Janus kinase signaling activation mediates peritoneal inflammation and injury in vitro and in vivo in response to dialysate

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& 2014 International Society of Nephrology

Janus kinase signaling activation mediates peritoneal inflammation and injury in vitro and in vivo in response to dialysate Tiane Dai1,3, Ying Wang1,3, Aditi Nayak1, Cynthia C. Nast2, Lan Quang1, Janine LaPage1, Ali Andalibi1 and Sharon G. Adler1 1

Division of Nephrology and Hypertension, Department of Medicine, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, California, USA and 2Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California, USA

Peritoneal membrane pathology limits long-term peritoneal dialysis (PD). Here, we tested whether JAK/STAT signaling is implicated and if its attenuation might be salutary. In cultured mesothelial cells, PD fluid activated, and the pan-JAK inhibitor P6 reduced, phospho-STAT1 and phosphoSTAT3, periostin secretion, and cleaved caspase-3. Ex vivo, JAK was phosphorylated in PD effluent cells from long-term but not new PD patients. MCP-1 and periostin were increased in PD effluent in long term compared with new patients. In rats, twice daily, PD fluid infusion induced phospho-JAK, mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity after 10 days of exposure to PD fluid. Concomitant instillation of a JAK1/2 inhibitor virtually completely attenuated these changes. Thus, our studies directly implicate JAK/STAT signaling in the mediation of peritoneal membrane pathology as a consequence of PD. Kidney International advance online publication, 9 July 2014; doi:10.1038/ki.2014.209 KEYWORDS: JAK/STAT; JAK inhibitor; MCP-1; periostin; peritoneal dialysis

Correspondence: Sharon G. Adler, Division of Nephrology and Hypertension, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 West Carson Street, Torrance, California 90502, USA. E-mail: [email protected] 3

These authors contributed equally to this work.

Received 7 April 2014; revised 17 April 2014; accepted 24 April 2014 Kidney International

Peritoneal dialysis (PD) is an alternative to hemodialysis as a treatment for end-stage renal disease. Despite clinical advantages,1–14 high rates of technique failure diminish PD utilization.15–17 Failure is mainly attributed to ultrafiltration loss, inadequate solute clearance, and recurrent peritonitis,18,19 with ultrafiltration failure and poor solute clearance emerging as major factors in the long term. The peritoneal membrane (P-membrane) of PD patients undergoes several structural and functional changes.20 Many begin before dialysate exposure. Renal failure per se is toxic to the peritoneum. However, in PD, the peritoneum is also injured by low PD fluid pH;21–24 glucose degradation product (GDP) and advanced glycosylation end products (AGE) in PD fluid;25–28 the PD catheter;29,30 and from peritonitis.31 In susceptible individuals, this may lead to membrane failure. The mesothelial cell monolayer undergoes recurrent denudation and regeneration. Some mesothelial cells adopt mesenchymal characteristics.23,32–34 The submesothelial interstitium expands from extracellular matrix accumulation22,35,36 and the influx of immune cells.32 Lymphangiogenesis and neovascularization occur,22 and milky spots (macrophage accumulation) around vessels increase in number.22,35–39 These structural changes are mediated by chemokines, cytokines, growth factors, adipokines, and adhesion molecules elaborated by activated peritoneal mesothelial cells, adipocytes, fibroblasts, endothelial cells, and infiltrating mononuclear leukocytes. Activated adhesion molecules on the surface of P-membrane cells32,40 bind infiltrating leukocytes. Signaling events mediating these peritoneal inflammatory changes are still being defined. Activated p38 mitogenactivated protein kinase in mesothelial cells in vitro41 and reactive oxygen species acting through protein kinase C signaling have been implicated.42 GDPs bind to the receptor for advanced glycation end products (RAGE) on mesothelial cells and activate downstream NFkB and mitogen-activated protein kinase signaling.24,43 In rats exposed to PD fluid, anti-RAGE antibody attenuated transforming growth factor-b (TGF-b) elaboration and peritoneal fibrosis but not hypervascularity.25 RAGE-deficient mice exposed to GDP-containing PD fluid experienced less peritoneal 1

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RESULTS In vitro, in cultured mesothelial cells, PDF induced, and the pan-JAKi P6 attenuated, STAT 1 and STAT 3 phosphorylation

Immortalized human mesothelial cells (Met-5A) were cultured in standard medium, and then switched to control medium, filter-sterilized PD fluid, or heat-sterilized PD fluid (30%:70%, medium:PDF) with or without GDPs and/or the pan-JAKi P6 for 1 h. Heat-sterilized and filter-sterilized PD fluids induced both STAT 1 and STAT 3 phosphorylation, which was prevented by P6 (Figure 1a and b; Po0.01). Heatsterilized and filter-sterilized PD fluid increased phosphoJAK1 by 11.1 and 9.4%, respectively. These were decreased by 4.8 and 1.2% with the pan-JAKi P6. Heat-sterilized and filtersterilized PD fluid induced phospho-JAK2 by 5.9 and 6.6%, respectively, which were decreased by 4.8 and 3.9% with the pan-JAKi P6 (NS, not shown). Adding GDP to filtersterilized PD fluid did not additionally activate JAK1/2 or STAT1/3 (Figure 1a and b). These data show that all PD fluids activated JAK/STAT signaling. The pan-JAKi P6 attenuated activation. The pan-JAKi P6 reduced the PD fluid–induced increment in periostin secretion by cultured mesothelial cells

Met-5A cells were cultured in standard medium, and then switched to control medium or heat-sterilized PD fluid (30%:70%, medium:PD fluid) with or without different concentrations of P6 for 24 h. Periostin was induced by 2

a

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GDP JAKi (250 nM)

