In normotensive rats, an increase in dietary salt leads to decreased arteriolar responsiveness to acetylcholine (ACh) because of suppressed local nitric oxide (NO) activity. We evaluated the possibility that generation of reactive oxygen species in the arteriolar wall is responsible for this loss of NO activity. Arteriolar responses to iontophoretically applied ACh were examined in the superfused spinotrapezius muscle of Sprague-Dawley rats fed a low-salt (LS; 0.45%) or high-salt diet (HS; 7%) for 4-5 wk. Responses to ACh were significantly depressed in HS rats but returned to normal in the presence of the oxidant scavengers superoxide dismutase + catalase or 2,2,6, 6-tetamethylpiperidine-N-oxyl (TEMPO) + catalase. Arteriolar responses to the NO donor sodium nitroprusside were similar in HS and LS rats. Arteriolar and venular wall oxidant activity, as determined by reduction of tetranitroblue tetrazolium, was significantly greater in HS rats than in LS rats. Exposure to TEMPO + catalase reduced microvascular oxidant levels to normal in HS rats. These data suggest that a high-salt diet leads to increased generation of reactive oxygen species in striated muscle microvessels, and this increased oxidative state may be responsible for decreased endothelium-dependent responses associated with high salt intake.

Autoimmune disease in MRL-Faslpr mice is characterized by fatal nephritis, systemic pathology, and autoantibodies, mimicking human lupus. We previously reported that 1) intrarenal IL-12 elicits nephritis by fostering the accumulation of intrarenal IFN-γ-secreting T cells, and 2) MRL-Faslpr mice deficient in the IFN-γ receptor were spared from nephritis. Therefore, we hypothesized that eliminating IL-12 in MRL-Faslpr mice reduces IFN-γ-secreting cells and thereby prevents systemic pathology. For this purpose, we constructed an IL-12p40-deficient MRL-Faslpr(IL-12−/−) strain. We determined that glomerular and interstitial, but not perivascular, renal pathology were decreased in IL-12−/− mice vs the wild-type (WT) strain (5 mo of age). Similarly, systemic pathology (lung, lacrimal and salivary glands, skin, and lymphadenopathy) was diminished. The intrarenal accumulation of T cells (CD4+, CD8+, CD4−CD8−B220+) and macrophages was dramatically reduced in IL-12−/− MRL-Faslpr kidneys. We determined that there were fewer IFN-γ transcripts (>70%) in the IL-12−/− protected kidneys compared with the WT kidneys. Similarly, cells propagated from IL-12−/− MRL-Faslpr kidneys generated substantially less IFN-γ when stimulated with IL-12 and IL-18 compared with those from WT kidneys, and we detected fewer CD8 and B220 T cells producing IFN-γ in these IL-12−/− MRL-Faslpr kidneys. Of note, survival was modestly extended in the IL-12−/− MRL-Faslpr mice. While lung and lacrimal and salivary gland pathology remained reduced in moribund IL-12−/− MRL-Faslpr mice, renal pathology and IFN-γ expression were equivalent to those in the WT strain. Thus, we suggest that IL-12 is a therapeutic target for multiple tissues in lupus; however blocking IL-12 alone is not sufficient to confer enduring protection from lupus nephritis.

