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One of the most common causes of peritoneal dialysis withdrawal is ultrafiltration failure which is characterized by peritoneal membrane thickening and fibrosis. Although previous studies have demonstrated the inhibitory effect of p38 MAPK inhibitors on peritoneal fibrosis in mice, it was unclear which specific cells contribute to peritoneal fibrosis. To investigate the role of p38 MAPK in peritoneal fibrosis more precisely, we examined the expression of p38 MAPK in human peritoneum and generated systemic inducible p38 MAPK knockout mice and macrophage-specific p38 MAPK knockout mice. Furthermore, the response to lipopolysaccharide (LPS) was assessed in p38 MAPK-knocked down RAW 264.7 cells to further explore the role of p38 MAPK in macrophages. We found that phosphorylated p38 MAPK levels were increased in the thickened peritoneum of both human and mice. Both chlorhexidine gluconate (CG)-treated systemic inducible and macrophage-specific p38 MAPK knockout mice ameliorated peritoneal thickening, mRNA expression related to inflammation and fibrosis, and the number of αSMA- and MAC-2-positive cells in the peritoneum compared to CG control mice. Reduction of p38 MAPK in RAW 264.7 cells suppressed inflammatory mRNA expression induced by LPS. These findings suggest that p38 MAPK in macrophages plays a critical role in peritoneal inflammation and thickening., (© 2024. The Author(s).)

Background: Aldosterone has been assumed to be one of aggravating factors in diabetic kidney disease (DKD). Natriuretic peptides/guanylyl cyclase-A/cGMP signalling has been shown to ameliorate aldosterone-induced renal injury in mice. Sacubitril/valsartan (SAC/VAL) is used clinically for chronic heart failure and hypertension, in part by augmenting natriuretic peptide bioavailability. The effects of SAC/VAL on renal pathophysiology including in DKD, however, have remained unclarified., Methods: Eight-week-old male db/db mice fed on a high-salt diet (HSD) were treated with vehicle or aldosterone (0.2 μg/kg/min), and divided into four groups: HSD control, ALDO (aldosterone), ALDO + VAL (valsartan), and ALDO + SAC/VAL group. After 4 weeks, they were analysed for plasma atrial natriuretic peptide (ANP) levels, renal histology, and haemodynamic parameters including glomerular filtration rate (GFR) by FITC-inulin and renal plasma flow (RPF) by para-amino hippuric acid., Results: The ALDO + SAC/VAL group showed significantly increased plasma ANP concentration and creatinine clearance, and decreased tubulointerstitial fibrosis and neutrophil gelatinase-associated lipocalin expression compared to ALDO and ALDO + VAL groups. SAC/VAL treatment increased GFR and RPF, and suppressed expression of Tgfb1, Il1b, Ccl2, and Lcn2 genes compared to the ALDO group. The percentage of tubulointerstitial fibrotic areas negatively correlated with the RPF and GFR., Conclusion: In a mouse model of type 2 diabetes with aldosterone excess, SAC/VAL increased RPF and GFR, and ameliorated tubulointerstitial fibrosis. Furthermore, RPF negatively correlated well with tubulointerstitial injury, suggesting that the beneficial effects of SAC/VAL could be through increased renal plasma flow with enhanced natriuretic peptide bioavailability., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