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Heated PDF – – – – – – + +

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+ + p-STAT1 STAT1

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inflammation, neoangiogenesis, and fibrosis, associated with downregulation of NFkB binding activity in visceral peritoneal tissue compared with controls.26 Anti-RAGE antibody attenuated the propensity for the supernatant of human peritoneal mesothelial cells exposed to glucose and the glycated protein N-epsilon-(carboxymethyl) lysine-human serum albumin in vitro to secrete substances (including vascular endothelial growth factor) that facilitated endothelial tube formation in cultured endothelial cells.27 The Janus kinase/signal transducer and activator of transcription (JAK/STAT) proteins mediate the actions of many cytokines, chemokines, hormones, and growth factors critical to cell proliferation, differentiation, migration, and apoptosis.44 JAK/STAT signaling may mediate P-membrane injury in PD,20,45 but direct evidence is limited. Leptin has been shown to activate JAK 2 and STAT 3 in cultured human peritoneal mesothelial cells, and the JAK2 inhibitor AG-490 abolished leptin-induced TGFb synthesis.46 Additional evidence supporting a role for JAK/STAT signaling in peritoneal injury is inferred from other clinical conditions and tissues.47–49 Downstream, STAT-regulated proteins such as periostin and MCP-1 may be transcribed, making them potential markers of JAK/STAT activation in vivo. This study was undertaken to test whether inflammation generated by bioincompatible PDF activates JAK/STAT signaling, and to determine whether JAK inhibitors (JAKi) enhance mesothelial cell survival, limit hypervascularity, and preserve P-membrane structure.

T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

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§

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 P61 h. 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 ¼ 4). (b) Representative western blots of phospho-STAT3 (p-STAT3) and quantitative evaluation of relative fold changes of phosphoSTAT3/total STAT3 by densitometry (n ¼ 4 for each condition).

heat-sterilized PD fluid, which was attenuated by P6 dose dependently (Figure 2a). The optimal P6 dose (250 nM) was then applied to Met-5A cells with filter-sterilized PD fluid with or without GDP added. Filter-sterilized PD fluid alone did not increase periostin, but the addition of GDP induced periostin, and P6 inhibited this increment (Figure 2b). The discrepancy between the effect of GDPs in filter-sterilized PD fluid on phospho-STAT1 and 3 and periostin implicate additional signaling pathways besides JAK/STAT in regulating periostin transcription and secretion. Periostin mRNA induced by heated PD fluid was also attenuated in a dose-dependent manner by P6 in vitro in the immortalized human peritoneal cell line LP9/TERT.bsd (Figure 2c). Kidney International

T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

labeled cleaved caspase-3 at indicated conditions, demonstrating activated caspase-3 consistent with the immunoblots. Together, the data show less apoptosis in PD fluid–exposed P6-treated mesothelial cells.

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Relarive fold change (periostin/GAPDH)

PD effluent cell lysate demonstrates JAK phosphorylation in long-term patients but not in new patients

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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) þ 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 panJAKi P6 for 24 h. 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 ¼ 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 P624 h. 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 ¼ 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 ¼ 3).

PD fluid–activated, and P6-inhibited, caspase-3 cleavage

Met-5A cells were cultured in standard medium, and then switched to control medium, heat-sterilized PD fluid, or filter-sterilized PD fluid (30%:70%, medium:PD fluid) with or without GDPs and/or P6 for 24 h. Cell lysates were immunoblotted for cleaved caspase-3. Heat-sterilized PD fluid (Figure 3a) and filter-sterilized PD fluid activated caspase-3; P6 inhibited this. GDPs added to filter-sterilized PD fluid marginally increased caspase-3 activation (NS). Figure 3b shows representative mesothelial cell fluorescenceKidney International

STAT-regulated proteins MCP-1 and periostin were higher in the PD effluent of long-term patients compared with new patients

# P <0.05 vs. control *P <0.05 vs. HPDF+JAKi 0 nM

#

1

Table 1 shows patients’ clinical summaries. In cell lysates centrifuged from long-term (n ¼ 4) and new (n ¼ 4) patients, phospho-JAK1 or phospho-JAK2 were each measureable in two long-term patients. No JAK phosphorylation was observed in new patients (Figure 4a).

There was an increase in MCP-1 (Po0.001; Figure 4b) and periostin in PD effluent in long-term (n ¼ 7) versus new (n ¼ 8) patients (Po0.05; Figure 4c). These studies show that mean MCP-1 and periostin, both with STAT consensus sequences in their promoter regions, were increased in the PD effluent of Long-term patients.

Heated PD fluid

Control Relarive fold change (periostin/18s)

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In vivo, infusion of 4.25% dextrose PD fluid, but not saline, activated JAK, mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity in nonuremic rats; the JAK1/2i Ruxolitinib (Incyte, Wilmington, DE) attenuated these changes

Nonuremic rats were dialyzed with normal saline, PD fluid with Dianeal 4.25%, or PD fluid with Dianeal 4.25% þ JAK1/2i BID for 10 days, killed, and tissues were examined after staining for phospho-JAK1, Masson’s trichrome, or hematoxylin and eosin (Figure 5), or the endothelial cell marker CD34 (Figure 6). In the parietal peritoneum, rats receiving saline showed no peritoneal JAK activation (Figure 5a). In rats receiving Dianeal 4.25%, there was phospho-JAK1 staining in the mesothelial and submesothelial compact zone cells (Figure 5b). Instillation of JAK1/2i with Dianeal 4.25% attenuated JAK activation (Figure 5c). In the visceral peritoneum, there was no JAK activation in rats receiving normal saline (Figure 5d), whereas exposure to Dianeal 4.25% induced marked phospho-JAK1 staining in hyperplastic mesothelial cells (Figure 5e). JAK1/2i with Dianeal 4.25% nearly completely abrogated JAK activation (Figure 5f). In the parietal peritoneum, saline dialysate caused minor thickening of the submesothelial compact zone from edema, fibrosis, and mild hypercellularity (Figure 5g). Rats receiving Dianeal 4.25% had prominently thickened parietal peritoneal submesothelial compact zones with infiltrating fibroblasts and mononuclear leukocytes (Figure 5h). In the Dianeal 4.25% þ Jak1/2i group, there was a marked reduction in submesothelial fibrosis and cellularity (Figure 5i). In rats dialyzed with saline, the visceral peritoneal mesothelial cell layer was undisturbed (Figure 5j), but dialysis with Dianeal 4.25% induced marked mesothelial reactive hyperplasia (Figure 5k). JAK1/2i infused with Dianeal 4.25% nearly 3

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T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

