Additional file 1 of Macrophage subpopulations in pediatric patients with lupus nephritis and other inflammatory diseases affecting the kidney

Additional file 1: Supplemental Table 1. Characteristics of adult LN cohort. Supplemental Table 2. Primary antibodies used for immunofluorescence microscopy. Supplemental Table 3. Secondary antibodies used for immunofluorescence microscopy (IF)

Oxidative DNA Damage in Kidneys and Heart of Hypertensive Mice Is Prevented by Blocking Angiotensin II and Aldosterone Receptors

<div><p>Introduction</p><p>Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied.</p><p>Methods</p><p>In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis.</p><p>Results</p><p>Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone.</p><p>Conclusion</p><p>Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress <i>in vivo</i>. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.</p></div

Primer sequences used for real-time PCR.

<p>NADPH oxidase isoform 1,</p><p>Nox2  =  NADPH oxidase isoform 2, Nox4  =  NADPH oxidase isoform 4.</p><p>Primer sequences used for real-time PCR.</p

Clinical parameters, parameters of kidney function as well as aldosterone levels of mice after 27 days of angiotensin II-infusion.

<p>* p≤0.05, ** p<0.01, *** p<0.001 vs. Control, ° p≤0.05, °° p<0.01, °°° p<0.001 vs. Ang II treatment.</p><p>Clinical parameters, parameters of kidney function as well as aldosterone levels of mice after 27 days of angiotensin II-infusion.</p

Histopathological changes of kidneys of control animals and animals treated with angiotensin II (AngII) with or without co-treatment with candesartan (+Cand), eplerenone (+Eple), or tempol (+Tem).

<p>A: Representative pictures of PAS-stained tissue, visualizing glomerular damage. B: Representative pictures of HE-stained tissue, visualizing regions of inflammation. C: Representative pictures of Sirius red-stained tissue visualizing regions of fibrosis (red). D: Representative pictures of HE-stained tissue, focussing on changes of the vasculature. Blue filled arrows: examples of mesangiolysis, orange filled arrows: examples of glomerulosclerosis, white filled arrows: examples of infiltrated leukocytes as a marker of inflammation, I–I: device illustrating the thickness of the vessel walls.</p

Multiple reaction monitoring parameters for the analysis of oxidized bases and internal standard and optimized conditions of the mass spectrometry measurement with the Q-Trap 2000.

<p>Multiple reaction monitoring parameters for the analysis of oxidized bases and internal standard and optimized conditions of the mass spectrometry measurement with the Q-Trap 2000.</p

Induction of a marker of renal damage.

<p>Western blot-analysis of the amount of the kidney injury marker KIM-1 (kidney injury molecule) in kidneys of control animals and animals treated with angiotensin II (AngII) with or without co-treatment with candesartan (+Cand), eplerenone (+Eple), or tempol (+Tem). Shown is a representative blot and the quantification of band densities of proteins of all animals.* p≤0.05 vs. Control.</p

Histopathological parameters.

<p>GSI: glomerular sclerosis index, MSI: mesangiolysis index, TSI: tubulointerstitial sclerosis index, VSI: vascular sclerosis index, each normalized to the Control values.</p><p>* p≤0.05, ** p<0.01, *** p<0.001 vs. Control, ° p≤0.05, °° p<0.01, °°° p<0.001 vs. Ang II treatment.</p><p>Histopathological parameters.</p

Induction of transcription factors and target proteins.

<p>Shown are representative pictures of paraffin-embedded kidney cortex and heart sections of control animals and animals treated with angiotensin II (AngII) with or without co-treatment with candesartan (+Cand), eplerenone (+Eple), or tempol (+Tem). Some examples of positive-stained cells are marked with red arrows. Representative pictures of the immunohistochemical detection of (<b>a</b>) Nrf2-positive cells on kidney sections. Quantification of cells positively stained for Nrf2 with the cell image analysis CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115715#pone.0115715-Lamprecht1" target="_blank">[15]</a> on kidney (<b>b</b>) and heart (<b>c</b>) tissue in 7 visual fields. Representative pictures of the immunohistochemical detection of Nrf2-positive cells on heart sections (<b>d</b>). Western blot-analysis of the amount of the Nrf2-target protein heme oxygenase-1 (HO-1) in protein extracts of kidney (<b>e</b>) and heart (<b>f</b>), and the Nrf2-target protein superoxide dismutase (SOD) in protein extracts of heart (<b>g</b>), related to the house-keeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Shown are representative blots and quantification of band densities of proteins of all animals. Representative pictures of the immunohistochemical detection of (<b>h</b>) NF-κB-positive cells on kidney sections. Quantification of cells positively stained for NF-κB with the cell image analysis CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115715#pone.0115715-Lamprecht1" target="_blank">[15]</a> on kidney tissue in 7 visual fields (<b>i</b>). Quantification of cells positively stained for NF-κB per mm² of heart tissue (<b>j</b>). Representative pictures of the immunohistochemical detection of (<b>k</b>) NF-κB-positive cells on heart sections. * p≤0.05, ** p<0.01,*** p<0.001 vs. Control, ° p≤0.05, °° p<0.01, °°° p<0.001 vs. AngII-treatment.</p

Markers of DNA damage and DNA repair in kidney, heart and urine.

<p>Shown are representative pictures of paraffin-embedded kidney cortex and heart sections of animals of the Control group, the angiotensin II group (AngII), the AngII/candesartan group, the AngII/eplerenone group, and the AngII/tempol group. Some examples of positive-stained cells are marked with white (<b>a</b>) or red (<b>a, e, h</b>) arrows. Representative pictures of the immunofluorescent detection of (<b>a</b>) γ-H2AX-positive cells on kidney sections. Quantification of cells positively stained for γ-H2AX per mm² of kidney (<b>b</b>) and heart (<b>c</b>) tissue. Representative pictures of the immunohistochemical detection of γ-H2AX -positive cells on heart sections (<b>d</b>). Representative pictures of the immunofluorescent detection of (<b>e</b>) poly(ADP-ribose) (PADPR)-positive cells on kidney sections. Quantification of cells positively stained for PADPR with the cell image analysis CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115715#pone.0115715-Lamprecht1" target="_blank">[15]</a> on kidney tissue (<b>f</b>) and heart tissue (<b>g</b>) in 7 visual fields. Representative pictures of the immunohistochemical detection of PADPR-positive cells on heart sections (<b>h</b>). As a marker of DNA oxidation, excreted 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) was measured in urine by mass spectrometry (<b>i</b>). As a marker of RNA oxidation, excreted 7,8-dihydro-8-oxo-guanosine (8-oxoGuo) was measured in urine by mass spectrometry (<b>j</b>). * p≤0.05, ** p<0.01,*** p<0.001 vs. Control, ° p≤0.05, °° p<0.01, °°° p<0.001 vs. AngII-treatment.</p