Matrix metalloproteinase 10 and the slow demise of the peritoneal membrane.

Matrix metalloproteinases (MMPs) cleave matrix components along with multiple other effects. They are integral to virtually all biological processes, including inflammation and wound healing. As such, MMPs have been studied in the context of peritoneal membrane injury. MMP10 is a stromelysin and is involved in the degradation of matrix proteoglycans. Ishimura et al. demonstrate that MMP10 is involved in peritoneal membrane fibrosis. The clinical implications of these observations are presently unknown.

Impact of Bioelectrical Impedance-Guided Fluid Management and Vitamin D Supplementation on Left Ventricular Mass in Patients Receiving Peritoneal Dialysis: A Randomized Controlled Trial.

RATIONALE & OBJECTIVE: Hypervolemia and vitamin D deficiency occur frequently in patients receiving peritoneal dialysis and may contribute to left ventricular (LV) hypertrophy. The effect of bioelectrical impedance analysis (BIA)-guided volume management or vitamin D supplementation on LV mass among those receiving peritoneal dialysis is uncertain. STUDY DESIGN: Two-by-two factorial randomized controlled trial. SETTING & PARTICIPANTS: Sixty-five patients receiving maintenance peritoneal dialysis. INTERVENTION: BIA-guided volume management versus usual care and oral cholecalciferol 50,000 U weekly for 8 weeks followed by 10,000 U weekly for 44 weeks or matching placebo. OUTCOME: Change in LV mass at 1 year measured by cardiac magnetic resonance imaging. RESULTS: Total body water decreased by 0.9 + 2.4 (SD) L in the BIA group compared with a 1.5 ± 3.4 L increase in the usual care group (adjusted between-group difference: -2.4 [95% CI, -4.1 to -0.68] L, P = 0.01). LV mass increased by 1.3 ± 14.3 g in the BIA group and decreased by 2.4 ± 37.7 g in the usual care group (between-group difference: +2.2 [95% CI, -13.9 to 18.3] g, P = 0.8). Serum 25-hydroxyvitamin D concentration increased by a mean of 17.2 ± 30.8 nmol/L in the cholecalciferol group and declined by 8.2 ± 24.3 nmol/L in the placebo group (between-group difference: 28.3 [95% CI, 17.2-39.4] nmol/L, P < 0.001). LV mass decreased by 3.0 ± 28.1 g in the cholecalciferol group and increased by 2.0 ± 31.2 g in the placebo group (between-group difference: -4.5 [95% CI, -20.4 to 11.5] g, P = 0.6). LIMITATIONS: Relatively small sample size with larger than expected variation in change in LV mass. CONCLUSIONS: BIA-guided volume management had a modest impact on volume status with no effect on the change in LV mass. Vitamin D supplementation increased serum vitamin D concentration but had no effect on LV mass. FUNDING: Unrestricted Baxter International extramural grant and the Kidney Foundation of Canada. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT01045980.

The role of WNT5A and Ror2 in peritoneal membrane injury.

Patients on peritoneal dialysis are at risk of developing peritoneal fibrosis and angiogenesis, which can lead to dysfunction of the peritoneal membrane. Recent evidence has identified cross-talk between transforming growth factor beta (TGFB) and the WNT/ß-catenin pathway to induce fibrosis and angiogenesis. Limited evidence exists describing the role of non-canonical WNT signalling in peritoneal membrane injury. Non-canonical WNT5A is suggested to have different effects depending on the receptor environment. WNT5A has been implicated in antagonizing canonical WNT/ß-catenin signalling in the presence of receptor tyrosine kinase-like orphan receptor (Ror2). We co-expressed TGFB and WNT5A using adenovirus and examined its role in the development of peritoneal fibrosis and angiogenesis. Treatment of mouse peritoneum with AdWNT5A decreased the submesothelial thickening and angiogenesis induced by AdTGFB. WNT5A appeared to block WNT/ß-catenin signalling by inhibiting phosphorylation of glycogen synthase kinase 3 beta (GSK3B) and reducing levels of total ß-catenin and target proteins. To examine the function of Ror2, we silenced Ror2 in a human mesothelial cell line. We treated cells with AdWNT5A and observed a significant increase in fibronectin compared with AdWNT5A alone. We also analysed fibronectin and vascular endothelial growth factor (VEGF) in a TGFB model of mesothelial cell injury. Both fibronectin and VEGF were significantly increased in response to Ror2 silencing when cells were exposed to TGFB. Our results suggest that WNT5A inhibits peritoneal injury and this is associated with a decrease in WNT/ß-catenin signalling. In human mesothelial cells, Ror2 is involved in regulating levels of fibronectin and VEGF.