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Figure 3 | PDF induced caspase-3 activation; induction was attenuated by the pan-JAKi p6. After 24 h 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 ¼ 4). (b) Representative fluorescence-labeled cleaved caspase-3 at indicated conditions.

completely abrogated the hyperplasia (Figure 5l). Parietal membrane submesothelial compact zone hyperplasia and fibrosis was quantified on digitized Masson’s trichrome– stained tissue sections (Figure 5g–i). Rats infused with Dianeal 4.25% had significant submesothelial compact zone thickening (Po0.01) compared with rats infused with saline. This was attenuated with JAK1/2i (Po0.01) (Figure 5m). The baseline parietal (Figure 6a–c) and visceral (Figure 6d–f) peritoneal vascularity observed in rats receiving saline (Figure 6a and d) was augmented in rats receiving Dianeal 4.25% (Figure 6b and d), and nearly completely attenuated in rats receiving Dianeal 4.25% with JAK1/2i (Figure 6c and f). Vascularity was morphometrically measured and expressed as the number of capillary structures with CD34 staining per mm length of parietal peritoneum. In rats receiving the JAK1/ 2i with Dianeal 4.25%, there was significantly less hypervascularity observed than in rats receiving Dianeal 4.25% alone (Po0.05). The rats receiving the Dianeal 4.25% with JAK1/2i 4

did have more vascularity than rats dialyzed with saline (Po0.05). Taken together, the JAK1/2i reduced virtually all of the features that characteristically lead to membrane failure, including mesothelial morphological injury, submesothelial compact zone expansion, fibrosis, and hypervascularity. DISCUSSION

Together, these studies show that in mesothelial cells in vitro, after long-term PD fluid exposure in patients ex vivo, and in a nonuremic rat PD model, the inflammation generated by bioincompatible PD fluid induces chemokine elaboration and activates JAK/STAT signaling and the appearance of potentially injurious proteins under STAT regulation. This is the first study to demonstrate a beneficial effect of JAK1/2i on the mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity resulting from PD fluid exposure. PD fluid alters the peritoneal expression of secreted chemokines, cytokines, adipokines, and growth factors. Kidney International

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T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

Table 1 | Clinical characteristics of long-term (LT) and new (N) patients

F F F F F M M M F F F M M M M

ESRD etiology

PD vintage

Nail patella/FSGS DN DN DN DN þ HTN Unclear etiology DN DN HTN DN DN HTN DN HTN DN

39 Months 7 Months 10 Months 56 Months 80 Months 16.5 Months 13 Months 2 Weeks 1st Exchange 1 Week 1st Exchange 2 Days 3 Days 1st Exchange 6 Days

130 kDa

Dextrose CAPD/CCPD concentration (%) CCPD CAPD CAPD CCPD CAPD CAPD CAPD CAPD CAPD CAPD CAPD CAPD CAPD CAPD CAPD

1.5 2.5 1.5 1.5 1.5 1.5 2.5 2.5 2.5 1.5 4.25 2.5 2.5 2.5 1.5

New patients Phospho JAK 2

Long-term patients

130 kDa New patients 600,000

Long-term patients

*

500,000 MCP-1 (pg/ml)

Gender

Phospho JAK 1

400,000 300,000 200,000

*P <0.001

100,000

Abbreviations: CAPD, continuous ambulatory peritoneal dialysis; CCPD, continuous cycling peritoneal dialysis; DN, diabetic nephropathy; ESRD, end-stage renal disease; F, female; FSGS, focal and segmental glomerulosclerosis; HTN, hypertension; M, male; PD, peritoneal dialysis.

0 New

LT

18,000 #

16,000

The milieu of cellular and soluble mediators contribute both anti-inflammatory and proinflammatory effects. The GDPs implicated in the proinflammatory state50 also inhibit the respiratory burst in peritoneal leukocytes51,52 and inhibit mesothelial cell IL6 and prostaglandin synthesis.53 On the other hand, the persistence of mononuclear leukocytes in peritoneal fluid and tissue, coupled with P-membrane matrix expansion and neoangiogenesis, indicate the presence of a chronic inflammatory state, believed to lead to ultrafiltration failure and reduced solute clearance.54–56 Key among the proinflammatory chemokines present in increased amounts in the reactive peritoneum include MCP-1, tumor necrosis factora, RANTES,57 interferong, interferong-inducible protein 10, angiotensin II, TGF-b, vascular endothelial growth factor, leptin,46 and the interleukins 1, 6, 8,45 and 15.58 In accordance with these reports, we found increments in the JAK/STAT-inducible proteins MCP-1 and periostin. We and others showed that periostin induces a mesenchymal phenotype in epithelial cells59–61 and it accumulates in encapsulating peritoneal sclerosis.62 MCP-1 has long been implicated in P-membrane inflammation.63,64 Glucose, AGEs, and GDPs are implicated in inciting this process. In mesangial cells, the JAK/STAT pathway mediates angiotensin II–induced cell proliferation from high-glucose exposure.48 Mesangial cell incubation with JAK/STATi prevented TGF-b and fibronectin induction by high-glucose medium.41 In rat kidney fibroblasts, high glucose–induced collagen synthesis was inhibitable by captopril, by the JAK2 inhibitor AG-490, and by STAT1 and STAT3 deoxyoligonucleotide decoys.49 JAK/STAT activation occurs in patients with diabetic nephropathy,65 and urine MCP-1 is a biomarker for diabetic nephropathy.66 Together, these findings suggest that JAK/STAT signaling may mediate the inflammation and fibrosis that contributes to P-membrane injury in PD. Kidney International

Periostin (pg/ml)

14,000 12,000 10,000 8000 6000 4000 2000

#

P <0.05

0 New

LT

Figure 4 | JAK phosphorylation was present in long-term (LT) (n ¼ 7) but not in new (n ¼ 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 ¼ 4) but not in new (n ¼ 4) patients (a). MCP-1 (b, Po0.001) and periostin (c, Po0.05) were higher in the PD effluent of long-term (n ¼ 7) versus new patients (n ¼ 8).

