Effect of microbiota on the physiology of blood-tissue barriers.

Las uniones estrechas (UE) son estructuras altamente complejas que se localizan en la porción más apical de la membrana basolateral y están compuestas por una serie de proteínas, como claudinas, ocludinas y proteínas de la familia ZO. Las UE restringen el paso de sustancias potencialmente dañinas o microorganismos a lo largo del espacio paracelular, y participan de manera importante en procesos de mecanotransducción y señalización intercelular. Aunque la ultraestructura de las UE les permiten funcionar como una barrera en varios tejidos, como en la barrera hematoencefálica y la barrera hematotesticular, estas son propensas a cambios en su composición, lo cual podría disminuir sus características de permeabilidad. En este sentido, se ha demostrado que ciertos microorganismos enteropatógenos son capaces de desensamblar o modificar las propiedades de permeabilidad de las UE en las barreras hematotisulares. En particular, se ha estudiado cómo la microbiota contribuye a la formación, la función y el mantenimiento de las UE en varios nichos inmunitariamente privilegiados, tales como el tracto gastrointestinal, el sistema nervioso central y los testículos. Por lo tanto, resulta primordial comprender los mecanismos fisiológicos por los cuales la microbiota puede modificar la función de las barreras hematotisulares, con el -objetivo de diseñar nuevas estrategias terapéuticas que mejoren los efectos dañinos de varias enfermedades sobre nichos inmunitariamente privilegiados en el humano.Tight-junctions (TJ) are a highly complex structure located in the most apical portion of the basolateral membrane, composed of series of proteins, such as claudins, occludins and proteins of the ZO family. TJ restrict the passage of potentially harmful substances or microorganisms through paracellular space and participate importantly in the mecanotransduction and intercellular signaling processes. Although the complex structure of TJ, allow them function as barrier in various tissues, such as brain-blood-barrier and testicular-blood-barrier, these barriers are prone to changes decreasing its permeability features. The contribution of microbiota in the formation, function and maintenance of TJ in various immunologically privileged niches, such as gastrointestinal tract, central nervous system and testicles have been recently studied. Nevertheless, it has been demonstrated that certain pathogenic microorganisms are able to disassemble or modify the permeability of TJ in blood-tissue barrier. Thereby, it is central to understand the physiological mechanisms of how microbiota could modify the function of epithelial blood barriers in order to design new therapeutic strategies to ameliorate harmful effects of many human diseases.

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway.

Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size.

All-trans retinoic acid prevents oxidative stress-induced loss of renal tight junction proteins in type-1 diabetic model.

We previously reported that diabetes decreased the expression of renal tight junction (TJ) proteins claudin-5 in glomerulus, and claudin-2 and occludin in proximal tubule through an oxidative stress dependent way. Now we investigated whether all-trans retinoic acid (atRA), a compound that plays a relevant role in kidney maintenance and that possesses antioxidant properties, prevents loss of TJ proteins in streptozotocin (STZ)-treated rats. atRA was administered daily by gavage (1mg/kg) from Days 3-21 after STZ administration. atRA attenuated loss of body weight, proteinuria and natriuresis but it did not prevent hyperglucemia. Other metabolic alterations, such as: increased kidney injury molecule (KIM)-1, oxidative stress, protein kinase C (PKC) beta 2, NADPH oxidase subunits (p47(phox) and gp91(phox)) expressions and endothelial nitric oxide synthase (eNOS) uncoupling, and decreased nitric oxide synthesis, nuclear factor-erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions were also attenuated by atRA. In vitro scavenging capacity assays showed that atRA scavenged peroxyl radicals (ROO•), singlet oxygen ((1)O2) and hypochlorous acid (HOCl) in a concentration-dependent manner. Decreased expressions of occludin, claudins-2 and -5 induced by diabetes were ameliorated by atRA. We also found that diabetes induced tyrosine nitration (3-NT), SUMOylation and phosphorylation in serine residues of claudin-2 and atRA prevented these changes. In conclusion, atRA exerted nephroprotective effects by attenuating oxidative stress and preventing loss of renal TJ proteins.

Tight junction proteins and oxidative stress in heavy metals-induced nephrotoxicity.

Kidney is a target organ for heavy metals. They accumulate in several segments of the nephron and cause profound alterations in morphology and function. Acute intoxication frequently causes acute renal failure. The effects of chronic exposure have not been fully disclosed. In recent years increasing awareness of the consequences of their presence in the kidney has evolved. In this review we focus on the alterations induced by heavy metals on the intercellular junctions of the kidney. We describe that in addition to the proximal tubule, which has been recognized as the main site of accumulation and injury, other segments of the nephron, such as glomeruli, vessels, and distal nephron, show also deleterious effects. We also emphasize the participation of oxidative stress as a relevant component of the renal damage induced by heavy metals and the beneficial effect that some antioxidant drugs, such as vitamin A (all-trans-retinoic acid) and vitamin E ( α -tocopherol), depict on the morphological and functional alterations induced by heavy metals.

Zona occludens-2 protects against podocyte dysfunction induced by ADR in mice.

