Nox2 and Cyclosporine-Induced Renal Hypoxia

Abstract

Background: We hypothesized that nicotinamide adenosine diphosphate oxidase 2 (Nox2) plays an important role in cyclosporine A (CsA)-induced chronic hypoxia.

Methods: We tested this hypothesis in Fisher 344 rats, C57BL/6 J wild type and Nox2-/- mice, and in liver transplant recipients with chronic CsA nephrotoxicity. We used noninvasive molecular imaging (blood oxygen level-dependent magnetic resonance imaging and dynamic contrast-enhanced magnetic resonance imaging) and molecular diagnostic tools to assess intrarenal oxygenation and perfusion, and the molecular phenotype of CsA nephrotoxicity.

Results: We observed that chemical and genetic inhibition of Nox2 in rats and mice resulted in the prevention of CsA-induced hypoxia independent of regional perfusion (blood oxygen level-dependent magnetic resonance imaging and dynamic contrast-enhanced magnetic resonance imaging, pimonidazole, HIF-1α). Nicotinamide adenosine diphosphate oxidase 2 knockout was also associated with decreased oxidative stress (Nox2, HIF-1α, hydrogen peroxide, hydroxynonenal), and fibrogenesis (α-smooth muscle actin, picrosirius red, trichrome, vimentin). The molecular signature of chronic CsA nephrotoxicity using transcriptomic analyses demonstrated significant changes in 40 genes involved in injury repair, metabolism, and oxidative stress in Nox2-/- mice. Immunohistochemical analyses of kidney biopsies from liver transplant recipients with chronic CsA nephrotoxicity showed significantly greater Nox2, α-smooth muscle actin and picrosirius levels compared with controls.

Conclusions: These studies suggest that Nox2 is a modulator of CsA-induced hypoxia upstream of HIF-1α and define the molecular characteristics that could be used for the diagnosis and monitoring of chronic calcineurin inhibitor nephrotoxicity.

Figure 1. CsA toxicity was associated with fibrogenesis and OS in rats

CsA toxicity was associated with fibrogenesis and OS in rats. Immunoblot analyses of whole kidney tissue lysates from rats treated with CsA or vehicle for 1 month. Panel (a) shows representative blots and Panel (b) represents the semiquantitative assessment of all protein expression normalized to GAPDH with Image J software analyses using the mean of three representative blots from three different animals. These studies determined an upregulation of biomarkers of oxidative stress (Nox2, Nox4, p22phox, HNE) and fibrogenesis (α-SMA, fibronectin, phosphorylated-Smad2).

Figure 2. Nox2 inhibition prevented CsA-induced hypoxia in rats-BOLD MRI studies

Nox2 inhibition prevented CsA-induced hypoxia in rats-BOLD MRI studies. Adult male Fisher 344 rats were randomized into four treatment groups: Castor oil vehicle, Cyclosporine (15mg/kg/day) alone, Cyclosporine (15mg/kg/day) + diphenyleneiodonium (DPI, 0.5mg/kg/day), or Cyclosporine (15mg/kg/day) + Apocynin (16mg/kg/day) (n=6–8 in each treatment group). Panel (A) represents Grayscale and BOLD color map images of a control rat kidney. The color map indicates the cortex and inner medullar have lower R2* values (1,3) than the outer medulla (2). Panel (B) displays the bar graph of mean (±SD) cortical and medullary R2* levels in the 4 treatment groups. CsA therapy resulted in a significant increase in R2* levels indicating cortical and medullary hypoxia. Co-treatment with DPI prevented cortical and medullary hypoxia (significantly lower R2* levels). Therapy with Apocynin did not result in a significant change in R2* levels.

Figure 3. Apocynin and DPI prevented CsA-induced Nox2 expression in rats

Apocynin and DPI prevented CsA-induced Nox2 expression in rats. Panels a-d display representative images of Nox2 staining experiments in rats treated with castor oil, CsA, CsA-Apocynin and CsA-DPI (n=6–8 in each group). Panel e represents the semiquantitative assessment of Nox2 staining using digital software analyses. The studies showed that CsA upregulated Nox2 expression in renal cortical tubules and interstitium. Nox2 expression was significantly inhibited by apocynin and DPI.

Figure 4. Nox2 inhibition prevented CsA-induced fibrosis in rats

Nox2 inhibition prevented CsA-induced fibrosis in rats. Panels a-d display representative images of trichrome blue staining experiments in rats treated with castor oil, CsA, CsA-Apocynin and CsA-DPI (n=6–8 in each group). Panel e represents the semiquantitative assessment of trichrome blue using digital software analyses. The studies showed that CsA increased interstitial trichrome staining compared to control. Fibrosis was significantly inhibited by DPI, while apocynin-associated changes did not reach statistical significance.

Figure 5. Apocynin and DPI prevented CsA-induced fibrosis and OS in rats

Apocynin and DPI prevented CsA-induced fibrosis and OS in rats. Panel a displays representative immunoblots of Nox2, vimentin, and GAPDH internal control from whole kidney tissue lysates from rats treated with castor oil, CsA, CsA-Apocynin and CsA-DPI. Panel b represents the semiquantitative assessment of Nox2 and vimentin expression normalized to GAPDH with Image J software analyses using the mean of three representative blots from three different rats. Panel c displays serum hydrogen peroxide measurements using the Amplex Red assay. The studies showed that CsA upregulation of Nox2 and vimentin protein levels was blunted by apocynin and DPI (Panels a,b). Similarly, the increase in systemic hydrogen peroxide levels (H2O2) was prevented by DPI. Apocynin related changes did not reach statistical significance (Panel c).