Pathogenetic commonality between diabetic nephropathy and P-membrane damage may reflect, at least in part, glucose/AGE/GDP-induced activation and reactive oxygen species–generated JAK/STAT-mediated inflammation. Proteins such as periostin and MCP-1, with STAT-binding sites in their promoter regions, may report, at least in part, an integrated reflection of glucose, AGE, and GDP-stimulated JAK/STAT activation. This work has limitations. We used immortalized human pleural mesothelial cells (Met-5A) in most of our in vitro studies. Met-5A cells are derived from human pleural mesothelial cells, and they have been used by many investigators to model P-membrane pathophysiology.67–77 Indeed, a recent review states that human pleural Met-5A cells ‘are increasingly 5

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a

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T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

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Figure 5 | Histological changes in rats receiving peritoneal dialysis with or without concomitant JAK1/2i. Nonuremic rats were dialyzed with either normal saline (n ¼ 7), Dianeal 4.25% (n ¼ 7), or Dianeal 4.25% þ JAK1/2 i (n ¼ 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% þ 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).

used in peritoneal mesothelial cell research, and the data obtained with Met-5A cells have shown much concordance with data obtained with primary peritoneal mesothelial cells’.77 We corroborated the results for periostin mRNA in human peritoneal mesothelial cells immortalized with human telomerase reverse transcriptase (LP9/TERT.bsd cells).78 This immortalized human peritoneal mesothelial cell line has been shown to phenotypically and functionally resemble normal human peritoneal mesothelial cells. There were few patients studied. However, our results are in agreement with other reports demonstrating increments in PD effluent MCP-164 in PD patients. In rats, we studied histopathlological change, not P-membrane function. Longer-term studies are required to determine whether the changes observed are the antecedents of functional changes. 6

However, associations between PD effluent inflammation and membrane function have previously been reported.56,79,80 Finally, we tested the effects of PD fluid with 4.25% Dianeal, the most bioincompatible of all PD fluids, as proofof-principle. We did not test more ‘biocompatible’ PD fluids. Whether these ‘biocompatible’ solutions better preserve P-membrane structure or solute clearance remains controversial.81–85 Long-term experiments using models employed herein are ideal to further test these questions. To summarize, we provide evidence that bioincompatible PD fluid induces JAK/STAT signaling and promotes the elaboration of injurious STAT-regulated proteins with the capacity to induce structural injury. A recent report demonstrates JAK/STAT signaling in an animal model of peritonitis, and in PD patients who experienced at least one Kidney International

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T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

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c 4.25% Dianeal with JAK 1/2 inhibitor

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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.

episode of peritonitis,86 lending further credence to our observations. MCP-1 has been reported previously as a biomarker of injury in PD. This study adds periostin as a novel indicator of JAK/STAT activation and P-membrane injury. Thus, this study groups together two biomarkers acting as reporters for JAK/STAT-mediated peritoneal injury. However, the major novel contribution of this work is that, to our knowledge, this is the first work to demonstrate in vivo that a JAK/STAT inhibitor substantially attenuates P-membrane inflammation, mesothelial cell injury, fibrosis, and hypervascularity induced by bioincompatible PD fluid. These findings provide a foundation for studies to determine whether JAKi can preserve both P-membrane structure and function in the long term.

MATERIALS AND METHODS Cell culture Immortalized human pleural (Met-5A, ATCC CRL 9444) mesothelial cells were cultured in Medium199 supplemented with 3.3 nM epidermal growth factor, 400 nM hydrocortisone, 870 nM insulin, 1% antibiotics, and 10% fetal bovine serum, in a humidified atmosphere with 95% air and 5% CO2, at 37 1C. Human peritoneal mesothelial LP9/TERT.bsd cells,75 an hTERT immortalized cell line phenotypically and functionally resembling normal human peritoneal mesothelial cells, were obtained from Dr James Rheinwald (Brigham and Women’s Hospital, Boston, MA). Cells were grown in M199/MCDB106 medium supplemented with 15% newborn Kidney International

calf serum, 10 ng/ml epidermal growth factor, and 0.4 mg/ml hydrocortisone. Test solutions Mesothelial cells were exposed to heat-sterilized PD fluid, Filtersterilized PD fluid, or GDPs added to filter-sterilized PD fluid. Test solutions were prepared to simulate commercially available PD fluid (g/l): NaCl 5.786, CaCl22H2O 0.257, MgCl26H2O 0.102, sodium DL-lactate 3.925, and anhydrous D-glucose 15.0. GDP solutions were prepared by dissolving acetaldehyde 1 mM, formaldehyde 1.7 mM, 2-furaldehyde 0.2 mM, glyoxal 5.8 mM, methylglyoxal 2.8 mM, and 5-hydroxymethyl 2-furaldehyde 8.3 mM in filtered PD fluid. Filtersterilized solutions were passaged through a 0.2-mm-pore filter. Heat-sterilized solutions were autoclaved (121 1C, 0.2 MPa, 20 min). Cells were starved in rest medium with 0.3% bovine calf serum overnight before treatment, and then treated with medium in a ratio of 70% test solution and 30% rest medium. In controls, the medium was mixed with HBSS to normalize for dilution of the culture medium component. The pan-JAKi pyridone (P6) was purchased from Millipore (Billerica, MA). To determine the optimal P6 concentration for JAK inhibition, a dose-titration study was performed (0, 50, 100, 250 nM). Afterward, cells were incubated with heatsterilized PD fluid or filter-sterilized PD fluid þ GDPs with or without 250 nM P6. Cells were harvested at 1 h and 24 h. Patients Sample processing. PD effluents were obtained from eight new (PD vintage p2 weeks) and seven long-term (PD vintage X6 months) patients after dwell times ranging from 1 to 7 h, and the 7