Zona occludens-2 (ZO-2) is a protein present at the tight junction and nucleus of epithelial cells. ZO-2 represses the transcription of genes regulated by the Wnt/ß-catenin pathway. This pathway plays a critical role in podocyte injury and proteinuria. Here, we analyze whether the overexpression of ZO-2 in the glomerulus, by hydrodynamics transfection, prevents podocyte injury mediated by the Wnt/ß-catenin pathway in the mouse model of adriamycin (ADR) nephrosis. By immunofluorescence and immunogold electron microscopy, we show that ZO-2 is present in mice glomerulus, not at the slit diaphragms where nephrin concentrates, but in the cytoplasm and at processes of podocytes. Our results indicate that in the glomeruli of mice treated with ADR, ZO-2 overexpression increases the amount of phosphorylated ß-catenin, inhibits the expression of the transcription factor snail, prevents nephrin and podocalyxin loss, reduces podocyte effacement and massive fusions, restrains proteinuria, and supports urea and creatinine clearance. These results suggest that ZO-2 could be a new target for the regulation of hyperactive Wnt/ß-catenin signaling in proteinuric kidney diseases.

Effects of febuxostat on metabolic and renal alterations in rats with fructose-induced metabolic syndrome.

Increased fructose consumption is associated with hyperuricemia, metabolic syndrome, and renal damage. This study evaluated whether febuxostat (Fx), an investigational nonpurine, and selective xanthine oxidase inhibitor, could alleviate the features of metabolic syndrome as well as the renal hemodynamic alterations and afferent arteriolopathy induced by a high-fructose diet in rats. Two groups of rats were fed a high-fructose diet (60% fructose) for 8 wk, and two groups received a normal diet. For each diet, one group was treated with Fx (5-6 mg.kg(-1).day(-1) in the drinking water) during the last 4 wk (i.e., after the onset of metabolic syndrome), and the other received no treatment (placebo; P). Body weight was measured daily. Systolic blood pressure and fasting plasma uric acid (UA), insulin, and triglycerides were measured at baseline and at 4 and 8 wk. Renal hemodynamics and histomorphology were evaluated at the end of the study. A high-fructose diet was associated with hyperuricemia, hypertension, as well as increased plasma triglycerides and insulin. Compared with fructose+P, fructose+Fx rats showed significantly lowered blood pressure, UA, triglycerides, and insulin (P < 0.05 for all comparisons). Moreover, fructose+Fx rats had significantly reduced glomerular pressure, renal vasoconstriction, and afferent arteriolar area relative to fructose+P rats. Fx treatment in rats on a normal diet had no significant effects. In conclusion, normalization of plasma UA with Fx in rats with metabolic syndrome alleviated both metabolic and glomerular hemodynamic and morphological alterations. These results provide further evidence for a pathogenic role of hyperuricemia in fructose-mediated metabolic syndrome.

Chronic inhibition of NOS-2 ameliorates renal injury, as well as COX-2 and TGF-beta 1 overexpression in 5/6 nephrectomized rats.

BACKGROUND: Chronic renal damage is associated with inflammatory infiltration, fibrosis and vascular lesion, coupled with increased expression of cyclo-oxygenase 2 (COX-2) and transforming growth factor-beta1 (TGF-beta1). However, the role of inducible nitric oxide synthase (NOS-2) is still controversial. Thus, we studied the contribution of NOS-2 to the expression levels of COX-2 and TGF-beta1, as well as the structural renal injury in rats with subtotal renal ablation (5/6 Nx). METHODS: Four groups of rats were studied: sham, 5/6 Nx, 5/6 Nx+aminoguanidine (AG) and 5/6 NX+L-NIL (L-N6-iminoethyl-lysine). Systolic blood pressure (SBP), proteinuria and creatinine (Cr) clearance were measured. NOS-2, COX-2 and TGF-beta1 gene expression was determined by real-time reverse transcription-polymerase-chain reaction. Protein expression was evaluated by western blot and ELISA (TGF-beta1). Immunohistochemistry and morphometry were performed for NOS-2, microvascular thickening and fibrosis. RESULTS: Systemic hypertension and marked proteinuria, increased expression of NOS-2, COX-2 and TGF-beta1, thickening of arteriolar wall and tubulointerstitial fibrosis were produced in 5/6 Nx rats. Chronic inhibition of NOS-2 did not prevent arterial hypertension or the fall in Cr clearance, but partially reduced proteinuria. Nevertheless, AG and L-NIL preserved arteriolar morphology and the administration of both selective inhibitors of inducible NOS (AG and L-NIL) prevented NOS-2 overexpression. CONCLUSION: This study shows that NOS-2 was markedly enhanced in renal tissue of 5/6 Nx rats. Moreover, treatment with AG and L-NIL prevented the morpho-functional changes induced by subtotal renal ablation, despite persistence of systemic hypertension, suggesting that high concentrations of nitric oxide produced by NOS-2 could act as a positive modulator of the proinflammatory and profibrotic pathways involved in the progression of renal disease.