Using bioimpedance analysis to assess intensive care unit patients with sepsis in the post-resuscitation period: a prospective multicentre observational study.

PURPOSE: Clinicians lack well-validated, non-invasive, objective tools to guide volume management in the post-resuscitative period. Bioimpedance analysis (BIA) represents a novel method for guiding fluid management. We studied the relationship of BIA vector length (VL), an indicator of volume status, to the need for mechanical ventilation in patients with sepsis. METHODS: This is a multicentre prospective observational study at four Canadian ICUs. We examined adult patients admitted to the ICU within 72 hr of a sepsis diagnosis. Patients underwent daily BIA measurements for 30 days, until discharge from the ICU, or until death. Our primary outcome was the ongoing need for invasive mechanical ventilation, and we examined the association with VL using a generalized estimating equation. Our secondary analyses were targeted to determine an association between VL and other measures of volume status and acute kidney injury (AKI). RESULTS: We enrolled 159 patients from four centres over 27 months. The mean (standard deviation [SD]) age was 64 (15) yr with a mean (SD) APACHE (acute physiology, age, chronic health evaluation) II score of 25 (10); 57% (n = 91) were male. A 50-unit (ohm·m) increase in VL over any time period was associated with a 30% decrease in the probability of requiring invasive mechanical ventilation (P < 0.03). Volume expansion, indicated by a shorter VL, correlated with higher edema scores (r = - 0.31; P < 0.001) and higher net 24-hr fluid balance (r = - 0.27, P < 0.001). Patients with AKI had a shorter overall VL (r = - 0.23; P = 0.003). CONCLUSIONS: An increase in VL over time is associated with a decrease in probability of requiring invasive mechanical ventilation. Vector length correlates with other commonly used volume assessment methods in post-resuscitation patients with sepsis.

SMAD3-dependent and -independent pathways in glomerular injury associated with experimental glomerulonephritis.

Glomerulonephritis (GN) is a common cause of end-stage kidney disease and is characterized by glomerular inflammation, hematuria, proteinuria, and progressive renal dysfunction. Transforming growth factor (TGF)-ß is involved in glomerulosclerosis and interstitial fibrosis. TGF-ß activates multiple signaling pathways, including the canonical SMAD pathway. We evaluated the role of SMAD signaling in renal injury and proteinuria in a murine model of GN. SMAD3+/+ or SMAD3-/- mice received anti-glomerular basement membrane antibodies to induce GN. We confirmed previous reports that demonstrated that SMAD3 is an important mediator of glomerulosclerosis and renal interstitial fibrosis. Proteinuria was highly SMAD3 dependent. We found differential effects of SMAD3 deletion on podocytes and glomerular endothelial cells. GN led to podocyte injury, including foot process effacement and loss of podocyte-specific markers. Interestingly, these changes were not SMAD3 dependent. Furthermore, there were significant changes to glomerular endothelial cells, including loss of fenestrations, swelling, and basement membrane reduplication, which were SMAD3 dependent. Despite ongoing markers of podocyte injury in SMAD3-/- mice, proteinuria was transient. Renal injury in the setting of GN involves TGF-ß and SMAD3 signaling. Cell populations within the glomerulus respond differently to SMAD3 deletion. Proteinuria correlated more with endothelial cell changes as opposed to podocyte injury in this model.

WNT signaling is required for peritoneal membrane angiogenesis.

The wingless-type mouse mammary tumor virus integration site family (WNT) signaling pathway is involved in wound healing and fibrosis. We evaluated the WNT signaling pathway in peritoneal membrane injury. We assessed WNT1 protein expression in the peritoneal effluents of 54 stable peritoneal dialysis (PD) patients and WNT-related gene expression in ex vivo mesothelial cell cultures from 21 PD patients. In a transforming growth factor-ß (TGF-ß)-mediated animal model of peritoneal fibrosis, we evaluated regulation of the WNT pathway and the effect of WNT inhibition on peritoneal fibrosis and angiogenesis. WNT1 and WNT2 gene expression were positively correlated with peritoneal membrane solute transport in PD patients. In the mouse peritoneum, TGF-ß-induced peritoneal fibrosis was associated with increased expression of WNT2 and WNT4. Peritoneal ß-catenin protein was significantly upregulated after infection with adenovirus expressing TGF-ß (AdTGF-ß) along with elements of the WNT signaling pathway. Treatment with a ß-catenin inhibitor (ICG-001) in mice with AdTGF-ß-induced peritoneal fibrosis resulted in attenuation of peritoneal angiogenesis and reduced vascular endothelial growth factor. Similar results were also observed with the WNT antagonist Dickkopf-related protein (DKK)-1. In addition to this, DKK-1 blocked epithelial-mesenchymal transition and increased levels of the cell adhesion protein E-cadherin. We provide evidence that WNT signaling is active in the setting of experimental peritoneal fibrosis and WNT1 correlates with patient peritoneal membrane solute transport in PD patients. Intervention in this pathway is a possible therapy for peritoneal membrane injury.