BK polyomavirus-associated nephropathy occurs in kidney transplant recipients under immunosuppressive treatment. BK polyomavirus is implicated in cancer development and invasion, and case reports of renal cell carcinoma and urothelial carcinoma possibly associated with BK polyomavirus has been reported. Further, it has been suggested that the immune responses of KT-related diseases could play a role in the pathogenesis and progression of renal cell carcinoma. Thus, we thought to examine the relationship between BK polyomavirus-associated nephropathy and renal cell carcinoma in terms of gene expression. To identify the common and specific immune responses involved in kidney transplantation-related diseases with a specific focus on BK polyomavirus-associated nephropathy, we performed consensus weighted gene co-expression network analysis on gene profile datasets of renal biopsy samples from different institutions. After the identification of gene modules and validation of the obtained network by immunohistochemistry of the marker across kidney transplantation-related diseases, the relationship between prognosis of renal cell carcinoma and modules was assessed. We included the data from 248 patients and identified the 14 gene clusters across the datasets. We revealed that one cluster related to the translation regulating process and DNA damage response was specifically upregulated in BK polyomavirus-associated nephropathy. There was a significant association between the expression value of hub genes of the identified cluster including those related to cGAS-STING pathway and DNA damage response, and the prognosis of renal cell carcinoma. The study suggested the potential link between kidney transplantation-related diseases, especially specific transcriptomic signature of BK polyomavirus associated nephropathy and renal cell carcinoma., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.P.M. is employed by the TMK Project, which is a collaboration project between Kyoto University and Mitsubishi Tanabe Pharma. M.Y. receives research grants from Mitsubishi Tanabe Pharma and Boehringer Ingelheim. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Sato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Osteocrin (OSTN), a bone-derived humoral factor, was reported to act on heart and bone by potentiating the natriuretic peptide (NP) system. Ostn gene polymorphisms have been associated with renal function decline, but its pathophysiological role in the kidney remains unclear., Methods: The role of endogenous OSTN was investigated using systemic Ostn-knockout (KO) mice. As a model for OSTN administration, liver-specific Ostn-overexpressing mice crossed with KO (KO-Tg) were generated. These mice were subjected to unilateral ischemia-reperfusion injury (IRI) and renal lesions after 21 days of insult were evaluated. A comprehensive analysis of the Wnt/β-catenin pathway was performed using a polymerase chain reaction (PCR) array. Reporter plasmid-transfected proximal tubular cells (NRK52E) were used to investigate the mechanism by which OSTN affects the pathway., Results: After injury, KO mice showed marginal worsening of renal fibrosis compared with wild-type mice, with comparable renal atrophy. KO-Tg mice showed significantly ameliorated renal atrophy, fibrosis and tubular injury, together with reduced expressions of fibrosis- and inflammation-related genes. The PCR array showed that the activation of the Wnt/β-catenin pathway was attenuated in KO-Tg mice. The downstream targets Mmp7, Myc and Axin2 showed similar results. MMP7 and Wnt2 were induced in corticomedullary proximal tubules after injury, but not in KO-Tg. In NRK52E, OSTN significantly potentiated the inhibitory effects of NP on transforming growth factor β1-induced activation of the Wnt/β-catenin pathway, which was reproduced by a cyclic guanosine monophosphate analog., Conclusions: Ectopic Ostn overexpression ameliorated subsequent renal injury following ischemia-reperfusion. OSTN could represent possible renoprotection in acute to chronic kidney disease transition, thus serving as a potential therapeutic strategy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the ERA.)

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury., (© 2021. The Author(s).)

Background: Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage., Methods: To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice., Results: Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa., Conclusions: These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Neutrophil gelatinase-associated lipocalin (NGAL, lipocalin 2 or LCN2) is an iron carrier protein whose circulating level is increased by kidney injury, bacterial infection and obesity, but its metabolic consequence remains elusive. To study physiological role of LCN2 in energy homeostasis, we challenged female Lcn2 knockout (KO) and wild-type (WT) mice with high fat diet (HFD) or cold exposure. Under normal diet, physical constitutions of Lcn2 KO and WT mice were indistinguishable. During HFD treatment, Lcn2 KO mice exhibited larger brown adipose tissues (BAT), consumed more oxygen, ate more food and gained less body weights as compared to WT mice. When exposed to 4 °C, KO mice showed higher body temperature and more intense 18 F-fluorodeoxyglucose uptake in BAT, which were cancelled by β3 adrenergic receptor blocker or iron-loaded (but not iron-free) LCN2 administration. These findings suggest that circulating LCN2 possesses obesity-promoting and anti-thermogenic effects through inhibition of BAT activity in an iron-dependent manner.

Although peptides are safe and useful as therapeutics, they are often easily degraded or metabolized. Dampening the clearance system for peptide ligands is a promising strategy for increasing the efficacy of peptide therapies. Natriuretic peptide receptor B (NPR-B) and its naturally occurring ligand, C-type natriuretic peptide (CNP), are potent stimulators of endochondral bone growth, and activating the CNP/NPR-B system is expected to be a powerful strategy for treating impaired skeletal growth. CNP is cleared by natriuretic peptide clearance receptor (NPR-C); therefore, we investigated the effect of reducing the rate of CNP clearance on skeletal growth by limiting the interaction between CNP and NPR-C. Specifically, we generated transgenic mice with increased circulating levels of osteocrin (OSTN) protein, a natural NPR-C ligand without natriuretic activity, and observed a dose-dependent skeletal overgrowth phenotype in these animals. Skeletal overgrowth in OSTN-transgenic mice was diminished in either CNP- or NPR-C-depleted backgrounds, confirming that CNP and NPR-C are indispensable for the bone growth-stimulating effect of OSTN. Interestingly, double-transgenic mice of CNP and OSTN had even higher levels of circulating CNP and additional increases in bone length, as compared with mice with elevated CNP alone. Together, these results support OSTN administration as an adjuvant agent for CNP therapy and provide a potential therapeutic approach for diseases with impaired skeletal growth.

