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. 2020 Jan:146:333-339.
doi: 10.1016/j.freeradbiomed.2019.11.014. Epub 2019 Nov 12.

NOX2-derived reactive oxygen species in immune cells exacerbates salt-sensitive hypertension

Justine M Abais-Battad  1 , Hayley Lund  2 , John Henry Dasinger  3 , Daniel J Fehrenbach  3 , Allen W Cowley Jr  2 , David L Mattson  3
Affiliations

NOX2-derived reactive oxygen species in immune cells exacerbates salt-sensitive hypertension

Justine M Abais-Battad et al. Free Radic Biol Med. 2020 Jan.

Abstract

Previous studies utilizing the SSp67phox-/- rat have demonstrated the importance of systemic NADPH oxidase NOX2-derived reactive oxygen species (ROS) production in the pathogenesis of Dahl Salt-Sensitive (SS) hypertension and renal damage. It is established that the immune system contributes to the development of SS hypertension and our laboratory has observed an enrichment of NOX2 subunits in infiltrating T cells. However, the contribution of immune cell-derived ROS in SS hypertension remains unknown. To assess the role of ROS in immune cells, SSp67phox-/- rats underwent total body irradiation and received bone marrow transfer from either SS (+SS) or SSp67phox-/- (+SSp67phox-/-) donor rats. Demonstrated in a respiratory burst assay, response to phorbol 12-myristate 13-acetate stimulus (135 μM) was 10.2-fold greater in peritoneal macrophages isolated from +SS rats compared to nonresponsive + SSp67phox-/- cells, validating that + SS rats were capable of producing NOX2-derived ROS in cells of hematopoietic origin. After 3 weeks of high salt challenge, there was an exacerbated increase in mean arterial pressure in +SS rats compared to + SSp67phox-/- control rats (176.1 ± 4.7 vs 147.9 ± 8.4 mmHg, respectively), which was accompanied by a significant increase in albuminuria (168.3 ± 23.7 vs 107.0 ± 20.4 mg/day) and renal medullary protein cast formation (33.2 ± 4.7 vs 8.1 ± 3.5%). Interestingly, upon analysis of renal immune cells, there was trending increase of CD11b/c + monocytes and macrophages in the kidney of +SS rats (4.7 ± 0.4 vs 3.5 ± 0.5 × 106 cells/kidney, +SS vs + SSp67phox-/-, p = 0.06). These data altogether demonstrate that immune cell production of NOX2-derived ROS is sufficient to exacerbate Dahl SS hypertension, renal damage, and renal inflammation.

Keywords: Hypertension; Immune cells; NADPH oxidase; Reactive oxygen species; Renal damage; p67phox.

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Figures

Figure 1.
Figure 1.. Attenuation of hypertension and renal damage in SSp67phox−/− rats.
(A) Daily mean arterial pressure (MAP) averages over the course of 3 weeks of 4.0% NaCl challenge. n=7-8. (B) Weekly determination of renal damages indices, urinary protein and albumin excretion. n=8-9. **p<0.005, ***p<0.001 SSp67phox−/− vs SS.
Figure 2.
Figure 2.. Attenuation of renal histological damage and renal immune cell infiltration in SSp67phox−/− rats.
(A) Percentage of medullary protein cast formation in the kidneys of SS and SSp67phox−/− rats after 3 weeks of 4.0% NaCl challenge. n=7-9. (B) Circulating immune cell profile between SS and SSp67phox−/− rats. (C) Absolute numbers of immune cells infiltrating into the kidneys at the conclusion of the experiment. CD45+: leukocytes, CD11b/c+: monocytes/macrophages, CD3+: T cells, CD4+: T-helper cells, CD8+: cytotoxic T cells, CD45R+: B cells. n=7-9. *p<0.05, **p<0.01 SSp67phox−/− vs SS.
Figure 3.
Figure 3.. Respiratory burst assay in isolated peritoneal macrophages from TBI/BMT rats demonstrates the ability of SS-derived hematopoetic cells to produce ROS.
Luminol derivative L-012 was used as an index of superoxide production. Superoxide production was detected in PMA-stimulated macrophages from isolated from rats received bone marrow from SS donors, which was not evident in macrophages from SSp67phox−/− donors. 1×106 peritoneal macrophages/well, PMA = 135 uM, SOD = 100 U/well. n=6-8. pGroup<0.001 SSp67phox−/− vs SS, ***p<0.001 PMA vs Ctrl (SS), †††p<0.001 PMA vs PMA+SOD (SS).
Figure 4.
Figure 4.. NADPH oxidase-derived ROS production in immune cells is necessary for the development of salt-sensitive hypertension and renal damage in SS rats.
(A) Exacerbated rise in mean arterial pressure (MAP) in SSp67phox−/− receiving SS bone marrow (+SS) after 3 weeks of 4.0% NaCl challenge. n=4-5. (B) Renal damage indices, urinary protein and albumin excretion, were also worsened in rats receiving SS bone marrow (+SS) compared to those receiving SSp67phox−/− bone marrow (+SSp67phox−/−). n=9-11. *p<0.05, **p<0.005, ***p<0.001 +SSp67phox−/− vs +SS.
Figure 5.
Figure 5.. Exacerbation of renal histological damage in SSp67phox−/− receiving SS bone marrow (+SS) rats.
(A) Percentage of medullary protein cast formation in the kidneys of +SS and +SSp67phox−/− rats after 3 weeks of 4.0% NaCl challenge. n=7-9. (B) Circulating immune cell profile between +SS and +SSp67phox−/− rats. (C) Absolute numbers of immune cells infiltrating into the kidneys at the conclusion of the experiment. CD45+: leukocytes, CD11b/c+: monocytes/macrophages, CD3+: T cells, CD4+: T-helper cells, CD8+: cytotoxic T cells, CD45R+: B cells. n=9-10. *p<0.05, **p<0.01 +SSp67phox−/− vs +SS.
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