Matrix metalloproteinase 9 is associated with peritoneal membrane solute transport and induces angiogenesis through ß-catenin signaling.

Background: For patients using peritoneal dialysis (PD), the peritoneal membrane can develop fibrosis and angiogenesis, leading to ultrafiltration failure, chronic hypervolemia and increased risk of technique failure and mortality. Matrix metalloproteinases (MMPs), and specifically the gelatinases (MMP2 and MMP9), may be involved in peritoneal membrane injury. Methods: From stable PD patients, mesothelial cells were assayed for MMP gene expression. MMP9 was overexpressed in mouse peritoneum by adenovirus, and MMP9 -/- mice were subjected to transforming growth factor ß (TGF-ß)-induced peritoneal fibrosis. Results: MMP9 mRNA expression correlated with peritoneal membrane solute transport properties. Overexpression of MMP9 in the mouse peritoneum induced submesothelial thickening and angiogenesis. MMP9 induced mesothelial cell transition to a myofibroblast phenotype measured by increased alpha smooth muscle actin and decreased E-cadherin expression. Angiogenesis was markedly reduced in MMP9 -/- mice treated with an adenovirus expressing active TGF-ß compared with wild-type mice. TGF-ß-mediated E-cadherin cleavage was MMP9 dependent, and E-cadherin cleavage led to ß-catenin-mediated signaling. A ß-catenin inhibitor blocked the angiogenic response induced by AdMMP9. Conclusions: Our data suggest that MMP9 is involved in peritoneal membrane injury possibly through cleavage of E-cadherin and induction of ß-catenin signaling. MMP9 is a potential biomarker for peritoneal membrane injury and is a therapeutic target to protect the peritoneal membrane in PD patients.

Gremlin promotes peritoneal membrane injury in an experimental mouse model and is associated with increased solute transport in peritoneal dialysis patients.

The peritoneal membrane becomes damaged in patients on peritoneal dialysis (PD). Gremlin 1 (GREM1) inhibits bone morphogenic proteins (BMPs) and plays a role in kidney development and fibrosis. We evaluated the role of gremlin in peritoneal fibrosis and angiogenesis. In a cohort of 32 stable PD patients, GREM1 concentration in the peritoneal effluent correlated with measures of peritoneal membrane damage. AdGrem1, an adenovirus to overexpress gremlin in the mouse peritoneum, induced submesothelial thickening, fibrosis, and angiogenesis in C57BL/6 mice, which was associated with decreased expression of BMP4 and BMP7. There was evidence of mesothelial cell transition to a mesenchymal phenotype with increased α smooth muscle actin expression and suppression of E-cadherin. Some of the GREM1 effects may be reversed with recombinant BMP7 or a pan-specific transforming growth factor ß (TGF-ß) antibody. Neovascularization was not inhibited with a TGF-ß antibody, suggesting a TGF-ß-independent angiogenic mechanism. Swiss/Jackson Laboratory (SJL) mice, which are resistant to TGF-ß-induced peritoneal fibrosis, responded in a similar fashion to AdGrem1 as did C57BL/6 mice with fibrosis, angiogenesis, and mesothelial-to-mesenchymal transition. GREM1 was associated with up-regulated TGF-ß expression in both SJL and C57BL/6 mice, but SJL mice demonstrated a defective TGF-ß-induced GREM1 expression. In summary, GREM1 induces fibrosis and angiogenesis in mouse peritoneum and is associated with increased solute transport in these PD patients.

Valsartan combined with clopidogrel and/or leflunomide for the treatment of progressive immunoglobulin A nephropathy.