Guanylyl cyclase-A (GC-A) signaling, a natriuretic peptide receptor, exerts renoprotective effects by stimulating natriuresis and reducing blood pressure. Previously we demonstrated massive albuminuria with hypertension in uninephrectomized, aldosterone-infused, and high salt-fed (ALDO) systemic GC-A KO mice with enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in podocytes. In the present study, we examined the interaction between p38 MAPK and GC-A signaling. The administration of FR167653, p38 MAPK inhibitor, reduced systolic blood pressure (SBP), urinary albumin excretion, segmental sclerosis, podocyte injury, and apoptosis. To further investigate the local action of natriuretic peptide and p38 MAPK in podocytes, we generated podocyte-specific (pod) GC-A conditional KO (cKO) mice. ALDO pod GC-A cKO mice demonstrated increased urinary albumin excretion with marked mesangial expansion, podocyte injury and apoptosis, but without blood pressure elevation. FR167653 also suppressed urinary albumin excretion without reducing SBP. Finally, we revealed that atrial natriuretic peptide increased phosphorylation of MAPK phosphatase-1 (MKP-1) concomitant with inhibited phosphorylation of p38 MAPK in response to MAPK kinase 3 activation, thereby resulting in decreased mRNA expression of the apoptosis-related gene, Bax, and Bax/Bcl2 ratio in cultured podocytes. These results indicate that natriuretic peptide exerts a renoprotective effect via inhibiting phosphorylation of p38 MAPK in podocytes.

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis., Competing Interests: The authors declare no competing financial interests.

The amount of albumin filtered through the glomeruli and reabsorbed at the proximal tubules in normal and in diabetic kidneys is debated. The megalin/cubilin complex mediates protein reabsorption, but genetic knockout of megalin is perinatally lethal. To overcome current technical problems, we generated a drug-inducible megalin-knockout mouse line, megalin(lox/lox);Ndrg1-CreER T2 (iMegKO), in which megalin expression can be shut off at any time by administration of tamoxifen (Tam). Tam administration in adult iMegKO mice decreased the expression of renal megalin protein by 92% compared with that in wild-type C57BL/6J mice and almost completely abrogated renal reabsorption of intravenously injected retinol-binding protein. Furthermore, urinary albumin excretion increased to 175 μg/d (0.46 mg albumin/mg creatinine) in Tam-treated iMegKO mice, suggesting that this was the amount of total nephron albumin filtration. By comparing Tam-treated, streptozotocin-induced diabetic iMegKO mice with Tam-treated nondiabetic iMegKO mice, we estimated that the development of diabetes led to a 1.9-fold increase in total nephron albumin filtration, a 1.8-fold increase in reabsorption, and a significant reduction in reabsorption efficiency (86% efficiency versus 96% efficiency in nondiabetic mice). Insulin treatment normalized these abnormalities. Akita;iMegKO mice, another model of type 1 diabetes, showed equivalent results. Finally, nondiabetic iMegKO mice had a glomerular sieving coefficient of albumin of 1.7×10 -5 , which approximately doubled in diabetic iMegKO mice. This study reveals actual values and changes of albumin filtration and reabsorption in early diabetic nephropathy in mice, bringing new insights to our understanding of renal albumin dynamics associated with the hyperfiltration status of diabetic nephropathy., (Copyright © 2016 by the American Society of Nephrology.)

We assessed the utility of two forms of osteopontin (OPN), OPN full and its cleaved form (OPN N-half), in plasma and urine as markers of disease activity in lupus nephritis (LN). Samples were collected from patients with systemic lupus erythematosus (SLE) (LN: N = 29, non-LN: N = 27), IgA nephropathy (IgAN) (N = 14), minimal change nephrotic syndrome (MCNS) (N = 5), diabetic nephropathy (DN) (N = 14) and healthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN full concentration between groups, urine OPN N-half concentration was significantly higher in patients with LN than HC (p < 0.05). Moreover, urine OPN N-half was higher in LN patients with overt proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimal proteinuria (P/C < 0.5, p < 0.0001), and also higher than in DN patients with overt proteinuria (P/C > 0.5, p < 0.01). Urine thrombin activity correlated with urine OPN N-half concentration (p < 0.0001), but not with urine OPN full concentration. These results suggest that urine OPN N-half concentration reflects renal inflammation. Thus, urine OPN N-half may be a novel disease activity marker for LN., Competing Interests: This work was supported by grants from the Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan. The Center was founded by a special coordination of the Ministry of Education, Culture, Sports, Science and Technology of Japan and Astellas Pharma Inc. Takuma Oku, Chiyomi Sasaki, and Yoshitaka Hirayama are employees of Astellas Pharma Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Aims/hypothesis: The accumulation of extracellular matrix (ECM) is a characteristic of diabetic nephropathy, and is partially caused by profibrotic proteins TGF-β and connective tissue growth factor (CTGF). We aimed to identify microRNAs (miRNAs) targeting CTGF on podocytes in diabetic nephropathy., Methods: We investigated miRNAs targeting CTGF on podocytes with miRNA array analysis and identified a candidate miRNA, miR-26a. Using overexpression and silencing of miR-26a in cultured podocytes, we examined changes of ECM and its host genes. We further investigated glomerular miR-26a expression in humans and in mouse models of diabetic nephropathy., Results: miR-26a, which was downregulated by TGF-β1, was expressed in glomerular cells including podocytes and in tubules by in situ hybridisation. Glomerular miR-26a expression was downregulated by 70% in streptozotocin-induced diabetic mice. Transfection of miR-26a mimics in cultured human podocytes decreased the CTGF protein level by 50%, and directly inhibited CTGF expression in podocytes, as demonstrated by a reporter assay with the 3'-untranslated region of the CTGF gene. This effect was abolished by a mutant plasmid. miR-26a mimics also inhibited TGF-β1-induced collagen expression, SMAD-binding activity and expression of its host genes CTDSP2 and CTDSPL. Knockdown of CTDSP2 and CTDSPL increased collagen expression in TGF-β-stimulated podocytes, suggesting that host genes also regulate TGF-β/SMAD signalling. Finally, we observed a positive correlation between microdissected glomerular miR-26a expression levels and estimated GFR in patients with diabetic nephropathy., Conclusions/interpretation: The downregulation of miR-26a is involved in the progression of diabetic nephropathy both in humans and in mice through enhanced TGF-β/CTGF signalling.

