X. Wang1, P. Duann1, C. Lu1, C. Moles1, H. Li1, M. Fahrenholtz1, M. Rae1, Y. Dhamija1, J. Cheng2, S. Balaji1, S. Keswani1 1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Baylor College Of Medicine,Medicine,Houston, TX, USA
Introduction:
Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), which affects nearly 700 million patients globally, and is a product of aberrant inflammation and extracellular matrix (ECM) deposition. Patients with CKD are associated with a three-fold or higher mortality rate compared to the general population. We have previously shown a novel role for interleukin-10 (IL-10) in dermal fibrosis, beyond its accepted anti-inflammatory role. In this role, IL-10 regulates the ECM, specifically hyaluronan (HA), and TGFβ isoforms, which are crucial for regenerative tissue repair. However, the roles of IL-10 and HA in renal fibrosis are not completely elucidated. We hypothesize that IL-10 might regulate HA and TGFβ expression in the kidney, and attenuate renal fibrosis in murine unilateral urethral obstruction (UUO) model.
Methods:
Primary renal fibroblasts (FB) were isolated from 8-10 week-old male C57BL/6J (WT) mice. IL-10 (200 ng/ml) with or without hyaluronidase (HYAL, 1.5 unit/ml) was added to cultures. HA matrices were analyzed by particle-exclusion assay at 24h. Gene expression of HA synthases 1, 2, and 3 (HAS1-3), hyaluronidases 1 and 2 (HYAL1-2) and TGFβ-1 were assessed by qPCR at 1, 2, 3 and 6 h. 8 weeks C57BL/6J (WT) and IL-10 KO male mice were injected with lenti-IL-10/ lenti-GFP (1×1010 IU) under the kidney capsule. Three days after the injection, unilateral ureteral obstruction (UUO) was performed. UUO/sham kidneys and serum were collected at 14 days after UUO for RNA, ELISA, and immunohistochemical (IHC) analysis. n=3/treatment group; p-values by ANOVA.
Results:
In vitro, IL-10 resulted in an upregulation of HAS-1,2, and 3 expression at 2h after treatment, and a significant downregulation of HYAL 1, 2 and TGFβ-1. IL-10 resulted in a 1.88-fold increase in HA-rich matrix formation at 24h, and the effect was abolished by HYAL treatment (p<0.05). In vivo, IL-10 KO mice demonstrated more fibrosis than WT mice. Lenti-IL-10 treatment resulted in less dilated tubules and decreased kidney fibrosis, as well as reduced α-SMA expression as compared to lenti-GFP treated kidneys in both WT and IL-10 KO mice. The HA level in serum was 1.7-fold higher in lenti-IL-10 treated mice as compared to lenti-GFP treated (p<0.05) (Fig.1).
Conclusion:
Our data demonstrates that IL-10 regulates HA metabolism and TGFβ expression of renal FB in vitro, and is effect of IL-10 is validated in the UUO model. The endogenous IL-10 is essential for normal kidney integrity against excessive fibrosis with UUO injury. This previously unreported mechanism for IL-10 regulation of ECM in the kidney may have a significant impact for future therapies to ameliorate kidney fibrosis.