AIM: The current standard treatment for IgA nephropathy relies on steroid and/or immunosuppressive therapy and angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blocker (ARB). This study examines the benefits and safety of combining valsartan with clopidogrel and leflunomide as a treatment for progressive IgA nephropathy. METHODS: Patients with primary IgA nephropathy, confirmed by renal biopsy, were recruited for this study. Patients were separated into four groups (n = 42 each) after 2 months of run-in period of valsartan treatment. All patients were treated with valsartan alone (Group 1) or valsartan and either clopidogrel (Group 2) or leflunomide (Group 3) or both clopidogrel and leflunomide (Group 4). Each group was followed up for their next 24 months for 24 h urinary protein excretion, serum creatinine and estimated glomerular filtration rate (eGFR) to assess the effect of the treatment. Adverse effects were recorded concurrently to evaluate the safety of the treatment. RESULTS: Of all 168 patients, 107 were males and 61 were females, with an average age of 33.8 ± 8.79 years. Baseline characteristics were comparable among the four groups (P > 0.05) prior to the experimental treatment. There was a significant (P < 0.05) decrease in 24 h urinary protein excretion after 4 months of experimental treatment. At the end of the 24 months, groups 3 and 4 showed a respective 62.35% and 69.47% reduction in proteinuria. The serum creatinine was significantly higher (P < 0.05) in group 1 and 2 at the end of the follow-up, and their respective eGFR was significantly lower. The incidence of cardiovascular complication was 11.9% and 9.5% for group 1 and 3, respectively. CONCLUSIONS: The treatment with Valsartan combined with Clopidogrel and Leflunomide can reduce the urinary proteins loss and renal function deterioration for IgA nephropathy patients and cause minimal adverse reactions. Our study suggests a new clinical treatment option for IgA nephropathy.

Estimated GFR reporting influences recommendations for dialysis initiation.

Automated reporting of estimated GFR (eGFR) with serum creatinine measurement is now common. We surveyed nephrologists in four countries to determine whether eGFR reporting influences nephrologists' recommendations for dialysis initiation. Respondents were randomly allocated to receive a survey of four clinical vignettes that included either serum creatinine concentration only or serum creatinine and the corresponding eGFR. For each scenario, the respondent was asked to rank his or her likelihood of recommending dialysis initiation on a modified 8-point Likert scale, ranging from 1 ("definitely not") to 8 ("definitely would"). Analysis of the 822 eligible responses received showed that the predicted likelihood of recommending dialysis increased by 0.55 points when eGFR was reported (95% confidence interval, 0.33 to 0.76), and this effect was larger for eGFRs >5 ml/min per 1.73 m(2) (P<0.001). Subgroup analyses suggested that physicians who had been in practice ≥13 years were more affected by eGFR reporting (P=0.03). These results indicate that eGFR reporting modestly increases the likelihood that dialysis is recommended, and physicians should be aware of this effect when assessing patients with severe CKD.

Implementation of a One-Day Living Kidney Donor Assessment Clinic to Improve the Efficiency of the Living Kidney Donor Evaluation: Program Report.

Purpose of program: A key barrier to becoming a living kidney donor is an inefficient evaluation process, requiring more than 30 tests (eg, laboratory and diagnostic tests), questionnaires, and specialist consultations. Donor candidates make several trips to hospitals and clinics, and often spend months waiting for appointments and test results. The median evaluation time for a donor candidate in Ontario, Canada, is nearly 1 year. Longer wait times are associated with poorer outcomes for the kidney transplant recipient and higher health care costs. A shorter, more efficient donor evaluation process may help more patients with kidney failure receive a transplant, including a pre-emptive kidney transplant (ie, avoiding the need for dialysis). In this report, we describe the development of a quality improvement intervention to improve the efficiency, effectiveness, and patient-centeredness of the donor candidate evaluation process. We developed a One-Day Living Kidney Donor Assessment Clinic, a condensed clinic where interested donor candidates complete all testing and consultations within 1 day. Sources of information: The One-Day Living Kidney Donor Assessment Clinic was developed after performing a comprehensive review of the literature, receiving feedback from patients who have successfully donated, and meetings with transplant program leadership from St. Joseph's Healthcare Hamilton. A multistakeholder team was formed that included health care staff from nephrology, transplant surgery, radiology, cardiology, social work, nuclear medicine, and patients with the prior lived experience of kidney donation. In the planning stages, the team met regularly to determine the objectives of the clinic, criteria for participation, clinic schedule, patient flow, and clinic metrics. Methods: Donor candidates entered the One-Day Clinic if they completed initial laboratory testing and agreed to an expedited process. If additional testing was required, it was completed on a different day. Donor candidates were reviewed by the nephrologist, transplant surgeon, and donor coordinator approximately 2 weeks after the clinic for final approval. The team continues to meet regularly to review donor feedback, discuss challenges, and brainstorm solutions. Key findings: The One-Day Clinic was implemented in March 2019, and has now been running for 4 years, making iterative improvements through continuous patient and provider feedback. To date, we have evaluated more than 150 donor candidates in this clinic. Feedback from donors has been uniformly positive (98% of donors stated they were very satisfied with the clinic), with most noting that the clinic was efficient and minimally impacted work and family obligations. Hospital leadership, including the health care professionals from each participating department, continue to show support and collaborate to create a seamless experience for donor candidates attending the One-Day Clinic. Limitations: Clinic spots are limited, meaning some interested donor candidates may not be able to enter a One-Day Clinic the same month they come forward. Implications: This patient-centered quality improvement intervention is designed to improve the efficiency and experience of the living kidney donor evaluation, result in better outcomes for kidney transplant recipients, and potentially increase living donation. Our next step is to conduct a formal evaluation of the clinic, measuring qualitative feedback from health care professionals working in the clinic and donor candidates attending the clinic, and measuring key process and outcome measures in donor candidates who completed the one-day assessment compared with those who underwent the usual care assessment. This program evaluation will provide reliable, regionally relevant evidence that will inform transplant centers across the country as they consider incorporating a similar one-day assessment model.

