible unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single cell level during renal injury and resolution of fibrosis. We generate a comprehensive catalogue of the changes induced during injury and repair, revealing significant myeloid cell heterogeneity, which would not have been identifiable by conventional flow cytometry. We identify new markers for the myeloid populations within the kidney as well as identification of novel subsets including an Arg1+ monocyte population specific to early injury and a Mmp12+ macrophage subset exclusive to repair. Finally, using paired blood exchange to track circulating immune cells, we confirm that monocytes are recruited to the kidney early after injury and are the source of Ccr2+ macrophages that accumulate in late injury. Our data demonstrate the utility of complementary technologies to identify novel myeloid subtypes that may represent therapeutic targets to inhibit progression or promote regression of kidney disease.