Fabian R. Reimold

Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis

BACKGROUND Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. METHODS We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. RESULTS Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). CONCLUSIONS EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EPS.

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization.

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO(3)(-) or more, mouse and rat ducts secrete ∼40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.

Pendrin function and regulation in Xenopus oocytes.

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K(1/2) (in mM) of 1.9 (Cl-), 1.8 (I-), and 0.9 (Br-). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).

Difference in the expression of hormone receptors and fibrotic markers in the human peritoneum--implications for therapeutic targets to prevent encapsulating peritoneal sclerosis.

♦ Objective: Encapsulating peritoneal sclerosis (EPS) is a rare but life-threatening complication of peritoneal dialysis (PD). The optimal management of patients with EPS is uncertain. In the present study, we investigated differences in the expression of nuclear receptors [progesterone (PR), androgen (AR), vitamin D (VDR), and glucocorticoid (GCR)] in the human peritoneum. We also investigated estrogen receptor (ER), matrix metalloproteinase 9 (MMP9), and transforming growth factor β1 (TGFβ1) in the context of their potential role in tamoxifen therapy. ♦ Methods: We analyzed clinical and histologic characteristics of 72 peritoneal biopsy specimens (22 from EPS patients, 11 from PD patients, 15 from uremic patients, and 24 from control subjects undergoing hernia repair). For immunophenotyping, we used antibodies against VDR, GCR, ER, PR, AR, MMP9, and TGFβ1. ♦ Results: In human peritoneum, VDR and GCR are highly expressed (98.6% and 87.3% respectively). Except in the case of VDR (p = 0.0012), we observed no significant difference in receptor expression between the groups. Expression of ER and PR was sparse (11.4% and 31% respectively), with higher expression in women, and AR was absent. Minimal MMP9 expression and moderate TGFβ1 expression were observed in all groups. The differences between the groups were nonsignificant. ♦ Conclusions: Nuclear receptors are present in human peritoneum. Except in the case of VDR, the pattern for any one group is nonspecific. Glucocorticoids, vitamin D, and angiotensin converting-enzyme inhibitors or angiotensin II receptor blockers (via the vitamin D/angiotensin II pathway) might be suitable interventions for preservation of the integrity of the peritoneal membrane. The mechanism of action of tamoxifen is still not elucidated, ER expression in the peritoneum is sparse, and data about the studied pathways (MMP9, TGFβ) are inconsistent.

Histological criteria for encapsulating peritoneal sclerosis - a standardized approach.

Background The two most relevant pathologies of long-term peritoneal dialysis (PD) are simple sclerosis and encapsulating peritoneal sclerosis (EPS). The histological differentiation of those two entities is difficult. The Aim of the study was to establish a method to standardize and facilitate the differentiation between simple sclerosis and EPS Methods We investigated 58 peritoneal biopsies - 31 EPS patients and 27 PD patients. Two blinded investigators analyzed 20 histological characteristics in EPS and PD patients. Results The following findings were significantly more common in EPS than in patients on PD without EPS: fibroblast like cells (FLC) (p<0.0001), mesothelial denudation (p<0.0001), decreased cellularity (p = 0.008), fibrin deposits (p<0.03), Fe deposits (p = 0.05), podoplanin vascular (p<0.0001), podoplanin avascular (p<0.0001). Using all predictor variables we trained the classification method Random Forest to categorize future cases. Podoplanin vascular and avascular were taken together (p<0.0001), FLC (p<0.0001), mesothelial denudation (p = 0.0005), calcification (p = 0.0026), acellular areas (p = 0.0094), and fibrin deposits (p = 0.0336) showed up as significantly important predictor variables. Estimated misclassification error rate when classifying new cases turned out to be 14%. Conclusion The introduced statistical method allows discriminating between simple sclerosis and EPS. The misclassification error will likely improve with every new case added to the database.

Fibrogenic Growth Factors in Encapsulating Peritoneal Sclerosis

Background: Increased local levels of fibrogenic growth hormones contribute substantially to the process of encapsulating peritoneal sclerosis (EPS) in animal models. Methods: We analyzed probes from patients with normal kidney function (n = 10), with normal kidney function and inflammation (n = 10), on PD without (n = 10) and with EPS (n = 9). We investigated the degree of fibrosis and the number of vessels and vasculopathy. Additionally, we investigated the expression of NFκB, TGFβ1, TGFβ1 receptor, TGFβ2, TGFβ2 receptor, FGF-BP, CTGF and VEGF by immunohistochemistry. Results: In EPS, we found an exclusive upregulation of VEGF (normal 0, appendicitis 1.0 ± 1.2, PD 1.7 ± 1.8 and EPS 5.7 ± 4.4; p < 0.0001), whereas in PD, CTGF was significantly increased (normal 6.0 ± 2.8, appendicitis 7.3 ± 2.5, PD 10.0 ± 1.8 and EPS 7.3 ± 2.1; p = 0.0059). The results for the TGFβ system and NFκB were not uniform, in EPS no increases were demonstrable. Vasculopathy was significantly more pronounced in EPS (normal 0.4 ± 0.5, appendicitis 0.2 ± 0.3, PD 1.0 ± 0.7 and EPS 1.6 ± 1.2; p < 0.0001) than in PD or inflammation (normal 30 ± 16, appendicitis 82 ± 48, PD 1,936 ± 952 and EPS 2,613 ± 1,209; p < 0.0001), whereas the density of vessels were decreased (normal 125 ± 114, appendicitis 817 ± 347, PD 81 ± 57 and EPS 36 ± 33; p < 0.0001). Conclusions: The process of EPS was associated with increased VEGF in the peritoneum. The reduced density of vessels compared with marked fibrosis could point to hypoxia as an inducer.

