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

Differentially expressed genes identified from supervised analyses.

<p><b>A.</b> Venn diagram comparing significantly differentially expressed genes identified from the pairwise comparisons Uremic vs. PD (yellow), PD vs. EPS (blue), and Uremic vs. EPS (red). The genes were selected using supervised analysis on the basis of the 90% lower confidence bound (LCB) of the fold change (FC) by pairwise comparison of the groups. The analysis was performed on preprocessed data by filtering out low-expressing probes on the basis of absolute intensity (Intensity <10 in 90% of samples). <b>B.</b> Heatmap of genes differentially expressed in both EPS and PD groups as compared to Uremic group (P<0.05). <b>C.</b> Heatmap of genes differentially expressed only in EPS as compared to both PD and Uremic groups (P<0.05). Columns represent the samples, with rows representing genes. Gene expression levels are presented in pseudocolor (scale −3 to 3), with red and green respectively denoting high and low expression levels.</p

Immunohistochemical expression patterns of collagen 1 α 1 (Col1a1) polypeptide in peritoneal biopsy samples.

<p>Peritoneal biopsy sections from (<b>A.</b>) EPS, (<b>B.</b>) PD, and (<b>C.</b>) Uremic groups were analyzed for Col1a1 immunolocalization. Each panel indicates location of peritoneal cavity adjacent to the tissue surface. <b>D.</b> Normalized ratios of staining intensities measured in rectangular areas of thickness depth 0–100 µm and 100–200 µm from the peritoneal cavity tissue surface. P<0.05 for EPS vs. PD (One Way ANOVA with Dunn's Multiple Comparison post-test).</p

Principal Component Analysis (PCA) of normalized expression data obtained from Uremic, PD and EPS samples.

<p>The first component with highest variance (42.2%) is on the X-axis separating Uremic and PD from EPS samples. The second highest (26.4%) is on the Y-axis depicting maximum variation between PD and Uremic samples. The Uremic, PD and EPS samples formed three separate clusters on the PCA plot.</p

mRNA expression levels of selected gene products determined through qRT-PCR correlate well with corresponding data from the DNA chip array.

<p>Collagen 1 α 1 (Col1a1), Fibronectin 1 (FN1), and thrombospondin 1 (THBS1) were highly upregulated in EPS compared to PD and Uremic groups, while retinol-binding protein 4 (RBP4) was highly downregulated in EPS and PD groups compared to the Uremic group. <b>A.</b>, <b>C.</b>, <b>E.</b>, and <b>G.</b> Normalized mRNA expression levels determined through qRT-PCR of indicated gene products. Individual gene expression levels were calculated using the equation 2^−ΔCT with β-actin (ACTB) as endogenous control. Mean gene expression levels of biological groups were normalized to the Uremic group, defining the relative gene expression in this group as 1.0. <b>B.</b>, <b>D.</b>, <b>F.</b>, and <b>H.</b> Mean raw signal intensity of indicated gene products calculated from DNA array probe signals.</p

Network representation of cellular functions differentially expressed specifically in EPS but not in PD or Uremic groups.

<p><b>A.</b> Network related to cell assembly and organization as well as to connective tissue and skeletal muscle tissue disorders. This network has NF-κB, collagen, and ERK1/2 genes as primary regulatory focus nodes. <b>B.</b> Network enriched with genes involved in cellular growth and proliferation, carbohydrate metabolism, and gastrointestinal and immunological diseases. This network has PI3K, IFNγ and MAPK1 genes as primary regulatory focus nodes. Ingenuity Pathways Analysis software was used to generate comprehensive gene networks that merged affected networks of related function. Downregulated genes are shown in green, upregulated genes in red. All networks shown were significantly affected in EPS with a score >15 (−log 10[Fisher's Exact Test]).</p

Patient characteristics.

<p>Clinical data and laboratory values of (n) patients, collected before the surgeries that allowed collection of peritoneal biopsy samples. Values are shown as means ± s.e.m. for the EPS group and as means for the smaller PD and Uremic groups. Smoking history was not stratified by duration. Uremic group 24 h urine output was higher than that of the combined PD and EPS groups (p<0.001). Arterial hypertension was defined as resting arterial blood pressure ≥140/90 mmHg. Acidic PD solutions were lactate-buffered with pH 5.0–5.5. Neutral (multicomponent) solutions were of pH 6.5. Icodextrin status was listed as positive if used at any time during the course of PD.</p><p>EPS, Encapsulating peritoneal sclerosis; PD, Peritoneal dialysis; CrP, C reactive protein; BUN, blood urea nitrogen.</p

Network representation of the cellular functions affected specifically in both EPS and PD groups, but not in the Uremic group.

<p><b>A.</b> Network related to inflammatory and immunological diseases, including NF-κB, JUN, and SP1 genes as primary regulatory focus nodes. <b>B.</b> Network enriched with genes involved in lipid metabolism, cellular assembly and movement, and cell death, including PI3K, AKT and TP53 genes as primary regulatory focus nodes. The Ingenuity Pathways Analysis tool was used to generate comprehensive gene networks that merged with affected networks of related function. Downregulated genes are shown in green, upregulated genes in red. Geometric shapes are associated with individual gene products according to Ingenuity definitions. All networks shown were significantly affected in EPS with a score >20 (−log 10[Fisher's Exact Test]).</p

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

The spectrum of podoplanin expression in encapsulating peritoneal sclerosis

Encapsulating peritoneal sclerosis (EPS) is a life threatening complication of peritoneal dialysis (PD). Podoplanin is a glycoprotein expressed by mesothelial cells, lymphatic endothelial cells, and myofibroblasts in peritoneal biopsies from patients with EPS. To evaluate podoplanin as a marker of EPS we measured podoplanin mRNA and described the morphological patterns of podoplanin-positive cells in EPS. Included were 20 peritoneal biopsies from patients with the diagnosis of EPS (n = 5), patients on PD without signs of EPS (n = 5), and control patients (uremic patients not on PD, n = 5, non-uremic patients n = 5). EPS patient biopsies revealed significantly elevated levels of podoplanin mRNA (p<0.05). In 24 peritoneal biopsies from patients with EPS, podoplanin and smooth muscle actin (SMA) were localized by immunohistochemistry. Four patterns of podoplanin distribution were distinguishable. The most common pattern (8 of 24) consisted of organized, longitudinal layers of podoplanin-positive cells and vessels in the fibrotic zone ("organized" pattern). 7 of 24 biopsies demonstrated a diffuse distribution of podoplanin-positive cells, accompanied by occasional, dense clusters of podoplanin-positive cells. Five biopsies exhibited a mixed pattern, with some diffuse areas and some organized areas ("mixed"). These contained cuboidal podoplanin-positive cells within SMA-negative epithelial structures embedded in extracellular matrix. Less frequently observed was the complete absence of, or only focal accumulations of podoplanin-positive fibroblasts outside of lymphatic vessels (podoplanin "low", 4 of 24 biopsies). Patients in this group exhibited a lower index of systemic inflammation and a longer symptomatic period than in EPS patients with biopsies of the "mixed" type (p<0.05). In summary we confirm the increased expression of podoplanin in EPS, and distinguish EPS biopsies according to different podoplanin expression patterns which are associated with clinical parameters. Podoplanin might serve as a useful adjunct to the morphological workup of peritoneal biopsies