Role of claudins in idiopathic hypercalciuria and renal lithiasis.

Paracellular transport in the kidney is mediated by a family of proteins located in the tight junctions called claudins which confers its ionic selectivity. Claudin-2 is highly expressed in the proximal tubule and descending limb of Henle and mediate paracellular reabsorption of sodium and calcium cations. In the thick ascending limb of Henle (TALH) calcium is reabsorbed by a paracellular channel formed by Claudin-16 and-19. Claudin-16 mediates cationic permeability while Claudin-19 increases the cationic selectivity of Claudin-16 by blocking anionic permeability. On the other hand, Claudin 14, that is also located in TALH, inhibits the paracellular permeability of Claudin-16 to calcium. Recent wide genomic association analysis studies have detected four common synonymous variants (genetic polymorphisms of a single nucleotide, SNPs) at the locus of Claudin-14 gene that were significantly associated with the presence of renal lithiasis. Another study of wide genomic association and nephrolithiasis was carried out in the general population but including chromosome X, where claudin-2 gene is located. They detected nine SNPs that had a significant association with renal lithiasis risk. A greater knowledge of the paracellular pathway controlled by claudins and its regulation will allow us to develop future new treatments for idiopathic hypercalciuria and renal lithiasis.

Intestinal Ca2+ absorption revisited: A molecular and clinical approach.

Ca2+ has an important role in the maintenance of the skeleton and is involved in the main physiological processes. Its homeostasis is controlled by the intestine, kidney, bone and parathyroid glands. The intestinal Ca2+ absorption occurs mainly via the paracellular and the transcellular pathways. The proteins involved in both ways are regulated by calcitriol and other hormones as well as dietary factors. Fibroblast growth factor 23 (FGF-23) is a strong antagonist of vitamin D action. Part of the intestinal Ca2+ movement seems to be vitamin D independent. Intestinal Ca2+ absorption changes according to different physiological conditions. It is promoted under high Ca2+ demands such as growth, pregnancy, lactation, dietary Ca2+ deficiency and high physical activity. In contrast, the intestinal Ca2+ transport decreases with aging. Oxidative stress inhibits the intestinal Ca2+ absorption whereas the antioxidants counteract the effects of prooxidants leading to the normalization of this physiological process. Several pathologies such as celiac disease, inflammatory bowel diseases, Turner syndrome and others occur with inhibition of intestinal Ca2+ absorption, some hypercalciurias show Ca2+ hyperabsorption, most of these alterations are related to the vitamin D endocrine system. Further research work should be accomplished in order not only to know more molecular details but also to detect possible therapeutic targets to ameliorate or avoid the consequences of altered intestinal Ca2+ absorption.

Glutamine protects both transcellular and paracellular pathways of chick intestinal calcium absorption under oxidant conditions.

Glutamine (GLN) avoids the inhibition of the intestinal Ca2+ absorption caused by menadione (MEN) through oxidative stress. The purpose of this study was to elucidate whether molecules of transcellular and/or paracellular pathways of intestinal Ca2+ absorption are involved in the GLN action and underlying mechanisms. One-month old chicks were divided in four groups: 1) controls, 2) MEN treated, 3) GLN treated and 4) GLN + MEN treated. The morphology of intestinal villi, the intestinal Ca2+ absorption and the molecules involved in the transcellular and paracellular pathways were analyzed. Markers of autophagy and inflammation were also evaluated. The data demonstrated that GLN protected both transcellular and paracellular pathways. GLN avoided morphological changes in the intestine caused by MEN. GLN protected the gene expression of transporters involved in the transcellular pathway and the gene and protein expression of molecules belonging to the paracellular pathways altered by MEN. GLN increased the LC3-II protein expression and the number of acidic vesicular organelles, markers of autophagy, and blocked an increase in the NFkB protein expression in the nuclei and in the IL-6 gene expression caused by MEN. In conclusion, GLN protects both transcellular and paracellular pathways of intestinal Ca2+ absorption by increasing autophagy and blocking inflammation.

Small intestinal epithelial permeability to water-soluble nutrients higher in passerine birds than in rodents.

