Purpose: Kidney fibrosis, a terminal pathway that is common among all CKD patients, represents an intractable condition in which myofibroblasts proliferate in the interstitium. This condition is induced by persistent damage and inflammation caused by the overproduction of extracellular matrix, eventually leading to renal failure, with no effective therapies available to date. Recent studies revealed that a reduction in nitric oxide (NO) is observed in the onset and progression of CKD and kidney fibrosis. In fact, mice with inhibited nitric oxide synthase (NOS) activity or eNOS-knockout mice were reported to show an enhanced level of renal impairment 1, and pathologic progression of the kidney was suppressed by an enhanced NO synthesis. Also, the decreased NO level is related with a reduction in a production of erythropoietin (EPO) 2 and an exacerbation of inflammation in the renal tissue1, leading to aggravation of the fibrosis3. These findings lead to the expectation that NO should exhibit therapeutic effects on kidney fibrosis. However, the biological half-life of NO is extremely short due to its high reactivity, therefore, developing an NO donor that can be used in clinical applications is an urgent issue. Based on the facts that HSA is the major endogenous NO carrier in plasma, we created S-nitrosated human serum albumin (SNO-HSA). In the present study, the therapeutic effect of SNO-HSA was evaluated using mice with unilateral ureteral obstruction (UUO)-induced kidney fibrosis.
Methods: Preparation of SNO-HSA: Cys-34 of HSA was reduced by incubating with DTT, an reducing agent. To carry out S-nitrosation of the reduced thiol at the Cys-34, the HSA was incubated with Isoamyl nitrite (NO donor) Afterward, Isoamyl nitrite was removed by dialysis.
Preparation of UUO model mice: Using ICR mice (male, 4 weeks old), the left ureter was exposed and ligated as UUO model. The SNO-HSA (48 nmol/mouse of NO), HSA or saline was administered intravenously just after the ligation (Day 0) and two days afterward (Day 2). Mice were sacrificed on Day 7 or Day 14 after surgery, and the obstructed kidneys were harvested.
Results: On Day 14, the UUO with the saline or HSA treatment caused an increase in the levels of hydroxyproline, a constituent of collagen, which is a component of the extracellular matrix, while the SNO-HSA treatment significantly reduced this elevation. Histological changes of kidney fibrosis were evaluated by Picrosirius red staining and Masson’s trichrome staining. In the UUO with saline or HSA-treated group, marked staining was observed in the fibrotic portions in the tubulointerstitium, but these stained portions were observed to be suppressed by the administration of SNO-HSA. These findings indicate that SNO-HSA, but not HSA, suppressed kidney fibrosis by inhibiting the production of an excess of extracellular matrix. This suggests that NO is the major contributor to the anti-fibrosis action of SNO-HSA. E-cadherin, an epithelial cell marker, was significantly decreased, while α-SMA, a myofibroblast marker, was significantly increased in UUO mice. Whereas, SNO-HSA significantly suppressed the changes in these fibrosis factors caused by the UUO treatment. Since the development of kidney fibrosis was accompanied with oxidative stress and microvascular damages, the expression of 8-OHdG, a marker of oxidative stress, and CD31, a marker of vascular endothelial cells, were also evaluated. As a result, SNO-HSA improved the elevation in the expression of 8-OHdG and the reduction in that of CD31 caused by the UUO-treatment. In the UUO model, fibrosis progresses rapidly before Day 7. Therefore, to explore the mechanism of the anti-fibrotic effect of SNO-HSA, the point of evaluation was changed from Day 14 to Day 7. Similar to Day 14, SNO-HSA significantly inhibited the accumulation of hydroxyproline and α-SMA. The UUO treatment resulted in a marked increase in TGF-β, IL-6 and mRNA levels in the kidney, while SNO-HSA significantly suppressed these elevations, suggesting anti-inflammatory effect of SNO-HSA. On the other hand, the levels of EPO mRNA in kidneys of UUO-treated mice were markedly increased as the result of the SNO-HSA treatment. To confirm the anti-fibrosis action of EPO, the ESA preparation was administered twice, on Day 0 and Day 2 after the UUO treatment and the levels of hydroxyproline in the kidney were measured on Day 7. Exogenous EPO caused a significant suppression in the accumulation of hydroxyproline.
Conclusion: The present study demonstrated that SNO-HSA, a long-lasting NO donor, exerts anti-kidney fibrosis effects on the UUO model. SNO-HSA ameliorated the pathological condition in the kidney such as the excess accumulation of the extracellular matrix, changes in the fibrotic markers, the increased oxidative stress and the loss of vascular endothelium via anti-inflammatory effect and increased EPO levels. Therefore, SNO-HSA has the potential to function as a therapeutic agent for the treatment of kidney fibrosis.
1. Nakayama T, et al. Am J Physiol Renal Physiol. (2009) 296(2):F317-327.
2. Yoshioka K, et al. Am J Physiol. (1995) 269(4 Pt 1):C917-22.
3. Meng XM, et al. Nat Rev Nephrol. (2014) 10(9):493-503.
Yu Ishima– Associate professor, Tokushima University
Hitoshi Maeda– Assistant professor, Kumamoto University, Kumamoto
Hiroshi Watanabe– Associate professor, Kumamoto University, Kumamoto
Masaki Otagiri– Professor, Sojo University, Kumamoto
Toru Maruyama– Professor, Kumamoto University, Kumamoto