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samples were processed immediately upon collection. PD effluent was centrifuged at 2,000 g for 20 min at 4 1C. Supernatant was aliquotted with protease inhibitors (167 ml of 1 M sodium azide, 250 ml of 100 mM PMSF, 100 ml of 10 mM leupeptin per 50 ml PDF) and stored at  80 1C in 10-ml aliquots. Cell pellets were suspended in RIPA buffer with protease inhibitors and stored at  80 1C. All patients provided informed consent. The study was approved by the John F. Wolf, M.D. Human Subjects Committee-1 at the LA Biomedical Research Institute at Harbor-UCLA Medical Center. Measurements of JAK/STAT activators and transcriptional target proteins by electrochemiluminescence. MCP-1 was measured by electrochemiluminescence (Meso Scale Discovery, Rockville, MD) on PD effluent after the first thaw. Data were expressed as analyte concentration. Western blotting Whole-cell extracts were prepared as per ref. 87. Protein concentration of cell extracts was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Standard immunoblotting was performed.87 Antibodies used were as follows: JAK1, JAK2, STAT1, STAT3, phosphorylated STAT1, STAT3, JAK2, cleaved caspase-3 (Cell Signaling, Danvers, MA), phosphorylated JAK1 (GenScript, Piscataway, NJ), and periostin (Biovendor, Oxford, UK). p-STAT1, p-STAT3, p-JAK1, and p-JAK2 were measured on cell lysates at 1 h. Periostin and cleaved caspase-3 were measured by western blotting at 24 h. RNA extraction and real-time PCR LP9/TERT.bsd cells were harvested after 24 h of treatment. Total RNA was extracted by Trizol (Life technology, Carlsbad, CA); real-time reverse transcriptase-PCR for periostin was described previously.59 Immunofluorescence microscopy After cells were incubated with test solutions with and without P6 for 24 h, cells were washed, fixed in 100% cold methanol, and labeled with cleaved caspase-3 antibody (1:400) overnight at 4 1C, followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (Life Technology) for 20 min. Nuclei were labeled with TO-PRO-3 iodide (1:2500, Life Technology) and slides were examined using a confocal laser scanning microscope. PD effluent periostin ELISA Six hundred microliters of PD effluent was applied to Microcon 10 ultrafiltration membrane units (Millipore). Concentrated PD effluent was washed with 10 mM Tris-HCl (pH 8.0), ultrafiltered twice to remove residual small molecules including salts, and purified samples were diluted in 10 mM Tris-HCl (pH 8.0) to adjust the final volume to 300 ml. PD effluent periostin levels were measured using human Periostin DuoSet ELISA Development Systems (R&D systems, Minneapolis, MN). Catheter implantation Male Sprague–Dawley rats weighing 275–300 g were anesthetized with isoflurane. An incision was made B0.75 to 1.5 cm below the xiphoid process, extended caudally B3 cm, followed by blunt abdominal wall dissection. The catheter tip was advanced into the cavity and the catheter cuff sutured to the superficial muscle layer. Then, a cervical neck incision was made, and the polyurethane 8

T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

heparin-coated catheter (Instech Solomon, Plymouth Meeting, MA) was tunneled over the left flank to the neck. The abdominal incision was then closed. A subcutaneous port (PMINA-CBAS-C30 Soloport) (Instech Solomon) was connected to the tunneled catheter, implanted in the neck fixed to subcutaneous tissue, and the incision was sutured. Rats were housed individually. The effects of PD fluid in nonuremic rats with and without the JAK1/2 inhibitor Ruxolitinib Control rats (PDC, n ¼ 7) were infused with normal saline (Baxter Healthcare Corporation, Deerfield, IL) intraperitoneally 10 ml BID for 10 days. The Dianeal treatment group (PDF 4.25%, n ¼ 7) received 10 ml of BID infusions of standard lactate-buffered 4.25% dextrose PDF, pH 5.5 (Dianeal, Baxter, Deerfield, IL). The JAKi group (PDFJAKi, n ¼ 7) received PDF 4.25% with Ruxolitinib at 5 mg/kg infused BID. Ruxolitinib powder (100 mg) (Selleckchem, Houston, TX) was dissolved in 2 ml of 100% ethanol for a stock solution. For use, it was further diluted into 10 ml of PD fluid for a final concentration of 5 mg/kg. Ceftazidime (500 mg/l; Sandoz GmbH for Hospira worldwide, Lake Forest, IL) was infused IP with the PD fluid. The port area was disinfected with ethanol before puncture. Dialysis was begun on postoperative day one. At the timing of killing on day 10, the mesenteric membrane from the distal loop of the small intestine (visceral) and the right or left side abdominal wall away from the linea alba (parietal) were separately harvested and fixed in 4% paraformaldehyde overnight at 4 1C. Animal experiments were approved by LA Biomed Research Institutional Animal Care and Use Committee. Morphometric analysis Paraffin-embedded parietal peritoneum blocks were sectioned at 4 mm and stained with Masson’s trichrome. Slides were digitized using a high-resolution whole slide scanner (Leica SCN400, Buffalo Grove, IL) at magnification 20 with image magnification available up to 40. Digitized slides were visualized and analyzed using the Slide Path 3.0 software. The submesothelial compact 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. Immunohistochemistry Four-mm sections of parietal and visceral peritoneum were cut from paraffin blocks. For phospo-JAK1, slides were deparaffinized, rehydrated in graded ethanol, processed with 10 mM citrate buffer (pH 6.0) in a microwave for 5 min at high power, and suffused with endogenous enzyme block solution (Dako, Carpinteria, CA) for 20 min. Slides were then incubated with rabbit anti-phospho-JAK1 (Santa Cruz, CA, 1:250) overnight at 4 1C. Dextran polymer conjugated with horseradish peroxidase and affinity-isolated immunoglobulin (Dako) were used as secondary antibodies for 30 min at room temperature. Immunoreactivity was detected by diaminobenzidine (Vector Laboratories, Burlingame, CA). Negative controls were performed using normal rabbit IgG antibody. Sections were counterstained with hematoxylin. For CD34, slides were deparaffinized, pretreated with UltraCC1 (Ventana, Tucson, AZ) at 100 1C for 64 min, and then run on automated immunohistochemistry instruments using anti-CD34 antibody (Epitomics, Burlingame, CA) at 1:100 at room temperature for 44 min by using the Optiview kit (Ventana). After automated staining, slides were counterstained with Mayers Hematoxylin and Kidney International

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T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

bluing reagent. Ten consecutive fields of the parietal peritoneum submesothelial layer and adjacent muscle were photographed at 40 magnification. The length of the parietal peritoneum was measured using Spot Advanced imaging software, and submesothelial vascularity was determined by counting all capillaries with open lumina identified by CD34 staining. Vascularity is expressed as the number of submesothelial capillaries/mm length of parietal peritoneum.

