Kidney Week

Abstract: TH-OR112

5-Lipoxygenase Promotes Renal Interstitial Injury and Fibrosis

Session Information

• Scarred for Life?
  November 02, 2017 | Location: Room 394, Morial Convention Center
  Abstract Time: 05:18 PM - 05:30 PM

Category: Chronic Kidney Disease (Non-Dialysis)

• 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

• Montford, John Ross, University of Colorado Denver, AMC, Aurora, Colorado, United States

John Ross Montford,

• Bauer, Colin D, University of Colorado Denver, AMC, Aurora, Colorado, United States

Colin D Bauer,

• Dobrinskikh, Evgenia, University of Colorado Denver, AMC, Aurora, Colorado, United States

Evgenia Dobrinskikh,

• Hopp, Katharina, University of Colorado Denver, AMC, Aurora, Colorado, United States

Katharina Hopp,

• Levi, Moshe, University of Colorado Denver, AMC, Aurora, Colorado, United States

Moshe Levi,

• Nemenoff, Raphael A., University of Colorado Denver, AMC, Aurora, Colorado, United States

Raphael A. Nemenoff,

• Furgeson, Seth B., University of Colorado Denver, AMC, Aurora, Colorado, United States

Seth B. Furgeson, MD

Background

Although macrophages promote renal fibrosis, the mechanism is incompletely understood. Macrophages may exert tissue damage through activation of 5-lipoxygenase (5-LO). 5-LO and 5-LO associated protein (FLAP) initiate leukotriene synthesis; the downstream enzymes LTA₄ hydrolase (Lta4h) and LTC₄ synthase (Ltc4s) catalyze the production of LTB₄ and cysteinyl leukotrienes, respectively. We hypothesized that leukotriene inhibition would decrease renal fibrosis after unilateral ureteral obstruction (UUO).

Methods

C57Bl/6 mice undergoing UUO were treated with zileuton (5-LO antagonist) or vehicle. UUO was also done in wild type, Flap knockout (ko), Lta4h ko and Ltc4s ko mice. Renal fibrosis was measured using both a biochemical assay (hydroxyproline) and microscopy to detect second harmonic generation (SHG) signal from collagen. To measure metabolic changes in renal tubular epithelial cells, fluorescent lifetime imaging microscopy (FLIM) was performed to determine alterations in free and bound NADH levels.

Results

We found a large induction in 5-LO-expressing interstitial leukocytes in kidneys of UUO mice. Treatment of mice with zileuton before UUO significantly decreased hydroxyproline content at 7 days (37% reduction vs. vehicle). SHG microscopy also confirmed a significant decrease in renal fibrosis in zileuton-treated mice (40% reduction). Compared to littermate controls, Flap ko mice had less interstitial fibrosis as measured by hydroxyproline assay (33% reduction). Ltc4s ko mice, but not Lta4h ko mice, were significantly protected from UUO-induced fibrosis (44% reduction in hydroxyproline content and 41% reduction in fibrotic area by SHG). We then performed FLIM for NADH to determine if 5-LO inhibition led to metabolic changes in renal tubular epithelial cells. We found that there was significant shift to glycolytic metabolism after UUO in control mice; this was partially abrogated in zileuton-treated mice. To validate these findings, we tested whether 5-LO is induced in other models of chronic kidney disease and found increased numbers of 5-LO expressing leukocytes in mice with polycystic kidney disease.

Conclusion

5-lipoxygenase and LTC₄ synthase are potent inducers of renal fibrosis after UUO. The pathologic effects of leukotrienes may be partially mediated through changes in renal tubular epithelial metabolism.

Funding

• Other NIH Support