Objectifs du programme: Devenir donneur de rein vivant est difficile, le principal obstacle étant le processus d'évaluation inefficace auquel les candidats doivent se soumettre. Ce processus comporte plus de 30 examens (p. ex. tests de laboratoire et tests diagnostiques), questionnaires et consultations avec des spécialistes. Les candidats donneurs font plusieurs visites dans les hôpitaux et cliniques, et passent souvent plusieurs mois à attendre des rendez-vous et des résultats de tests. En Ontario (Canada), le délai médian pour l'évaluation d'un candidat au don est de près d'un an. Les temps d'attente plus longs sont associés à de moins bons résultats pour les receveurs d'une greffe rénale, ainsi qu'à des coûts de soins de santé plus élevés. Un processus d'évaluation plus court et plus efficace des donneurs potentiels permettrait à un plus grand nombre de patients atteints d'insuffisance rénale de recevoir une greffe, y compris une greffe préventive (c.-à-d. permettant d'éviter la dialyse). Cet article décrit une intervention d'amélioration de la qualité visant à augmenter l'efficience, l'efficacité et la personnalisation du processus d'évaluation des candidats au don. Nous avons développé une clinique d'un jour pour l'évaluation des donneurs de reins vivants (One-Day Living Kidney Donor Assessment Clinic), soit une clinique condensée où les candidats passent tous les tests et consultent un spécialiste dans la même journée. Sources de l'information: La clinique d'un jour pour l'évaluation des donneurs de reins vivants a été développée à la suite d'un examen approfondi de la littérature, de la consultation des commentaires de patients ayant donné avec succès et de rencontres avec les dirigeants du programme de transplantation du St Joseph's Healthcare d'Hamilton. Une équipe multipartite a été formée; celle-ci réunit du personnel soignant en néphrologie, chirurgie de transplantation, radiologie, cardiologie, travail social et médecine nucléaire, ainsi que des patients ayant une expérience vécue du don de rein. L'équipe s'est réunie régulièrement pendant les étapes de planification pour déterminer les objectifs, les paramètres et le calendrier de la clinique, ainsi que les critères de participation et le flux de patients. Méthodologie: Les donneurs potentiels qui avaient complété les tests de laboratoire initiaux et qui acceptaient de se soumettre à un processus accéléré ont été évalués à la clinique d'un jour. Si des tests supplémentaires étaient nécessaires, ceux-ci étaient effectués un autre jour. Les candidats ont été rencontrés par le néphrologue, le chirurgien de transplantation et le coordonnateur des dons environ deux semaines après leur visite à la clinique pour l'approbation finale. L'équipe multipartite continue de se réunir régulièrement pour examiner les commentaires des donneurs, discuter des défis et trouver des solutions. Principaux résultats: La clinique d'un jour, mise sur pied en mars 2019, est en activité depuis quatre ans et permet des améliorations itératives grâce à la rétroaction continue des patients et des soignants. À ce jour, plus de 150 candidats au don ont été évalués à la clinique. Les commentaires des donneurs sont quasi unanimement positifs (98 % des candidats ont déclaré être très satisfaits de la clinique), la plupart soulignant l'efficacité de la clinique et les conséquences minimes du processus sur les obligations professionnelles et familiales. La direction de l'hôpital, tout comme les professionnels de la santé des services participants, continue d'appuyer la clinique d'un jour et de collaborer à la création d'une expérience fluide pour les donneurs potentiels qui la fréquentent. Limites: Les places à la clinique sont limitées; ainsi, certains candidats au don d'un rein vivant pourraient ne pas pouvoir être admis dans le mois où ils se présentent à la clinique. Conclusion: Cette intervention d'amélioration de la qualité axée sur les patients est conçue pour augmenter l'efficacité du processus d'évaluation et bonifier l'expérience des donneurs de rein vivants. Elle vise également à améliorer les résultats des receveurs d'une greffe rénale et, potentiellement, augmenter le don vivant. La prochaine étape sera une évaluation formelle de la clinique, c'est-à-dire la mesure de la rétroaction qualitative des professionnels de la santé qui y travaillent et des candidats au don qui la fréquentent, et l'analyse des processus clés et des résultats des candidats évalués à la clinique d'un jour par rapport à ceux qui suivent le processus d'évaluation habituel. Cette évaluation du programme fournira des données probantes fiables et propres à la région qui pourront informer les centres de transplantation de tout le pays qui envisagent d'intégrer un processus d'évaluation similaire.