Figure 6. Nox2^−/− prevented CsA-induced intrarenal hypoxia in mice-BOLD MRI Studies

Nox2^−/− prevented CsA-induced intrarenal hypoxia in mice-BOLD MRI Studies. CsA (30mg/kg/day) was administered i.p. to C57BL/6J mice and B6.129S-CybbTm1Din/J (Nox2−/−) mice. Animals were treated for 4–8 weeks for time course studies (6–10 animals in each group). BOLD MRI grayscale gradient echo anatomical image (Panel a) with correlative color R2* map (Panel b). The color map indicates the cortex has a lower R2* values (2) than the outer medulla (1). Panels c and d display bar graphs of mean cortical and medullary R2* levels at 4 and 8 weeks in control and CsA treated wild type and Nox2−/− mice. Panel e is a Table summarizing the p values for the difference between various groups. CsA therapy in wild type animals resulted in cortical and medullary hypoxia as evidenced by increased R2* compared to vehicle (Panels c,d,e). CsA therapy had no significant effect on intrarenal oxygenation in Nox2−/− animals compared to vehicle at 8 weeks. CsA-induced hypoxia was significantly prevented in the cortex and medulla of Nox2−/− mice at 8 weeks (Panels c,d,e).

Figure 7. Nox2^−/− prevented CsA-induced intrarenal hypoxia measured by pimonidazole

Nox2^−/− prevented CsA-induced intrarenal hypoxia measured by pimonidazole. Animals received pimonidazole i.p. (60mg/kg) 2 hours prior to terminal exsanguination. Panels and b display representative pimonidazole staining (brown color) in wild type (a) and Nox2−/− mice (b). Intensity of staining is greater in panel (a) and the distinction between cortex and outer medulla is not as clear in wild type animals as in Nox2−/− mice. Panel c is a bar graph representing semiquantitative pimonidazole staining intensity in the 4 groups using digital software analyses (n=6–10 in each group). These studies determined that CsA therapy was associated with a significant increase in pimonidazole staining in wild type mice. However, this staining was significantly reduced in Nox2−/− animals, to baseline control levels.

Figure 8. Renal Perfusion was not affected by CsA or Nox2

Regional perfusion was not affected by CsA and Nox2. Using DCE imaging, we fit the renal contrast uptake as a single, first pass vascular compartment for this analysis. This gave the best separation between the groups. We used an average arterial input function (AIF) calculated from each mouse. Panel (a) shows grayscale pre-contrast and peak-contrast, and perfusion color map (ml/min/g) images in a control mouse at baseline. Panel (b) shows measured and fitted vascular compartment kinetic maps in cortex and medulla. Panels (c) and (d) represent bar graphs of renal cortical and medullary perfusion of experimental animals at baseline, 4W, and 8W. These studies showed no significant effect of CsA or Nox2 on regional perfusion. Panel (e) is a summary table of the p values comparing perfusion values.

Figure 9. Nox2^−/− prevented CsA-induced Fibrogenesis and OS in mice

Nox2^−/− prevented CsA-induced Fibrogenesis and OS in mice. We performed immunoblot analyses examining matrix remodeling (vimentin), hypoxia (HIF-1α), and OS (HNE) at baseline and 8 weeks in vehicle and CsA-treated wild type and Nox2−/− mice. We observed reduced vimentin, HIF-1α, and HNE levels in Nox2−/− animals compared to wild type. Bar graphs represent semiquantitative assessment of protein expression normalized to GAPDH with Image J software analyses using the mean of three representative blots from three different mice at baseline, 8 wks vehicle and 8 wks CsA groups.

Figure 10. CsA-induced picrosirius red staining was prevented in Nox2−/− mice

CsA-associated picrosirius staining was prevented in Nox2−/− mice. High dose CsA (15 mg/kg/d x 8 weeks) resulted in significantly greater collagen deposition measured by picrosirius digital quantification (Panels a–d). Picrosirius staining was significantly reduced in CsA treated Nox2−/− animals (Panels a, and b).

Figure 11. Molecular classifiers defining the top 40 genes characterizing CsA nephrotoxicity in mice and the effect of Nox2^−/− on CsA nephrotoxicity

Molecular classifiers defining the top 40 genes characterizing CsA nephrotoxicity in mice and the effect of Nox2^−/− on CsA nephrotoxicity. Heat maps displaying the top 40 genes with most significant p values, differentially expressed in kidneys from wild type mice treated with CsA compared to controls. These genes were isolated out of a pool of 410 transcripts, all differentially expressed, with a p value < 0.05 (Panel a). Panel b displays the top 40 genes (out of 662 significantly different transcripts) characterizing the molecular effect of Nox2 on chronic CsA nephrotoxicity. Most (85%) of these transcripts were decreased in the absence of Nox2.

Figure 12. Nox2 was associated with CNI induced renal fibrosis in liver transplant recipients

Nox2 was associated with CNI induced renal fibrosis in liver transplant recipients. We determined Nox2, α-SMA, heat shock factor (HSF), and picrosirius expression in clinical biopsies from liver transplant patients diagnosed with chronic CsA nephrotoxicity compared to pre-implantation biopsies from donor kidneys (n=5 in each group). These studies revealed higher tubulointerstitial expression of Nox2 and α-SMA, and greater interstitial deposition of collagen noted by picrosirius staining.