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Ernesto Sabath

Publications and source records attributed to Ernesto Sabath.

6 recordsLinked to original sources
PubMed2006

Cell-cell interactions in the kidney: inducible expression of mutant G protein alpha-subunits in Madin-Darby canine kidney cells for studies of epithelial cell junction structure and function.

Disrupted epithelial cell junctions are a hallmark of numerous disease processes. The signaling mechanisms regulating barrier function and re-establishment of intact junctions after injury and during development are complex and tightly regulated. We have shown that heterotrimeric G proteins regulate assembly and maintenance of epithelial cell barrier in cultured Madin-Darby canine kidney (MDCK) cells. The inducible expression of mutant signaling molecules (constitutively active or dominant negative) in polarized cells is a useful strategy for elucidating the role(s) for specific proteins. Using tetracycline-off inducible expression of wild-type and constitutively active Galpha12, we have demonstrated a fundamental role for Galpha12 in regulating the junction of MDCK cells. Inducible expression permits the comparison of the identical cell line in the presence and absence of the protein of interest and minimizes variation arising from distinct clones. The methods described here are applicable to virtually any protein that may regulate maintenance or assembly of the epithelial cell junctional complex.

Animals↗
PubMed2004

Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.

Alkalosis↗
PubMed2003

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4.

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a "mirror image" of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

Animals↗
PubMedDate not supplied

[Molecular physiopathology of Bartter's syndrome].

Bartter's syndrome is an autosomic recessive disease characterized by hypokalemic metabolic alkalosis accompanied with hypercalciuria, polyuria and hypotension due to volume depletion. The pathophysiology of this hereditary disease was largely unknown until the last few years in which inactivating mutations in up to five different genes have been shown to produce or be associated with the development of this syndrome. All the involved proteins are expressed either in the apical or basolateral membrane of the thick ascending limb of Henle's loop. These clinical and molecular findings have increased our understanding of the Bartter's disease and also of the thick ascending limb physiology.

Bartter Syndrome↗
PubMedDate not supplied

[Early referral to the nephrologist: impact on initial hospitalization and the first 6 months of continuous ambulatory peritoneal dialysis].

BACKGROUND: The prognosis and outcome in patients with end stage renal disease is significantly related to the predialysis care and early or late nephrological referral. Late nephrological referral has been associated with more hospital admissions and mortality at six months after the dialysis was initiated. We lack information about the role of early nephrological referral and its impact in biochemical variables and first hospitalization in our country. METHODS: The charts of all patients that were admitted to peritoneal dialysis in the period 01/1999-06/2001 were reviewed. We divided the population in group A with patients referred to nephrologist more than 3 months before the start of dialysis (early referral) and group B with those patients referred for a period shorter than three months or no referred before the start of dialysis (late referral or no referral). The patient's characteristics, laboratory values, characteristics of the first hospitalization and drugs were analyzed when dialysis was initiated and after six months of follow-up. RESULTS: Patients in group A were older and the percentage of patients with diabetes and hypertension was higher. The patients with early referral started dialysis with higher levels of hemoglobin (9.2 vs. 8.3 g/dL, p = 0.01) and with lower serum levels of BUN (91 vs. 122.5 mg/dL, p = 0.0001), creatinine (8.4 vs. 12.8 mg/dL, p = 0.0002) and phosphorus (6.4 vs. 7.7 mg/dL, p = 0.01). The length of the first hospitalization was shorter in patients of Group A (5.7 vs. 10.5 days, p = 0.004) and the emergency department was less used to peritoneal catheter placement in this group (46.3 vs. 86.9%, p = 0.01). There were not differences in hospitalization between the two groups after six months. CONCLUSION: Patients with early referral to a nephrologist show better biochemical variables, shorter first hospitalization length and higher percentage of elective placement of catheter.

Adult↗