Adenovirus-mediated gene transfer of TGF-ß1 to the renal glomeruli leads to proteinuria.

The mechanism of proteinuria in many common kidney diseases involves glomerular hemodynamic effects and local expression of angiogenic, fibrogenic, and vasoactive factors. Transforming growth factor (TGF)-ß has been associated with many diseases involving proteinuria and renal fibrosis. TGF-ß has been shown to induce podocyte dedifferentiation in vitro, but its in vivo effects on the glomerular filtration barrier are not well described. In this study, we used an adenovirus vector to transfer active TGF-ß1 to the glomeruli of rat kidneys. Transient TGF-ß1 overexpression induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGF-ß1. Increased glomerular expression of Snail, suggestive of an in vivo dedifferentiation process, was associated with a loss of podocyte epithelial markers. The expression of angiopoietin-1 and angiopoietin-2 was significantly increased in TGF-ß1-transfected glomeruli, and TGF-ß1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. TGF-ß1 down-regulated nephrin and synaptopodin expression in podocytes in cell culture; this effect was reversed by the blockade of both angiopoietin and Tie2 activities. These findings suggest that locally produced TGF-ß1 can cause podocyte dedifferentiation marked by a loss of synaptopodin, nephrin, and foot process effacement, partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.

Transforming growth factor ß-induced peritoneal fibrosis is mouse strain dependent.

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of peritoneal dialysis. The etiology is unclear, but genetic predisposition may be a contributing factor. We used adenovirus-mediated gene transfer of transforming growth factor (TGF) ß1 to the peritoneum in four genetically distinct laboratory mouse strains to assess differences in fibrogenic response. METHODS: Mice from four genetic backgrounds (C57BL/6J, DBA/2J, C3H/HeJ and SJL/J) received an intraperitoneal injection of an adenovirus expressing TGFß1 (AdTGFß1) or control adenovirus (AdDL) and were assessed 4 and 10 days after infection. Submesothelial thickening, angiogenesis and gene expression were quantified from peritoneal tissue. Protein was extracted from omental tissue and assessed for collagen, E-cadherin and TGFß signaling pathway proteins. RESULTS: There was a graded response among the mouse strains to the peritoneal overexpression of TGFß1. TGFß1 induced a significant fibrogenic response in the C57BL/6J mice, whereas the SJL/J mice were resistant. The DBA/2J and the C3H/HeJ mice had intermediate responses. A similar graded response was seen in collagen protein levels in the omental tissue and in fibrosis-associated gene expression. TGFß type 1 receptor and SMAD signaling pathways appeared to be intact in all the mouse strains. CONCLUSIONS: There were significant differences in mouse strain susceptibility to peritoneal fibrosis, suggesting that genetic factors may play a role in the development of peritoneal fibrosis and possibly EPS. As early TGFß1 signaling mechanisms appear to be intact, we hypothesize that fibrosis resistance in the SJL/J mice lies further down the wound-healing cascade or in an alternate, non-SMAD pathway.

Effect of tangnaikang on TGF-beta1-induced transdifferentiation of human renal tubular epithelial HK-2 cells.