In the small intestine transcellular and paracellular pathways are implicated in water-soluble nutrient absorption. In small birds the paracellular pathway is quantitatively important while transcellular pathway is much more important in terrestrial mammals. However, there is not a clear understanding of the mechanistic underpinnings of the differences among taxa. This study was aimed to test the hypothesis that paracellular permeability in perfused intestinal segments is higher in passerine birds than rodents. We performed in situ intestinal perfusions on individuals of three species of passerine birds (Passer domesticus, Taeniopygia guttata and Furnarius rufus) and two species of rodents (Mus musculus and Meriones ungiculatus). Using radio-labelled molecules, we measured the uptake of two nutrients absorbed by paracellular and transcellular pathways (L-proline and 3-O-methyl-D-glucose) and one carbohydrate that has no mediated transport (L-arabinose). Birds exhibited ~2 to ~3 times higher L-arabinose clearance per cm2 epithelium than rodents. Moreover, paracellular absorption accounted for proportionally more of 3-O-methyl-D-glucose and L-proline absorption in birds than in rodents. These differences could be explained by differences in intestinal permeability and not by other factors such as increased retention time or higher intestinal nominal surface area. Furthermore, analysis of our results and all other existing data on birds, bats and rodents shows that insectivorous species (one bird, two bats and a rodent) had only 30% of the clearance of L-arabinose of non-insectivorous species. This result may be explained by weaker natural selection for high paracellular permeability in animal- than in plant-consumers. Animal-consumers absorb less sugar and more amino acids, whose smaller molecular size allow them to traverse the paracellular pathway more extensively and faster than glucose.

Entamoeba histolytica EhCP112 Dislocates and Degrades Claudin-1 and Claudin-2 at Tight Junctions of the Intestinal Epithelium.

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease.

Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded ß-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with ß-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with ß-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.

La diabetes mellitus experimental produce aumento en la expresión de iNOS y altera la vía paracelular de la absorción intestinal de calcio / Experimental diabetes mellitus produces increased iNOS expression and alters the paracellular pathway of intestinal calcium absorption

Previamente hemos demostrado que la diabetes mellitus tipo 1 experimental (D.m.1) inducida por estreptozotocina (STZ) produce estrés oxidativo intestinal en las primeras etapas de la enfermedad, lo que conduce a la inhibición de la absorción intestinal de Ca+2, alterando la vía transcelular del transporte del catión. El objetivo de este trabajo fue estudiar la vía paracelular del transporte del Ca+2 y analizar si la D.m.1 induce estrés nitrosativo a nivel duodenal. Se utilizaron ratas Wistar machos a las que se inyectaron 60 mg STZ/kg de peso corporal; se sacrificaron a los 30 días postratamiento. Se determinó la expresión génica y proteica de claudina 2 y 12, proteínas involucradas en el transporte paracelular del Ca+2. En la mucosa duodenal se determinó el contenido de óxido nítrico (NO) y la expresión proteica de óxido nítrico sintasa inducible (iNOS). Los resultados revelaron que la expresión génica de claudina 2 en las ratas diabéticas fue más del doble en comparación con la de los controles, mientras que la expresión génica de claudina 12 fue similar en ambos grupos. La expresión proteica de claudina 2 y 12 aumentó en las ratas diabéticas. El contenido de NO fue similar en ambos grupos; sin embargo, la expresión proteica de iNOS fue mayor en las ratas diabéticas en comparación con la de las ratas controles. En conclusión, la D.m.1 experimental se acompaña de estrés oxidativo y de aumento en la expresión proteica de iNOS, alterándose la vía paracelular de absorción de Ca+2 como un mecanismo compensatorio. (AU)

We have previously shown that experimental type 1 diabetes mellitus (D.m.1) produced by streptozotocin (STZ) in rats causes intestinal oxidative stress in the early stages of the disease, which leads to the inhibition of intestinal Ca2+ absorption, altering the transcellular Ca2+ pathway. The aim of this work was to study the paracellular Ca2+ pathway and analyze if D.m.1 induces duodenal nitrosative stress. The animals were assigned to two groups: 1) control rats, and 2) STZ-induced diabetic rats (60 mg/kg b.w.). Rats were sacrificed 30 days after induction of diabetes. The gene and protein expression of claudin 2 and 12, proteins involved in paracellular Ca2+ pathway, was determined as well as the nitric oxide (NO) content and protein expression of iNOS in rat duodenum mucosa. The results revealed that claudin 2 expression was more that double in diabetic rats compared to control rats at 30 days, while the gene expression of claudin 12 was similar in both groups. The protein expression of claudin 2 and 12 increased in the diabetic rats. NO content was similar in both groups, but the iNOS protein expression was enhanced in diabetic rats. To conclude, the experimental type I D.m.1 is accompanied by duodenal oxidative stress, increase iNOS protein expression and alteration of the paracellular Ca2+ pathway as a compensatory mechanism. (AU)