And qRT-PCR analysis showed Irinotecan Camptosar a dose- Independent increase in EGR1 expression after exposure to glucose. Similar results were obtained with HK 160 human epithelial kidney cells. The agreement was a significant increase in EGR1 mRNA and protein levels in the kidneys of diabetic mice M Of C57BL / 6 M Use found 10 weeks after induction of diabetes by STZ, compared to healthy mice M, Which best CONFIRMS the relevance of the results obtained in vitro. This increase was always EGR1 levels with induction of the expression of heparanase both in vitro and in kidney tissue of diabetic M Mice, as described by real time PCR, immunohistochemistry, immunoblotting, and determination of enzyme activity demonstrated t. These observations are also used to support our hypothesis that Similar to how some immune and cancer cells in the renal tissue acts as a transcriptional activator EGR1 heparanase. To test this hypothesis, we used the ChIP approach to assess the effect of high glucose levels on an occupation of heparanase promoter by EGR1 study in HEK 293 cells. To this end, HEK 293 cells were incubated in the presence of increasing concentrations of glucose. After chemical cross-linking, chromatin was isolated, treated with ultrasound, fragments of 500 bp and immunpr Zipitiert with anti-EGR1 Antique Body. DNA from the immunpr Zipitierten chromatin was obtained using primers promoter heparanase, and a set of primers specific for the normal gene sequence The unbound of 19, by the quantities of chromatin to the same. As shown in Figure 3E, obtained hte Significantly the occupancy heparanase gene promoter by EGR1 was detected in cells that were incubated with high glucose. No enrichment was observed when Antik Was used directed against a protein body irrelevant for chip analysis. Closing Lich, to validate the F Ability to induce EGR1 heparanase expression in our system, HEK293 cells were transfected with luciferase plasmids cotransfected entered Born with heparanase promoter by EGR1 vector, or empty vector pcDNA3 and incubated in the absence or presence of 4.5 g / l glucose.
Luciferase activity t in cell lysates was measured 24 h after transfection, and normalized b-galactosidase. A tripling of the heparanase promoter activity t was in cells that were co-transfected with the pEgr1, compared to cells detected with the empty vector co-transfected. It should be noted, both co-transfection with pEgr1 or incubation activated with high glucose levels heparanase promoter in a Hnlichen Ausma, In further support of r Of the EGR1 in glucose induced heparanase expression. These results indicate in its entirety, that binds directly in the presence of high glucose levels, EGR1 promoter of heparanase and stimulates the Transkriptionsaktivit t. Heparanase inhibitor reduces albuminuria in specific mouse models of DN. The above results led us to the inhibition of heparanase on shu Hypothesis of the progression of DN in a way Similar to that can prevent knockout Mice observed HPSE. To test this hypothesis, we investigated the effect of specific inhibitors of heparanase SST0001 development of proteinuria in diabetic mice M. Compound SST0001 is 100% acetylated N, 25% glycol-split heparin, which effectively inhibits heparanase enzymatic activity of t in vitro and in contrast to unmodifie.