OBJECTIVE: To explore the function of Tangnaikang (TNK) in the prevention and treatment of renal interstitial fibrosis through transdifferentiation of the human renal tubular epithelial cell line HK-2 induced by transforming growth factor-beta1 (TGF-beta1). METHODS: HK-2 cells cultured in dulbecco's modified eagle medium/F12 (1 : 1) with 10% fetal calf serum were divided into six groups: blank control group, TGF-beta1 group (TGF-beta1 10 ng/mL), serum control group (TGF-beta1 10 ng/mL + 10% serum), treatment group 1 (TGF-beta1 10 ng/mL + 5% TNK serum), treatment group 2 (TGF-beta1 10 ng/mL + 10% TNK serum), and treatment group 3 (TGF-beta1 10 ng/mL + 20% TNK serum). Cell proliferation was detected by 4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin were observed by immunohistochemical assay. The contents of collagen I (Col I), collagen III (Col III), and fibronectin (FN) in the culture medium supernatant were detected by ELISA. RESULTS: E-cadherin was expressed and alpha-SMA was not expressed in normal HK-2 cells. In HK-2 cells cultured with TGF-beta1, alpha-SMA expression significantly increased, HK-2 cells significantly proliferated, and secretion of Col I, Col III, and FN significantly increased compared with the blank control group (all P < 0.05). In the HK-2 cells cultured with TGF-beta1 and TNK serum, the expression of alpha-SMA significantly decreased, the expression of E-cadherin significantly increased, and the cell proliferation and the secretion of Col I, Col III and FN were significantly inhibited compared with the TGF-beta1 group (all P < 0.05). CONCLUSION: TNK can inhibit cell proliferation and reduce secretion of Col I, Col III, and FN. This indicates that TNK can inhibit transdifferentiation of human renal tubular epithelial cells induced by TGF-beta1, with the effect of preventing and treating renal interstitial fibrosis.

Rapamycin inhibits transforming growth factor ß-induced peritoneal angiogenesis by blocking the secondary hypoxic response.

Patients with end-stage kidney disease on peritoneal dialysis often develop progressive scarring of the peritoneal tissues. This manifests as submesothelial thickening and is associated with increased vascularization that leads to ultrafiltration dysfunction. Hypoxia induces a characteristic series of responses including angiogenesis and fibrosis. We investigated the role of hypoxia in peritoneal membrane damage. An adenovirus expressing transforming growth factor (TGF) ß was used to induce peritoneal fibrosis. We evaluated the effect of the mTOR inhibitor rapamycin, which has been previously shown to block hypoxia-inducible factor (HIF) 1α. We also assessed the effect of HIF1α independently using an adenovirus expressing active HIF1α. To identify the TGFß1-independent effects of HIF1α, we expressed HIF1α in the peritoneum of mice lacking the TGFß signalling molecule Smad3. We demonstrate that TGFß-induced fibroproliferative tissue is hypoxic. Rapamycin did not affect the early angiogenic response, but inhibited angiogenesis and submesothelial thickening 21 days after induction of fibrosis. In primary mesothelial cell culture, rapamycin had no effect on TGFß-induced vascular endothelial growth factor (VEGF) but did suppress hypoxia-induced VEGF. HIF1α induced submesothelial thickening and angiogenesis in peritoneal tissue. The fibrogenic effects of HIF1α were Smad3 dependent. In summary, submesothelial hypoxia may be an important secondary factor, which augments TGFß-induced peritoneal injury. The hypoxic response is mediated partly through HIF1α and the mTOR inhibitor rapamycin blocks the hypoxic-induced angiogenic effects but does not affect the direct TGFß-mediated fibrosis and angiogenesis.

TDAG51 mediates epithelial-to-mesenchymal transition in human proximal tubular epithelium.

Epithelial-to-mesenchymal transition (EMT) contributes to renal fibrosis in chronic kidney disease. Endoplasmic reticulum (ER) stress, a feature of many forms of kidney disease, results from the accumulation of misfolded proteins in the ER and leads to the unfolded protein response (UPR). We hypothesized that ER stress mediates EMT in human renal proximal tubules. ER stress is induced by a variety of stressors differing in their mechanism of action, including tunicamycin, thapsigargin, and the calcineurin inhibitor cyclosporine A. These ER stressors increased the UPR markers GRP78, GRP94, and phospho-eIF2α in human proximal tubular cells. Thapsigargin and cyclosporine A also increased cytosolic Ca(2+) concentration and T cell death-associated gene 51 (TDAG51) expression, whereas tunicamycin did not. Thapsigargin was also shown to increase levels of active transforming growth factor (TGF)-ß1 in the media of cultured human proximal tubular cells. Thapsigargin induced cytoskeletal rearrangement, ß-catenin nuclear translocation, and α-smooth muscle actin and vinculin expression in proximal tubular cells, indicating an EMT response. Subconfluent primary human proximal tubular cells were induced to undergo EMT by TGF-ß1 treatment. In contrast, tunicamycin treatment did not produce an EMT response. Plasmid-mediated overexpression of TDAG51 resulted in cell shape change and ß-catenin nuclear translocation. These results allowed us to develop a two-hit model of ER stress-induced EMT, where Ca(2+) dysregulation-mediated TDAG51 upregulation primes the cell for mesenchymal transformation via Wnt signaling and then TGF-ß1 activation leads to a complete EMT response. Thus the release of Ca(2+) from ER stores mediates EMT in human proximal tubular epithelium via the induction of TDAG51.

