Selective inhibition of arginase-2 in endothelial cells but not proximal tubules reduces renal fibrosis

Abstract

Fibrosis is the final common pathway in the pathophysiology of most forms of chronic kidney disease (CKD). As treatment of renal fibrosis still remains largely supportive, a refined understanding of the cellular and molecular mechanisms of kidney fibrosis and the development of novel compounds are urgently needed. Whether arginases play a role in the development of fibrosis in CKD is unclear. We hypothesized that endothelial arginase-2 (Arg2) promotes the development of kidney fibrosis induced by unilateral ureteral obstruction (UUO). Arg2 expression and arginase activity significantly increased following renal fibrosis. Pharmacologic blockade or genetic deficiency of Arg2 conferred kidney protection following renal fibrosis, as reflected by a reduction in kidney interstitial fibrosis and fibrotic markers. Selective deletion of Arg2 in endothelial cells (Tie2Cre/Arg2fl/fl) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2fl/fl) failed to reduce renal fibrosis after UUO. Furthermore, arginase inhibition restored kidney nitric oxide (NO) levels, oxidative stress, and mitochondrial function following UUO. These findings indicate that endothelial Arg2 plays a major role in renal fibrosis via its action on NO and mitochondrial function. Blocking Arg2 activity or expression could be a novel therapeutic approach for prevention of CKD.

Keywords: Chronic kidney disease; Mitochondria; Nephrology; Nitric oxide.

Figure 1. Arginases expression and activity increased in the UUO fibrosis model.

The left ureter was ligated and kidneys were collected after 7 days. Arg1 (A and C) and Arg2 (B and C) protein levels were determined using Western blot (n = 4 each group). Kidney arginase activity was determined as described in the Methods (D) (n = 5 each group). Values are shown as mean ± SEM. *P < 0.05, **P < 0.01 compared with sham-operated mice using unpaired t test.

Figure 2. Arginase inhibition reduces kidney fibrosis and fibrotic markers following UUO.

Representative images and quantitation of Masson’s trichrome– (A and B) and Sirius red–stained (C and D) left kidney sections. RT-PCR analysis of left kidneys from indicated mice subjected to UUO with or without BEC treatment for fibronectin (E) or smooth muscle actin (F). Values are shown as mean ± SEM. *P < 0.05, **P < 0.001 compared with vehicle sham; ^#P < 0.05 compared with vehicle UUO using 1-way ANOVA (n = 3–7 each group). Scale bar: 50 μm (A and C).

Figure 3. Arg2 deficiency or arginase inhibition reduces kidney fibrosis and fibrotic markers following UUO.

Representative images and quantitation of Masson’s trichrome– (A and B) and Sirius red–stained (C and D) left kidney sections. RT-PCR analysis of left kidneys from indicated mice for fibronectin (E) or smooth muscle actin (F). Values are shown as mean ± SEM. *P < 0.05, **P < 0.01 compared with WT sham; ^#P < 0.05, ^##P < 0.01 compared with WT UUO using 1-way ANOVA (n = 4–7 each group). Scale bar: 50 μm (A and C).

Figure 4. Arg2 deficiency or arginase inhibition does not reduce kidney macrophage infiltration following UUO.

Representative images (A) and quantitation of F4/80 stained left kidney sections (B). Values are shown as mean ± SEM. *P < 0.05 compared with WT sham-operated mice using 1-way ANOVA (n = 4–8 each group). Scale bar: 50 μm.

Figure 5. Arg2 deficiency, specifically in endothelial cells, but not proximal tubular epithelial cells, reduces kidney fibrosis and fibrotic markers following UUO.

Representative images and quantitation of Masson’s trichrome– (A and B) and Sirius red–stained (C and D) left kidney sections. RT-PCR analysis of left kidneys from indicated mice for smooth muscle actin (E). Values are shown as mean ± SEM. *P < 0.05 compared with Arg2^fl/fl sham; ^#P < 0.05 compared with Arg2^fl/fl UUO using 1-way ANOVA (n = 5–9 each group). Scale bar: 50 μm (A and C).

Figure 6. Arginase inhibition restored kidney NO and restored mitochondrial functions following UUO.

(A) Levels of nitrate and nitrite were determined from whole left kidney tissue 7 days after UUO. (B) TBARS assay following UUO in whole-kidney tissue. (C) Levels of mitochondrial ATP from indicated left kidney tissue. (D) Complex I activity levels from indicated left kidney tissue. (E and F) Protein was isolated form left kidney sections for WT mice subjected to UUO for 7 days. Western blotting for MCU normalized to Tomm20 as a mitochondrial marker, while β actin show a comparable protein loading. Values are shown as mean ± SEM. *P < 0.05 compared with vehicle sham; ^#P < 0.05 compared with vehicle UUO using 1-way ANOVA (n = 5–6 each group).