Volume 86, Issue 6, Pages 1187-1196 (December 2014) Janus kinase signaling activation mediates peritoneal inflammation and injury in vitro and in vivo in response to dialysate  Tiane Dai, Ying Wang, Aditi Nayak, Cynthia C. Nast, Lan Quang, Janine LaPage, Ali Andalibi, Sharon G. Adler  Kidney International  Volume 86, Issue 6, Pages 1187-1196 (December 2014) DOI: 10.1038/ki.2014.209 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Heat-sterilized or filter-sterilized peritoneal dialysis fluid (PDF) induced STAT 1 and STAT3 phosphorylation, which was prevented by the pan-JAKi P6. Met-5A was cultured in standard medium, and then switched to control medium or filter-sterilized or heat-sterilized PDF (30%:70%, medium:PDF) with or without glucose degradation product (GDP) and/or the pan-JAKi P6 × 1h. Cells were then collected and immunoblotted with indicated antibodies. (a) Representative western blots of phospho-STAT1 (p-STAT1) and quantitative evaluation of relative fold changes of phospho-STAT 1/total STAT1 by densitometry (n=4). (b) Representative western blots of phospho-STAT3 (p-STAT3) and quantitative evaluation of relative fold changes of phospho-STAT3/total STAT3 by densitometry (n=4 for each condition). Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Heat-sterilized peritoneal dialysis fluid (HPDF) induced periostin expression that was reduced by the pan-JAKi P6 in a dose-dependent manner. Filter-sterilized PDF (FPDF)+glucose degradation products (GDPs) but not FPDF alone induced periostin expression that was inhibited by the pan-JAKi P6. (a) Met-5A was cultured in control medium or heat-sterilized PDF (30%:70%, medium:PDF) with or without different concentrations of the pan-JAKi P6 for 24h. The upper panel shows representative western blots of periostin; the lower panel shows the quantitative analysis of relative fold change of periostin/GAPDH by densitometry (n=4). (b) Met-5A cells were cultured in control medium or filter-sterilized or heat-sterilized PDF (30%:70%, medium:PDF) with or without GDP and/or the pan-JAKi P6 × 24h. The upper panel shows representative western blots of periostin; the lower panel is quantitative analysis of relative fold change of periostin/GAPDH by densitometry with indicated condition (n=4). (c) Relative expression of periostin mRNA was measured by reverse transcriptase-PCR and normalized to 18S in the immortalized human peritoneal cell line LP9/TERT.bsd (n=3). Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 PDF induced caspase-3 activation; induction was attenuated by the pan-JAKi p6. After 24h of treatment, Met-5A cells were then harvested for western blot analysis of cleaved caspase-3. (a) The upper panel shows representative western blots of cleaved caspase-3; the lower panel shows quantitative analysis of relative fold change of cleaved caspase-3/GAPDH by densitometry with indicated conditions (n=4). (b) Representative fluorescence-labeled cleaved caspase-3 at indicated conditions. Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 JAK phosphorylation was present in long-term (LT) (n=7) but not in new (n=8) patients’ PD effluent cells. STAT transcriptional target proteins MCP-1 and periostin were increased in peritoneal dialysis effluent (PDE) of long-term versus new patients. Western blots were performed with PD effluent cell lysates centrifuged from long-term and new patients. Phospho-JAK1 or phospho-JAK2 were present in long-term patients (n=4) but not in new (n=4) patients (a). MCP-1 (b, P<0.001) and periostin (c, P<0.05) were higher in the PD effluent of long-term (n=7) versus new patients (n=8). Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Histological changes in rats receiving peritoneal dialysis with or without concomitant JAK1/2i. Nonuremic rats were dialyzed with either normal saline (n=7), Dianeal 4.25% (n=7), or Dianeal 4.25%+JAK1/2 i (n=7). Parietal and visceral peritonea were harvested and stained with anti-phospo-JAK1 (a–f), Masson’s trichrome (g–i), and hematoxylin and eosin (j–l). JAK1 staining (a–f). In parietal (a–c) and visceral (d–f) peritoneum, there was no JAK activation in rats receiving saline (a, d). There was phospho-JAK1 staining in the mesothelial cell layer and the submesothelial compact zone, most likely in fibroblasts and mononuclear infiltrating cells (b) and in hyperplastic mesothelial cells (e) in rats receiving Dianeal 4.25%. Instillation of the JAK1/2i attenuated JAK activation in rats that received Dianeal 4.25% (c, f). Peritoneal histology (g–l). In the parietal peritoneum of rats dialyzed with saline, there is thickening of the submesothelial area owing to edema and fibrosis with mild increase in cellularity (g). Rats receiving Dianeal 4.25% had prominently thickened submesothelial areas with infiltrating fibroblasts and mononuclear leukocytes (h). In rats dialyzed with Dianeal 4.25%+JAK1/2i, there is marked reduction in submesothelial fibrosis and cellularity (i). In the visceral peritoneum, dialysis with saline did not disrupt the delicate architecture of the mesothelial cell layer (j). Dialysis with Dianeal 4.25% induced marked mesothelial reactive hyperplasia and fibrosis (k). The JAK1/2i nearly completely inhibited the hyperplastic response (l). The submesothelial zone thickness was measured from the abdominal wall muscle to the mesothelial layer, and determined as the average distance of eight measurements from each peritoneal section (m). Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Peritoneal capillary staining with CD34 is increased in rats receiving peritoneal dialysis with Dianeal 4.25% compared with those receiving saline, and the JAK1/2i attenuated this hypervascularity. (a, d) Saline dialysate was associated with minimal vascularity in the parietal (a) and visceral (d) peritoneum. (b, e) Dianeal 4.25% resulted in a significant increase in vascularity shown as CD34-stained capillaries in the parietal peritoneum (b), with increased vascularity also in the visceral peritoneum (e). (c, f) Dianeal 4.25% with JAK 1/2i reduced, but did not eliminate, increased vascularity in the parietal (c) and visceral (f) peritoneum. Kidney International 2014 86, 1187-1196DOI: (10.1038/ki.2014.209) Copyright © 2014 International Society of Nephrology Terms and Conditions