Twist: a new player in the epithelial-mesenchymal transition of the peritoneal mesothelial cells.

BACKGROUND: The peritoneal membrane is a vital structure for peritoneal dialysis (PD) patients. It has been increasingly recognized that the transition of the peritoneal lining mesothelial cells into a more fibroblastic phenotype is a key step in peritoneal membrane injury. METHODS: Relevant literature was reviewed and summarized. RESULTS: Epithelial-to-mesenchymal transition (EMT) is a basic cellular process that occurs in a variety of physiologic and pathologic processes. The hallmark of this process is a loss of epithelial markers, and E-cadherin is a prototypical epithelial transmembrane protein. E-cadherin expression is suppressed at many levels and the gene is regulated by a family of transcription factors. Twist is one of the lesser studied E-cadherin regulatory factors, which belongs to a larger family of basic helix-loop-helix DNA-binding proteins. In this issue of Nephrology Dialysis Transplantation, Cuixiang Li reports on in vitro experiments where the expression of Twist led to a decreased expression of E-cadherin and the evidence of EMT. In an in vivo model of dialysate exposure, Li demonstrates that Twist expression is increased in the injured peritoneal tissues. CONCLUSIONS: These important observations are the first to link Twist to mesothelial cell EMT and peritoneal membrane injury. Like most novel observations, this paper leaves many questions unanswered. Twist is only one of several transcription factors involved in EMT and how these factors interact will require further investigations. Furthermore, the question of whether Twist interacts at multiple levels in the EMT process, or simply gives an initial push to the process, is left unanswered. Finally, to bring these early significant findings to the bedside as potential therapies for PD patients will require further innovation.

Differentiation of bone marrow-derived cells into regenerated mesothelial cells in peritoneal remodeling using a peritoneal fibrosis mouse model.

Marked thickening of the peritoneum and vasculopathy in the submesothelial compact zone have been reported in long-term peritoneal dialysis patients. Bone marrow (BM)-derived cell lines are considered to be useful tools for therapy of various diseases. To clarify the role of BM-derived cells in the peritoneal fibrosis (PF) model, we analyzed several lineages of cells in the peritoneum. BM cells from green fluorescent protein (GFP) transgenic mice were transplanted into naïve C57Bl/6 mice. Chlorhexidine gluconate (CG) was injected intraperitoneally to induce PF. Immunohistochemical analysis was performed with parietal peritoneum using anti-Sca-1 or -c-Kit and -GFP antibodies. Isolated BM cells were also transplanted into the CG-stimulated peritoneum. BM-derived cells from GFP transgenic mice appeared in the submesothelium from days 14 to 42. Both GFP- and stem cell marker-positive cells were observed in the submesothelium and on the surface. Isolated c-Kit-positive cells, transplanted into the peritoneal cavity, differentiated into mesothelial cells. In this study, we investigated whether or not BM-derived cells play a role in the repair of PF and immature cells have the potential of inducing repair of the peritoneum. The findings of this study suggest a new concept for therapy of PF.

Characterization of proteins, mRNAs, and miRNAs of circulating extracellular vesicles from prostate cancer patients compared to healthy subjects.

Prostate cancer (PC) is the fifth leading cause of death in men globally. Measurement of the blood PSA level is still considered the gold-standard biomarker test for PC despite its high rate of delivering false positives and negatives that result in an inappropriate medical response, including overtreatment. We collected extracellular vesicles (EVs) from the blood plasma of PC patients with organ-confined, extracapsular-invading, and seminal vesicle-invading tumors and from healthy subjects. We examined the protein, mRNA, and miRNA content of these EVs using mass spectrometry (MS), a human PC PCR array, and a miScript miRNA PCR array, respectively. The proteomic analysis showed distinct groups of proteins that are differently expressed in each group of patients, as well as in healthy subjects. Samples from healthy subjects and each tumor type were used for both mRNA and miRNA arrays. The mRNA analysis showed distinct groups of mRNAs that were overexpressed in healthy or in one of the three tumor types but not in the EVs of the other groups. The miRNA analysis showed distinct groups of miRNAs as well. The fold of regulation in the expression of the identified mRNA and miRNA of each stage of the disease from healthy subjects showed that various mRNAs and miRNAs could discriminate the disease stage. Overall, our data suggest many molecular marker candidates for distinguishing between healthy subjects and PC patients using the cargo of circulating vesicles, as well as markers to discriminate between the different tumor types. Once verified, these markers might have a diagnostic value for PC.