Abstract
Background and Aims
Obstructive nephropathy is the result of functional or anatomic lesions located in the urinary tract, and renal interstitial fibrosis is a common finding associated with long-term nephropathy. Many factors are suggested to be involved in the pathogenesis of renal fibrosis, such as infiltration of macrophages, growth factors, oxidative stress and cytokines. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, accumulates markedly in serum and renal tissue of cisplatin- or ischemia/reperfusion-induced acute kidney injury model animals, thereby inducing generation of oxidative stress. However, the relationship between IS and obstructive nephropathy or renal fibrosis remains unclear. IS is produced in the liver by CYP2A6/2E1-dependent oxidative metabolism of dietary protein-derived indole, followed by sulfotransferase 1a1 (SULT1A1)-mediated sulfate conjugation of indoxyl. IS in the blood circulation is efficiently taken up by renal proximal tubules via basolateral membrane-localized organic anion transporters, OAT1 and OAT3, and excreted into urine via unidentified apical membrane-located transporter. Thus, we established SULT1A1 gene-deficient (SULTKO) mice and developed UUO mice to investigate the pathological role of IS in UUO-induced renal fibrosis.
Method
The left ureter of C57BL/6J mice (wild type (WT), 8 weeks-old) and SULTKO mice (8 weeks-old) were obstructed last for 2 weeks. IS concentration in serum and kidney was determined by LC-MS/MS. Changes in histology and interstitial fibrosis were examined with PAS staining and Sirius red staining, respectively. Quantitative PCR was applied for determining expression levels of col1a1 encoding the major component of type I collagen, fibronectin, plasminogen activator inhibitor (PAI)-1, the activator of plasminogen and hence fibrinolysis, pro-inflammatory cytokine interleukin 6 (IL-6), Wnt4 encoding one protein of Wnt and Sfrp5, a gene that codes for antagonist of Wnt pathway. Renal fibrosis also evaluated through the expression of alpha smooth muscle actin (SMA) by Western blotting.
Results
By UUO treatment, the concentration of IS in serum, kidney and liver were elevated, which were suppressed in SULTKO mice. Ureter dilation was obviously observed in the obstructed kidney of WT mice, which was slightly prevented in SULTKO mice with UUO. Sirius red staining revealed that severe collagen deposition was found in the interstitium of WT kidney with UUO, but it was partly prevented in the kidney of KO mice with UUO along with the decrease in IS accumulation. The high expression of SMA, col1a1 and fibronectin in the kidney of WT mice with UUO were significantly suppressed in the kidney of SULTKO mice, 2.3-fold, 1.4-fold and 2.3-fold, respectively, suggesting that renal fibrotic responses were ameliorated in SULTKO mice. The expression of PAI-1, which was upregulated in WT mice with UUO, was also suppressed (1.8-fold) in SULTKO mice with UUO. The elevated expression of IL-6 in the kidney of WT mice with UUO was inhibited (1.8-fold) in SULTKO mice with UUO, indicating the possibility that inflammation-related signalling pathway also participated in the IS-exacerbated renal fibrosis. The enhanced expression of Wnt4 in the kidney of WT mice with UUO was suppressed (1.8-fold) in SULTKO mice with UUO, and the gene of Sfrp5 exhibited a higher expression level (3.2-fold) in the kidney of SULTKO mice with UUO compared with WT UUO mice.
Conclusion
Sult1a1-deficient mice showed the suppressed accumulation of IS in the kidney with UUO. Renal IS accumulation during pathological progression of obstructive nephropathy could enhance interstitial fibrosis through the activation of Wnt signalling pathway. Hepatic SULT1A1 could be a therapeutic target for preventing the progression of renal interstitial fibrosis by suppressing IS production during obstructive nephropathy.
Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3