Use of SLC26A4 Mutation Testing for Unilateral Enlargement of the Vestibular Aqueduct

IMPORTANCE Approximately one-half of all subjects with unilateral or bilateral hearing loss with enlargement of the vestibular aqueduct (EVA) will have SLC26A4 gene mutations. The number (0, 1, or 2) of mutant alleles of SLC26A4 detected in an individual subject with EVA is each associated with a distinct combination of diagnostic and prognostic information as well as probability of recurrence of EVA in siblings. OBJECTIVE To evaluate the results of SLC26A4 mutation testing in subjects with unilateral EVA. (The study objective was formulated before data were collected.) DESIGN Prospective cross-sectional study of cohort ascertained between 1998 and 2012. SETTING National Institutes of Health Clinical Center, a federal biomedical research facility. PARTICIPANTS Twenty-four subjects (10 males, 14 females) with unilateral EVA, defined as a midpoint diameter greater than 1.5 mm, who were referred or self-referred to participate in a study about the clinical and molecular analysis of EVA. Twenty-one (87.5%) of 24 subjects were white. Mean age was 10.3 years (age range, 5-39 years). INTERVENTION SLC26A4 mutation analysis. MAIN OUTCOMES AND MEASURES Audiometric results, the presence or absence of EVA, and the number of mutant alleles of SLC26A4. RESULTS Approximately 8.3% of the subjects with unilateral EVA had 2 mutant SLC26A4 alleles, 16.7% had 1 mutant allele, and 75.0% had 0 mutant alleles. CONCLUSIONS AND RELEVANCE Unilateral EVA can be associated with all possible SLC26A4 genotype results. The distinct combination of prognoses and recurrence probability associated with each genotype supports the clinical use of testing for SLC26A4 mutations in subjects with unilateral EVA.

Transcriptional patterns in peritoneal tissue of encapsulating peritoneal sclerosis, a complication of chronic peritoneal dialysis.

Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD), characterized by marked inflammation and severe fibrosis of the peritoneum, and associated with high morbidity and mortality. EPS can occur years after termination of PD and, in severe cases, leads to intestinal obstruction and ileus requiring surgical intervention. Despite ongoing research, the pathogenesis of EPS remains unclear. We performed a global transcriptome analysis of peritoneal tissue specimens from EPS patients, PD patients without EPS, and uremic patients without history of PD or EPS (Uremic). Unsupervised and supervised bioinformatics analysis revealed distinct transcriptional patterns that discriminated these three clinical groups. The analysis identified a signature of 219 genes expressed differentially in EPS as compared to PD and Uremic groups. Canonical pathway analysis of differentially expressed genes showed enrichment in several pathways, including antigen presentation, dendritic cell maturation, B cell development, chemokine signaling and humoral and cellular immunity (P value<0.05). Further interactive network analysis depicted effects of EPS-associated genes on networks linked to inflammation, immunological response, and cell proliferation. Gene expression changes were confirmed by qRT-PCR for a subset of the differentially expressed genes. EPS patient tissues exhibited elevated expression of genes encoding sulfatase1, thrombospondin 1, fibronectin 1 and alpha smooth muscle actin, among many others, while in EPS and PD tissues mRNAs encoding leptin and retinol-binding protein 4 were markedly down-regulated, compared to Uremic group patients. Immunolocalization of Collagen 1 alpha 1 revealed that Col1a1 protein was predominantly expressed in the submesothelial compact zone of EPS patient peritoneal samples, whereas PD patient peritoneal samples exhibited homogenous Col1a1 staining throughout the tissue samples. The results are compatible with the hypothesis that encapsulating peritoneal sclerosis is a distinct pathological process from the simple peritoneal fibrosis that accompanies all PD treatment.

Atypical patterns of segregation of familial enlargement of the vestibular aqueduct.

Hearing loss and enlarged vestibular aqueduct (EVA) can be inherited as an autosomal recessive trait caused by mutant alleles of the SLC26A4 gene. In some other families, EVA does not segregate in a typical autosomal recessive pattern. The goal of this study was to characterize the SLC26A4 genotypes and phenotypes of extended families with atypical segregation of EVA.

Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl−/HCO−3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.