Expression levels of the five associated genes.

<p>Rt-PCR for <i>Spata17, Gpatch2, Esrrg, Ush2a,</i> and <i>Kctd3</i> (n = 3–5). The results represent the mean ± SE. Comparing all animals (regardless of strain) with and without deposits using a t-test showed no significant difference for any of the genes.</p

Intracapillary glomerular deposits.

<p>Representative example of an unaffected NZW 6-month-old, an affected NZW 20-month-old and an unaffected C57BLKS 20-month-old male mouse using PAS (A–C), apoE (D–F), apoB (G–I), and apoA-IV (J–L) stainings. Deposits are seen in the glomerular capillary lumina (arrows). Immunohistochemistry staining shows that these deposits are strongly positive for apoE (E, arrows) compared to the younger NZW mouse (D) and a mouse of a strain negative for glomerular deposits (C57BLKS) (I). The deposits are weakly positive for apoB (H, arrows) and apoA-IV (K, arrows) compared to the 6-month old NZW mouse (G, J) and C57BLKS control (I, L).</p

Genome wide HAM results.

<p>Genome-wide scan for intracapillary glomerular deposits. A peak reaching the significant threshold of <i>P</i><10⁻⁶ is seen on Chr 1 (A). The haplotype block of six SNPs between rs48848476 and rs46020199 is associated with glomerular deposits. The region spanning 1 Mb on each site from this area includes the genes <i>Spata17, Gpatch2, Esrrg, Ush2a,</i> and <i>Kctd3</i> (B).</p

Characterization of the glomerular deposits.

<p>Elektron microscopy of an affected glomerulus of a 20-month-old NOD.B10 mouse shows filling of the intracapillary lumina with optically lucent areas, suggestive of lipid vacuoles. Intracapillary lumina (closed arrow), nucleus (*) and podocyte (dashed arrow).</p

Glomerular deposits express a lipid droplet surface protein.

<p>Glomeruli with intracapillary deposits show positive staining for a protein that binds to the surface of lipid droplets, perilipin-2 (B, D) in contrast to glomeruli without deposits, which are negative (A, C).</p

High concentrations of H₂S induce a state of hypometabolism.

<p>Within 15 minutes (A) CO₂-production decreased by 72% (***p<0.001) in mice (n = 11) subjected to 100 ppm H₂S. Exposure to 10 ppm H₂S (n = 11) did not induce a reduction in CO₂-production in these animals. Cessation of gaseous H₂S resulted in rapid recovery, within 30 minutes CO₂-levels returned back to baseline concentrations (***p<0.001). Administration of 100 ppm H₂S (n = 4) resulted in a 14% decrease in (B) mean arterial pressure and a 25% decrease in (C) heart rate (*p<0.05). However exposure to 10 ppm H₂S (n = 4) had no effect on mean arterial pressure or heart rate.</p

H₂S reduces ROS production in cultured cardiomyoblasts.

<p>(A) Antimycin (50 µg/ml) significantly induced ROS production in H9c2 cells from 24 minutes onwards compared to untreated control cells. Addition of 1 mM NaHS prevented the increase in ROS production. (^#p<0.001 vs. control; *p<0.001 vs. NaHS) (B) Representative photomicrographs of DHE stained H9c2 cells treated with antimycin and NaHS showing less DHE staining (red) in the NaHS treated cells as compared to cells treated with only antimycin.</p

H₂S reduces cardiac IRI induced inflammation.

<p>There was a marked increase in granulocyte influx after cardiac ischemia (p<0.001) compared to sham animals. Exposure to 10 ppm and 100 ppm H₂S significantly reduced the influx of granulocytes in the infarct area (10 ppm p<0.05; 100 pm p<0.001). Below are representative images of these stainings: (A) Sham (B) IRI, 0 ppm H₂S (C) IRI, 10 ppm H₂S (D) IRI, 100 ppm H₂S.</p

Cardiac damage is reduced by 10 and 100 ppm H₂S at 7 days of reperfusion.

<p>(A) Representative photomicrographs of Masson stained cardiac sections with fibrotic area artificially colored red, indicating the extent of fibrotic damage found in each group at 7 days of reperfusion. (B) Cardiac IR induced a significant amount of fibrosis in IRI animals exposed to 0 ppm H₂S (^###p<0.001 vs. sham). In animals treated with 10 ppm and 100 ppm H₂S fibrosis was significantly reduced (**p<0.01, *p<0.05 vs. IRI). (C) Expression of fibronectin at 7 days of reperfusion was increased in IRI animals (^##p<0.01 vs. sham). Treatment with 10 and 100 ppm H₂S reduced the expression of fibronectin (*p<0.05, **p<0.01 vs. IRI). (D) Seven days post-reperfusion hs Troponin-T levels were elevated in IRI animals exposed to 0 ppm H₂S (^#p<0.01 vs. sham). Both 10 and 100 ppm H₂S reduced hs Troponin-T levels by 59% and 75%, respectively, as compared to animals treated with 0 ppm (*p<0.05, **p<0.01 vs. IRI). (E) Expression of ANP mRNA at 7 days of reperfusion was increased in IRI animals (^##p<0.01 vs. sham). Treatment with 10 and 100 ppm H₂S reduced the expression of ANP (*p<0.05 vs. IRI).</p

H₂S attenuates NOX2 and NOX4 upregulation.

<p>Expression of (A) NOX2 and (B) NOX4 mRNA was increased in IRI animals at 7 days post-reperfusion (NOX2: ^#p<0.05, NOX4: ^##p<0.01 vs. sham). Treatment with 10 and 100 ppm H₂S reduced the expression of both genes (*p<0.05 vs. IRI). After 1 day of reperfusion no differences were observed between all groups.</p