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Statistical analysis The data are presented as mean±s.e.m. The normality of the distribution of data was established using the Wilks–Shapiro test, and outcome measures between groups were compared using Student’s t-test. For multiple group comparisons, one-way analysis of variance was used. Individual group comparisons within analysis of variance were performed using the Newman–Keul’s post hoc test. For nonparametric electrochemiluminescence and ELISA data, the Mann–Whitney U-test (two-tailed) was used. Significance was assigned at Pp0.05. DISCLOSURE

All the authors declared no competing interests.

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20. 21.

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ACKNOWLEDGMENTS

We gratefully acknowledge DaVita Clinical Research and an anonymous donor for their support of this work. We gratefully acknowledge electrochemiluminescence measurements by Meso Scale Discovery, Rockville, MD.

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REFERENCES

26.

1. 2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

Tokgoz B. Clinical advantages of peritoneal dialysis. Perit Dial Int 2009; 29(Suppl 2): S59–S61. Fenton SS, Schaubel DE, Desmeules M et al. Hemodialysis versus peritoneal dialysis: a comparison of adjusted mortality rates. Am J Kidney Dis 1997; 30: 334–342. Heaf JG, Lkkegaard H, Madsen M. Initial survival advantage of peritoneal dialysis relative to haemodialysis. Nephrol Dial Transplant 2002; 17: 112–117. Termorshuizen F, Korevaar JC, Dekker FW et al. Hemodialysis and peritoneal dialysis: comparison of adjusted mortality rates according to the duration of dialysis: analysis of the Netherlands Cooperative Study on theAdequacy of Dialysis 2. J Am Soc Nephrol 2003; 14: 2851–2860. Barendse SM, Speight J, Bradley C. The Renal TreatmentSatisfaction Questionnaire (RTSQ): a measure of satisfaction with treatment for chronic kidney failure. Am J Kidney Dis 2005; 45: 572–579. Rubin HR, Fink NE, Plantinga LC et al. Patient ratings of dialysis care with peritoneal dialysis vs. hemodialysis. JAMA 2004; 291: 697–703. Juergensen E, Wuerth D, Finkelstein SH et al. Hemodialysis and peritoneal dialysis: patients’ assessment of their satisfaction with the therapy and the impact of the therapy on their lives. Clin J Am Soc Nephrol 2006; 1: 1191–1196. Churchill DN, Taylor DW, Keshaviah PR et al. Adequacy of dialysis andnutrition in continuous peritoneal dialysis: associationwith clinical outcomes. J Am Soc Nephrol 1996; 7: 198–207. Misra M, Vonesh E, Van Stone JC et al. Effect of cause and time of dropout on the residual GFR: a comparative analysis of the decline of GFR on dialysis. Kidney Int 2001; 59: 754–763. Lang SM, Bergner A, To¨pfer M et al. Preservation of residual renal function in dialysis patients: effects of dialysis-technique–related factors. Perit Dial Int 2001; 21: 52–57. Van Biesen W, Veys N, Vanholder R et al. The impact of the pre-transplant renal replacement modality on outcome after cadaveric kidney transplantation: the Ghent experience. Contrib Nephrol 2006; 150: 254–258. Goldfarb–Rumyantzev AS, Hurdle JF, Scandling JD et al. The role of pretransplantation renal replacement therapy modality in kidney allograf t and recipient survival. Am J Kidney Dis 2005; 46: 537–549. Pe´rez Fonta´n M, Rodrı´guez–Carmona A, Bouza P et al. Delayed graft function after renal transplantation in patients undergoing peritoneal dialysis and hemodialysis. Adv Perit Dial 1996; 12: 101–104.

Kidney International

25.

27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

37. 38.

39.

