X. Wang1, S. Balaji1, H. Li1, E. Steen1, P. Bollyky3, J. Cheng2, S. Keswani1 1Baylor College Of Medicine,Pediatric Surgery/Surgery,Houston, TX, USA 2Baylor College Of Medicine,Nephrology/Medicine,Houston, TX, USA 3Stanford University,Infectious Diseases/Medicine,Palo Alto, CA, USA
Introduction: Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), and is a product of aberrant inflammation, extracellular matrix (ECM) deposition and peritubular capillary loss. We have demonstrated a novel role for interleukin-10 (IL-10) in abrogating dermal fibrosis by regulating hyaluronan (HA) through dermal fibroblasts. We therefore hypothesized that hyaluronan attenuate renal fibrosis via its molecular weight variation to influence extracellular matrix remodeling, promoting angiogenesis and reducing inflammation.
Methods: In vivo: We performed unilateral ureteral obstruction(UUO) as a renal fibrosis model with/without IL-10 overexpression through the injection under the kidney capsule with normal diet or diets with 5% HA inhibitor, 4-methylumbelliferone(4MU). UUO/sham kidneys were collected at d3, d7, d21 for RNA, ELISA, and/or immunohistochemistry. HA synthesis and degradation enzyme levels were assessed by qPCR and Western blot. HA molecular weight was assessed by size-exclusion chromatography. In vitro: Renal fibroblasts (FB) were isolated from C57BL/6J mice to determine the effect of IL-10(100 ng/ml) on gene expression of HAS1-3 and hyaluronidases (HYAL1-2) at 24h. Alpha-SMA, HAS1, HAS2 and p-STAT3 expression were assessed by western blotting at 48h. Data mean+/-SD; p-values by ANOVA.
Results:
In vivo, the up-regulation of HAS1 and HAS2 expression in normal and 4-MU diets UUO mice compared to control mice from day 3; ELISA showed that total HA is steady increased from day 3 up to day 14 for UUO mice, and up to day 21 for IL-10 treated UUO mice. In normal diet mice, lenti-IL-10 treatment resulted in less dilated tubules, decreased kidney fibrosis, and preserved tubular integrity in kidneys, compared to control treated mice; IL-10 treated 4-MU diet mice were not able to achieve attenuated fibrosis.). HA gel electrophoresis showed Day 3, 7 and 14 UUO kidneys have a previously unreported 1.5×106kD HA variant compared to control/sham kidneys. In vitro: HAS1, 2&3 and HYAL1 in renal FB (6.62±0.89, 1.83±0.54, 1.84±0.92) was also significantly dysregulated (p<0.05) after 24h IL-10 treatment by qPCR.With western blotting, HAS2, a-SMA and STAT3 were significantly upregulated. A 1.88-fold increase in HA-rich matrix formation was shown with 24h IL-10 stimulation, and the effect was abrogated by HYAL (p<0.05).
Conclusion:
Our study provides the first evidence that injured kidney tissues have the capacity to express increased levels of a high molecular weight HA variant, which contrasts to that found in normal, uninjured kidneys. This finding suggesting that HA plays important roles in kidney, development, homeostasis, architectural integrity and function.Moreover, our discovery of mechanisms behind the HA-attenuated fibrosis could inspire novel therapeutics.