Background: Neutrophil gelatinase-associated lipocalin (NGAL or LCN2) is an iron-transporting factor which possesses various activities such as amelioration of kidney injury and host defense against pathogens. Its circulating concentrations are elevated in acute and chronic kidney diseases and show a positive correlation with poor renal outcome and mortality, but its clinical significance in maintenance hemodialysis (HD) patients remains elusive., Methods: Serum NGAL levels were determined by enzyme-linked immunosorbent assay in out-patient, Japanese HD subjects. Their correlation to laboratory findings and morbidity (as development of severe infection or serum albumin reduction) was investigated using linear regression analysis and χ2 test., Results: Pre-dialysis serum NGAL levels in HD patients were elevated by 13-fold compared to healthy subjects (n=8, P<0.001). In a cross-sectional study of 139 cases, serum NGAL concentrations were determined independently by % creatinine generation rate (an indicator of muscle mass, standardized coefficient β=0.40, P<0.001), peripheral blood neutrophil count (β=0.38, P<0.001) and anion gap (which likely reflects dietary protein intake, β=0.16, P<0.05). Iron administration to anemic HD patients caused marked elevation of peripheral blood hemoglobin, serum ferritin and iron-regulatory hormone hepcidin-25 levels, but NGAL levels were not affected. In a prospective study of 87 cases, increase in serum albumin levels a year later was positively associated to baseline NGAL levels by univariate analysis (r=0.36, P<0.01). Furthermore, within a year, patients with the lowest NGAL tertile showed significantly increased risk for marked decline in serum albumin levels (≥0.4 g/dl; odds ratio 5.5, 95% confidence interval 1.5-20.3, P<0.05) and tendency of increased occurrence of severe infection requiring admission (odds ratio 3.1, not significant) compared to the middle and highest tertiles., Conclusion: Low serum NGAL levels appear to be associated with current malnutrition and also its progressive worsening in maintenance HD patients.

Aims/hypothesis: Hyperlipidaemia is an independent risk factor for the progression of diabetic nephropathy, but its molecular mechanism remains elusive. We investigated in mice how diabetes and hyperlipidaemia cause renal lesions separately and in combination, and the involvement of Toll-like receptor 4 (TLR4) in the process., Methods: Diabetes was induced in wild-type (WT) and Tlr4 knockout (KO) mice by intraperitoneal injection of streptozotocin (STZ). At 2 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Functional and histological analyses were carried out 6 weeks later., Results: Compared with treatment with STZ or HFD alone, treatment of WT mice with both STZ and HFD markedly aggravated nephropathy, as indicated by an increase in albuminuria, mesangial expansion, infiltration of macrophages and upregulation of pro-inflammatory and extracellular-matrix-associated gene expression in glomeruli. In Tlr4 KO mice, the addition of an HFD to STZ had almost no effects on the variables measured. Production of protein S100 calcium binding protein A8 (calgranulin A; S100A8), a potent ligand for TLR4, was observed in abundance in macrophages infiltrating STZ-HFD WT glomeruli and in glomeruli of diabetic nephropathy patients. High-glucose and fatty acid treatment synergistically upregulated S100a8 gene expression in macrophages from WT mice, but not from KO mice. As putative downstream targets of TLR4, phosphorylation of interferon regulatory factor 3 (IRF3) was enhanced in kidneys of WT mice co-treated with STZ and HFD., Conclusions/interpretation: Activation of S100A8/TLR4 signalling was elucidated in an animal model of diabetic glomerular injury accompanied with hyperlipidaemia, which may provide novel therapeutic targets in progressive diabetic nephropathy.

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.