Snyder JJ, Foley RN, Gilbertson DT et al. Hemoglobin levels and erythropoietin doses in hemodialysis and peritoneal dialysis patients in the United States. J Am Soc Nephrol 2004; 15: 174–179. Gokal R, King J, Bogle S et al. Outcome in patients on continuous ambulatory peritoneal dialysis and hemodialysis: 4-year analysis of a prospective multicentre study. Lancet 1987; 330: 1105–1109. Serkes KD, Blagg CR, Nolph KD et al. Comparison of patient and technique survival in continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis: a multicenter study. Perit Dial Int 1990; 10: 15–19. Maiorca R, Cancarini GC, Zubani R et al. CAPD viability: a long-term comparison with hemodialysis. Perit Dial Int 1996; 16: 276–287. Gokal R. Peritoneal dialysis in the 21st century: an analysis of current problems and future developments. J Am Soc Nephrol 2002; 13(Suppl 1): S104–S115. Kawaguchi Y. Peritoneal dialysis as long-term treatment: comparison of technique survival between Asian and Western populations. Perit Dial Int 1999; 19(Suppl 2): S327–S328. Schilte MN, Celie JW, Wee PM et al. Factors contributing to peritoneal tissue remodeling in peritoneal dialysis. Perit Dial Int. 2009; 29: 605–617. Albrektsson A, Bazargani F, Wieslander A et al. Peritoneal dialysis fluidinduced angiogenesis in rat mesentery is increased by lactate in the presence or absence of glucose. ASAIO J 2006; 52: 276–281. Zareie M, Hekking LH, Welten AG et al. Contribution of lactate buffer, glucose and glucose degradation products to peritoneal injury in vivo. Nephrol Dial Transplant 2003; 18: 2629–2637. Hekking LHP, Zareie M, Driesprong BAJ et al. Better preservation of peritoneal morphologic features and defense in rats after long-term exposure to a bicarbonate/lactate-buffered solution. J Am Soc Nephrol 2001; 12: 2775–2786. Witowski J, Korybalska K, Wisniewska J et al. Effect of glucose degradation products on human peritoneal mesothelial cell function. J Am Soc Nephrol 2000; 11: 729–739. De Vriese AS, Flyvbjerg A, Mortier S et al. Inhibition of the interaction of AGE–RAGE prevents hyperglycemia-induced fibrosis of the peritoneal membrane. J Am Soc Nephrol 2003; 14: 2109–2118. Schwenger V, Morath C, Salava A et al. Damage to the peritoneal membrane by glucose degradation products is mediated by the receptor for advanced glycation end-products. J Am Soc Nephrol 2006; 17: 199–207. Boulanger E, Grossin N, Wautier MP et al. Mesothelial RAGE activation by AGEs enhances VEGF release and potentiates capillary tube formation. Kidney Int 2006; 71: 126–133. Boulanger E, Wautier M-P, Wautier J-L et al. AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression. Kidney Int 2002; 61: 148–156. Flessner MF, Credit K, Henderson K et al. Peritoneal changes after exposure to sterile solutions by catheter. J Am Soc Nephrol. 2007; 18: 2294–2302. Flessner MF, Credit K, Richardson K et al. Peritoneal inflammation after twenty-week exposure to dialysis solution: effect of solution versus catheter-foreign body reaction. Perit Dial Int 2010; 30: 284–293. Verger C, Luger A, Moore HL et al. Acute changes in peritoneal morphology and transport properties with infectious peritonitis and mechanical injury. Kidney Int 1983; 23: 823–831. Zareie M, De Vriese AS, Hekking LH et al. Immunopathological changes in a uraemic rat model for peritoneal dialysis. Nephrol Dial Transplant 2005; 20: 1350–1361. Yanez-Mo M, Lara-Pezzi E, Selgas R et al. Peritoneal dialysis and epithelialto-mesenchymal transition of mesothelial cells. N Engl J Med 2003; 348: 403–413. Schilte MN, Loureiro J, Keuning ED et al. Long-term intervention with heparins in a rat model for peritoneal dialysis. Perit Dial Int 2009; 29: 26–35. Fusshoeller A. Histomorphological and functional changes of the peritoneal membrane during long-term peritoneal dialysis. Pediat Nephrol 2008; 23: 19–25. Zareie M, Keuning ED, Wee PM et al. Improved biocompatibility of bicarbonate/lactate-buffered PDF is not related to pH. Nephrol Dial Transplant 2006; 21: 208–216. Di Paolo N, Sacchi G, Garosi G et al. Omental milky spots and peritoneal dialysis—review and personal experience. Perit Dial Int 2005; 25: 48–57. Williams JD, Craig KJ, Ruhland CV et al. The natural course of peritoneal membrane biology during peritoneal dialysis. Kidney Int 2003; 64(Suppl 88): S43–S49. Williams JD, Craig KJ, Topley N et al. Morphologic changes in the peritoneal membrane of patients with renal disease. J Am Soc Nephrol 2002; 13: 470–479.

9

basic research

40.

41.

42.

43. 44. 45. 46.

47. 48.

49.

50. 51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

10

Liang Y, Sasaki K. Expression of adhesion molecules relevant to leukocyte migration on the microvilli of liver peritoneal mesothelial cells. Anat Rec 2000; 258: 39–46. Xu ZG, Kim KS, Park HC et al. High glucose activates the p38 MAPK pathway in cultured human peritoneal mesothelial cells. Kidney Int 2003; 63: 958–968. Lee HB, Yu MR, Song JS et al. Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells. Kidney Int 2004; 65: 1170–1179. Schwenger V. GDP and AGE receptors: mechanisms of peritoneal damage. Contrib Nephrol 2006; 150: 77–83. Hebenstreit D, Horejs-Hoeck J, Duschl A. JAK/STAT-dependent gene regulation by cytokines. Drug News Perspect 2005; 18: 243–249. Lai KN, Tang SC, Leung JC. Mediators of inflammation and fibrosis. Perit Dial Int 2007; 27(Suppl 2): S65–S71. Leung JC, Chan LY, Tang SC et al. Leptin induces TGF-beta synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell. Kidney Int 2006; 69: 2078–2086. Rawlings JS, Rosler KM, Harrison DA. The JAK/STAT signaling pathway. J Cell Sci 2004; 117: 1281–1283. Amiri F, Shaw S, Wang X et al. Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose. Kidney Int 2002; 61: 1605–1616. Huang JS, Guh JY, Chen HC et al. Role of receptor for advanced glycation end-product (RAGE) and the JAK/STAT-signaling pathway in AGE-induced collagen production in NRK-49F cells. J Cell Biochem 2001; 81: 102–113. Himmele R, Sawin DA, Diaz-Buxo JA. GDPs and AGEs: impact on cardiovascular toxicity in dialysis patients. Adv Perit Dial 2011; 27: 22–26. Jonasson P, Braide M. Acute in vivo toxicity of heat-sterilized glucose peritoneal dialysis fluids to rat peritoneal macrophages. Perit Dial Int 1998; 18: 376–381. Jonasson P, Braide M. Kinetics and dose response of the effects of heated glucose peritoneal dialysis fluids on the respiratory burst of rat peritoneal leukocytes. ASAIO J 2000; 46: 469–473. Witowski J, Topley N, Jo¨rres A et al. Effect of lactate-buffered peritoneal dialysis fluids on human peritoneal mesothelial cell interleukin-6 and prostaglandin synthesis. Kidney Int 1995; 47: 282–293. Bos EN, van Bronswijk H, Helmerhorst TJM et al. Distinct populations of elicited human macrophages in peritoneal dialysis patients and women undergoing laparoscope. A study of peroxidatic activity. J Leukocyte Biol 1988; 43: 172–178. Baroni G, Schuinski A, de Moraes TP et al. Inflammation and the peritoneal membrane: causes and impact on structure and function during peritoneal dialysis. Mediators Inflamm 2012; 2012: 912595. Lambie M, Chess J, Donovan KL et al. Independent effects of systemic and peritoneal inflammation on peritoneal dialysis survival. J Am Soc Nephrol 2013; 24: 2071–2080. Robson RL, McLoughlin RM, Witowski J et al. Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo. J Immunol 2001; 167: 1028–1038. Hausmann MJ, Rogachev B, Weiler M et al. Accessory role of human peritoneal mesothelial cells in antigen presentation and T-cell growth. Kidney Int 2000; 57: 476–486. Satirapoj B, Wang Y, Chamberlin MP et al. Periostin: novel tissue and urinary biomarker of progressive renal injury induces a coordinated mesenchymal phenotype in tubular cells. Nephrol Dial Transplant 2012; 27: 2702–2711. Yan W, Shao R. Transduction if a mesenchyme- specific gene periostin into 293T cells induces cell invasive activity through epithelial to mesenchymal transformation. J Biol Chem 2006; 281: 19700–19708. Hamilton DW. Functional role of periostin in development and wound repair: implications for connective tissue disease. J Cell Commun Signal 2008; 2: 9–17. Braun N, Sen K, Alscher MD et al. Periostin: a matricellular protein involved in peritoneal injury during peritoneal dialysis. Perit Dial Int 2013; 33: 515–528. Tekstra J, Visser CE, Tuk CW et al. Identification of the major chemokines that regulate cell influxes in peritoneal dialysis patients. J Am Soc Nephrol 1996; 7: 2379–2384.

