Fig 2: Effect of IL-1β siRNA on nuclear DNaseI translocation in RPTEC stimulated with TNFα.Confocal microscopy of sham-stimulated RPTEC (A, upper panels), cells stimulated with 20ng/ml of TNFα for 48 hrs (A, middle panels) and cells transfected with IL-1β specific siRNA and stimulated with TNFα for 48 hrs was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). While incubation of RPTEC with TNFα induced nuclear DNaseI translocation (A, middle panels), in RPTEC transfected with IL-1β siRNA and then stimulated with TNFα this did not result in translocation of DNaseI into the nuclei (A, lower panels). mRNA levels of DNaseI and IL-1β were increased in non-transfected RPTEC after stimulation with TNFα (B). Transfection with IL-1β siRNA inhibited mRNA level of IL-1β in RPTEC (C) but did not affect an increase in DNaseI gene expression induced by TNFα stimulation (C). Efficiency of the transfection procedure in RPTEC was controlled by transfection of P62 siRNA. As demonstrated in D, mRNA level of P62 was markedly decreased in transfected cells. Significances: *<0.05; **<0.005.

Fig 3: Cytoplasmic and nuclear DNaseI and Trap 1 expression in tubular cells after stimulation with TNFα.Confocal microscopy of sham-stimulated human renal proximal tubule epithelial cells (RPTEC) (A, upper panels), and cells stimulated with 20ng/ml of TNFα for 48 hrs (A, lower panels) was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). No nuclear staining of DNaseI was observed in sham-stimulation of the cells (A, upper panels). After stimulation of RPTEC with TNFα, expression of DNaseI generally increased in staining intensity, and DNaseI translocated into the nucleus (A, lower panels). Nuclear location of DNaseI was confirmed by co-staining with DAPI (A, blue). The data demonstrate that DNaseI and DAPI were in confocus (violet in the merged picture, A, lower panel), thus confirming that DNaseI indeed was translocated to the nucleus. Correspondingly, by western blots both the 40 kDa and the 55 kDa bands increased in response to increasing TNFα stimulation (0, 10, 20ng/ml TNFα) (B). In the same cells, Trap 1 expression was reduced after TNFα stimulation, as determined by confocal microscopy (A, lower panels versus upper panels, green staining) and by western blot (B). Nuclear and cytoplasmic protein fractions were isolated from RPTEC after 48 hrs stimulation by TNFα (C). The 40 kDa and 55 kDa bands were detected in cytoplasmic fraction (C, lane 1), while weak 52 kDa band was observed in nuclear fraction of sham-stimulated RPTEC (C, lane 2). After 48 hrs stimulation with 20ng of TNFα, an increased DNaseI expression in cytoplasm (C, lane 3) and remarkably strong expression of the 52 kDa DNaseI in the nuclei was apparent (C, lane 4). Thus data from western blot analysis corresponded with data from confocal microscopy with respect to TNFα-induced nuclear translocation of DNaseI. Only nuclear fractions of RPTEC contained histone H1 protein as shown in D, confirming controlled separation of nuclear and cytoplasmic fractions in the cells. Actin was used as a loading control in all western blot analyses.

Fig 4: Nuclear DNaseI translocation in RPTEC was not associated with caspase 3 activation or DNA fragmentation.Western blot analysis was performed to detect presence of activated caspase 3 in cell lysates from RPTEC stimulated or non-stimulated with 20ng/ml of TNFα for 48 hrs (A). Resting Jurkat cells or cytochrome c treated Jurkat cells were used as a negative and positive control for activated caspase 3 (A, lane 1 and 2, respectively). As demonstrated in A, lane 4, TNFα-induced nuclear DNaseI translocation was not accompanied by activation of caspase 3 since only inactive pro-caspase 3 was found in RPTEC stimulated with TNFα. Only in cytochrome c treated cells activated caspase 3 was detected by western blot as shown in A, lane 2. Equal loading was controlled by staining with actin. Location of DNaseI in the nuclei of RPTEC did not result in chromatin fragmentation as demonstrated by DNA gel electrophoresis in B and TUNEL assay in C. DNA from murine spleen incubated with 5μM camptothecin for 6 hrs was used as a positive control for apoptotic DNA fragmentation (B, lane 2). No apoptotic DNA fragmentation was observed in DNA isolated from sham-stimulated RPTEC cells (B, lane 3) or in DNA isolated from RPTEC after stimulation with 20ng/ml of TNFα for 48 hrs (B, lane 4). TUNEL-positive staining in RPTEC cells was detected only after treatment with 10U of rhDNaseI (C, green). RPTEC cells stimulated with TNFα were TUNEL–negative as well as sham-stimulated cells as demonstrated in C. Lane 1 in B represent DNA size markers.

Fig 5: Pro-inflammatory cytokines profile in lupus nephritis and their effect on DNaseI expression in tubular cells.Renal mRNA expression of pro-inflammatory cytokines in (NZBxNZW)F1 mice during development of lupus nephritis demonstrated in A. The data demonstrate significant up-regulation of TNFα and IL-1β in Group 2 and Group 3 mice when compared to Group 1. IL-10 mRNA levels were significantly increased in Group 3 BW mice compared to Group 1. One-way ANOVA with Dunett post hoc test was performed to assess the difference in gene expression between the groups when compared to group 1 and 2. Effect of pro-inflammatory cytokines and hypoxia on DNaseI and Trap 1 mRNA levels in human renal proximal tubule epithelial cells (RPTEC) are shown in B. Significant elevation of DNaseI mRNA levels in RPTEC was found only upon TNFα stimulation and stress by hypoxia (B). No change in DNaseI gene expression was observed when RPTEC were stimulated by IL-1β, IFNγ, IL-6 or IL-10 (B). Trap 1 mRNA was significantly reduced after stimulations with IL-1β and hypoxia. Significances: *<0.05; **<0.005; ***<0.0005.