Kidney tubular epithelial cell (TEC) death may be dependent on the number and activation state of macrophages (Mφ) during inflammation. Our prior studies indicate that activated Mφ release soluble mediators that incite TEC death, and reducing intrarenal Mφ during kidney disease diminishes TEC apoptosis. CSF-1 is required for Mφ proliferation and survival. We hypothesized that in the absence of CSF-1, Mφ-mediated TEC apoptosis would be prevented during renal inflammation. To test this hypothesis, we evaluated renal inflammation during unilateral ureter obstruction in CSF-1-deficient (Csf1op/Csf1op) mice. We detected fewer Mφ and T cells and less apoptotic TEC in the obstructed kidneys of Csf1op/Csf1op mice compared with wild-type (WT) mice. The decrease in intrarenal Mφ resulted from diminished recruitment and proliferation, not enhanced apoptosis. CSF-1 enhanced Mφ activation. There were far fewer activated (CD69, CD23, Ia, surface expression) Mφ in obstructed CSF-1-deficient compared with WT obstructed kidneys. Similarly, bone marrow Mφ preincubated with anti-CSF-1 receptor Ab or anti-CSF-1 neutralizing Ab were resistant to LPS- and IFN-γ-induced activation. We detected fewer apoptotic-inducing molecules (reactive oxygen species, TNF-α, inducible NO synthase) in 1) Mφ propagated from obstructed Csf1op/Csf1op compared with WT kidneys, and 2) WT bone marrow Mφ blocked with anti-CSF-1 receptor or anti-CSF-1 Ab compared with the isotype control. Furthermore, blocking CSF-1 or the CSF-1 receptor induced less TEC apoptosis than the isotype control. We suggest that during renal inflammation, CSF-1 mediates Mφ recruitment, proliferation, activation, and, in turn, TEC apoptosis.

Increased salt intake attenuates the endothelium-dependent dilation of skeletal muscle arterioles by abolishing local nitric oxide (NO) activity. There is evidence of oxidative stress in arteriolar and venular walls of rats fed a high-salt diet, and depressed arteriolar responses to acetylcholine (ACh) in these rats are reversed by scavengers of reactive oxygen species (ROS). In this study, we tested the hypothesis that this salt-dependent increase in microvascular ROS and the resulting attenuation of endothelium-dependent dilation are due to increased expression and/or activity of oxidant enzymes in the microvascular wall. Resting arteriolar and venular wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction, was consistently higher in the spinotrapezius muscle of rats fed a high-salt diet (7% NaCl, HS) for 4-5 wk than in those fed a normal diet (0.45% NaCl, NS) for the same duration. Western analysis of protein from isolated microvessels showed no difference between HS and NS rats in the expression of NAD(P)H oxidase or xanthine oxidase. Inhibition of NAD(P)H oxidase and/or xanthine oxidase with diphenyleneiodonium chloride and oxypurinol, respectively, reduced resting arteriolar wall oxidant activity to normal levels in HS rats but had no effect in NS rats, suggesting that the basal activities of NAD(P)H oxidase and xanthine oxidase are increased in HS microvessels. However, inhibition of these enzymes in HS rats did not restore normal arteriolar responses to ACh, suggesting that this stimulus activates an alternate source of ROS that eliminates the role of NO in the subsequent dilation.

High dietary salt intake decreases the endothelium-dependent dilation of skeletal muscle arterioles by inhibiting local nitric oxide (NO) activity without changing vascular smooth muscle responsiveness to NO. Under these conditions, microvascular walls show evidence of oxidative stress, and scavengers of reactive oxygen species (ROS) abolish this oxidative stress and restore normal arteriolar responses to acetylcholine (ACh). We tested the hypothesis that the salt-dependent appearance of microvascular ROS, and accompanying reduction in endothelium-dependent dilation, is due to a decrease in antioxidant enzyme expression or activity. We studied spinotrapezius muscle microvessels in rats fed normal (NS) (0.45%) or high (HS) (7%) salt diets for 4–5 weeks. Western analysis of arteriolar and venular protein showed no difference between groups in the content of superoxide dismutase (Cu/Zn SOD), catalase, or glutathione peroxidase. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) increased arteriolar and venular oxidant activity (assessed by tetranitroblue tetrazolium reduction) by the same amount in both groups, suggesting similar levels of catalase activity. 3AT did not affect arteriolar responses to ACh in either group. The Cu/Zn SOD inhibitor diethyldithiocarbamate increased arteriolar and venular oxidant activity to a lesser extent in HS rats, suggesting reduced Cu/Zn SOD activity in this group. Cu/Zn SOD inhibition decreased arteriolar responses to ACh only in NS rats. These findings suggest that endogenous Cu/Zn SOD preserves the endothelium-dependent control of arteriolar tone in NS rats, and that a reduction in Cu/Zn SOD activity contributes to the loss of arteriolar NO activity in HS rats.