T Dai et al.: Activated JAK/STAT pathway in peritoneal dialysis

64.

65.

66.

67.

68.

69.

70. 71.

72.

73.

74.

75.

76.

77. 78.

79.

80.

81.

82. 83.

84. 85.

86. 87.

Visser CE, Tekstra J, Brouwer-Steenbergen JJ et al. Chemokines produced by mesothelial cells: huGRO-alpha, IP-10, MCP-1 and RANTES. Clin Exp Immunol 1998; 112: 270–275. Berthier CC, Zhang H, Schin M et al. Enhanced expression of Janus kinasesignal transducer and activator of transcription pathway members in human diabetic nephropathy. Diabetes 2009; 58: 469–477. Tesch GH. MCP-1/CCL2: a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol 2008; 294: F697–F701. Arbeiter K, Bidmon B, Endemann M et al. Peritoneal dialysate fluid composition determines heat shock protein expression patterns in human mesothelial cells. Kidney Int 2001; 60: 1930–1937. Chan TM, Leung JK, Tsang RC et al. Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells. Kidney Int 2003; 64: 519–533. Bidmon B, Endemann M, Arbeiter K et al. Overexpression of HSP-72 confers cytoprotection in experimental peritoneal dialysis. Kidney Int 2004; 66: 2300–2307. Kinashi H, Ito Y, Mizuno M et al. TGF-b1 promotes lymphangiogenesis during peritoneal fibrosis. J Am Soc Nephrol 2013; 24: 1627–1642. Kratochwill K, Boehm M, Herzog R et al. Alanyl-glutamine dipeptide restores the cytoprotective stress proteome of mesothelial cells exposed to peritoneal dialysis fluids. Nephrol Dial Transplant 2012; 27: 937–946. Washida N, Wakino S, Tonozuka Y et al. Rho-kinase inhibition ameliorates peritoneal fibrosis and angiogenesis in a rat model of peritoneal sclerosis. Nephrol Dial Transplant 2011; 26: 2770–2779. Ogata R, Hiramatsu N, Hayakawa K et al. Impairment of MCP-1 expression in mesothelial cells exposed to peritoneal dialysis fluid by osmotic stress and acidic stress. Perit Dial Int 2011; 31: 80–89. Strippoli R, Benedicto I, Foronda M et al. p38 maintains E-cadherin expression by modulating TAK1-NF-kappa B during epithelial-tomesenchymal transition. J Cell Sci 2010; 123: 4321–4331. Kratochwill K, Lechner M, Siehs C et al. Stress responses and conditioning effects in mesothelial cells exposed to peritoneal dialysis fluid. J Proteome Res 2009; 8: 1731–1747. Endemann M, Bergmeister H, Bidmon B et al. Evidence for HSP-mediated cytoskeletal stabilization in mesothelial cells during acute experimental peritoneal dialysis. Am J Physiol Renal Physiol 2007; 292: F47–F56. Yung S, Li FK, Chan TM. Peritoneal mesothelial cell culture and biology. Perit Dial Int 2006; 26: 162–173. Murphy JE, Rheinwald JG. Intraperitoneal injection of genetically modified, human mesothelial cells forsystemic gene therapy. Hum Gene Ther 1997; 8: 1867–1879. Pecoits-Filho R, Arau´jo MR, Lindholm B et al. Plasma and dialysate IL-6 and VEGF concentrations are associated with high peritoneal solute transport rate. Nephrol Dial Transplant 2002; 17: 1480–1486. Oh KH, Jung JY, Yoon MO et al. Intra-peritoneal interleukin-6 system is a potent determinant of the baseline peritoneal solute transport in incident peritoneal dialysis patients. Nephrol Dial Transplant 2010; 25: 1639–1646. Johnson DW, Brown FG, Clarke M et al. Effects of biocompatible versus standard fluid on peritoneal dialysis outcomes. J Am Soc Nephrol 2012; 23: 1097–1107. Gotloib L, Wajsbrot V, Shostak A. Mesothelial dysplastic changes and lipid peroxidation induced by 7.5% icodextrin. Nephron 2002; 92: 142–155. Moriishi M, Kawanishi H, Watanabe H et al. Effect of icodextrin-based peritoneal dialysis solution on peritoneal membrane. Adv Perit Dial 2005; 21: 21–24. Cho Y, Badve SV, Hawley CM et al. Biocompatible peritoneal dialysis fluids: clinical outcomes. Int J Nephrol 2012; 2012: 812609. Srivastava S, Hildebrand S, Fan SL. Long-term follow-up of patients randomized to biocompatible or conventional peritoneal dialysis solutions show no difference in peritonitis or technique survival. Kidney Int 2011; 80: 986–991. Fielding CA, Jones GW, McLoughlin RM et al. Interleukin-6 signaling drives fibrosis in unresolved inflammation. Immunity 2014; 40: 40–50. Dai T, Natarajan R, Nast CC et al. Glucose and diabetes: effects on podocyte and glomerular p38MAPK, heat shock protein 25, and actin cytoskeleton. Kidney Int 2006; 6: 806–814.

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