Gabriela Coux

Renal function and cortical (Na(+)+K(+))-ATPase activity, abundance and distribution after ischaemia-reperfusion in rats.

The effects of ischaemic injury and reperfusion on renal function, cortical ATP content, alkaline phosphatase activity and (Na(+)+K(+))-ATPase activity and abundance in cortical homogenates and isolated basolateral and apical membranes were examined. Rats were submitted to 5 or 40 min of right renal artery occlusion and 60 min of reperfusion. Renal function of the ischaemic-reperfused kidney was studied by conventional clearance techniques. Our results show that 1 h of reperfusion after a short period of renal ischaemia (5 min) allows the complete restoration of the biochemical features of cortical cells and functional properties of the injured kidney. A longer period of ischaemia, such as 40 min, followed by 1 h of reperfusion showed functional and biochemical alterations. ATP recovered from 26% after 40 min of ischaemia to 50% of control values after 1 h reperfusion. However, renal function was strongly impaired. Brush border integrity was compromised, as suggested by AP excretion and actin appearance in urine. Although total cortical (Na(+)+K(+))-ATPase activity was not different from controls, its distribution in isolated apical and basolateral membranes was abnormal. Remarkably, we detected an increase in alpha-subunit protein abundance that may suggest that (Na(+)+K(+))-ATPase synthesis is promoted by ischaemia-reperfusion. This increase may play an important role in the pathophysiology of ischaemic acute renal failure.

Cortical Na+,K+-ATPase activity, abundance and distribution after in vivo renal ischemia without reperfusion in rats.

The aim of our work was to study the changes in activity, abundance and distribution of sodium, potassium-adenosine triphosphatase (Na⁺,K⁺-ATPase) in membranes of cortical tubular cells in an in vivo model of ischemic injury without reperfusion. Na⁺,K⁺-ATPase, alkaline phosphatase (AP) activities and their distribution in membranes isolated from renal cortex using a Percoll gradient were studied after different ischemic periods. Na⁺,K⁺-ATPase α-subunit protein abundance was analysed by Western-blot. Plasma urea and cortical adenosine 5’triphosphate (ATP) were also measured. In cortical homogenates 5 min of ischemia promoted a diminution in ATP content. Na⁺,K⁺-ATPase activity diminished after 40 min and AP after 100 min of ischemia. Na⁺,K⁺-ATPase activity in the Percoll gradient fractions after 5 min peaked at a higher density and was significantly decreased after 40 min. AP activity was decreased in typically enriched apical membranes after both times of ischemia. At each time studied Na⁺,K⁺-ATPase abundance was increased in cortical homogenates and membranes. Our results showed opposite effects of ischemia on Na⁺,K⁺-ATPase activity and abundance. Increased levels of Na⁺,K⁺-ATPase protein were observed. The enzyme would be rapidly delivered to membrane domains and become inactivated as ischemia persists.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patients fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.

The integrin binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (amphibia) oocytes.

Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

Amphibian Oocytes Release Heat Shock Protein A During Spawning: Evidence for a Role in Fertilization

ABSTRACT Heat shock proteins A (HSPAs, previously known as HSP70s) are widely distributed proteins originally linked with heat shock but now associated with several normal cellular functions. We recently found indirect evidence suggesting a role for HSPAs in sperm-oocyte interaction in the amphibian Bufo arenarum. In the present study our aim was to study its expression, subcellular distribution, and role during fertilization. By Western blot analysis using two different antibodies we detected HSPAs present in B. arenarum oocytes in the absence of any stress. We performed two-dimensional electrophoresis and detected two isoforms with isoelectric points of 5.25 and 5.45. We studied its subcellular distribution isolating total membranes, cytosol, and plasma membranes. HSPAs were present in all of these fractions. We confirmed these results by immunofluorescence microscopy and also found that the HSPA signal was present in the vitelline envelope. To further test this, we performed Western blot analysis in isolated vitelline envelopes and in egg water (diffusible material from deposited oocytes). HSPAs were present in these two fractions. Moreover, human recombinant his-tagged HSPA (HSPA1A) was able to specifically bind to sperm in vitro (midpiece) and enhance sperm membrane integrity. In vitro fertilization assays in the presence of anti-HSPA polyclonal antibodies showed diminished fertilization scores at low sperm concentrations (105 cells per milliliter). Our results suggest that HSPAs are present in intracellular and extracellular structures of nonstressed B. arenarum oocytes and participates in fertilization by and that their release during spawning plays a role in sperm membrane integrity.

Cnbp ameliorates Treacher Collins Syndrome craniofacial anomalies through a pathway that involves redox-responsive genes.

Treacher Collins Syndrome (TCS) is a rare congenital disease (1:50 000 live births) characterized by craniofacial defects, including hypoplasia of facial bones, cleft palate and palpebral fissures. Over 90% of the cases are due to mutations in the TCOF1 gene, which codifies the nucleolar protein Treacle. Here we report a novel TCS-like zebrafish model displaying features that fully recapitulate the spectrum of craniofacial abnormalities observed in patients. As it was reported for a Tcof1+/− mouse model, Treacle depletion in zebrafish caused reduced rRNA transcription, stabilization of Tp53 and increased cell death in the cephalic region. An increase of ROS along with the overexpression of redox-responsive genes was detected; furthermore, treatment with antioxidants ameliorated the phenotypic defects of craniofacial anomalies in TCS-like larvae. On the other hand, Treacle depletion led to a lowering in the abundance of Cnbp, a protein required for proper craniofacial development. Tcof1 knockdown in transgenic zebrafish overexpressing cnbp resulted in barely affected craniofacial cartilage development, reinforcing the notion that Cnbp has a role in the pathogenesis of TCS. The cnbp overexpression rescued the TCS phenotype in a dose-dependent manner by a ROS-cytoprotective action that prevented the redox-responsive genes’ upregulation but did not normalize the synthesis of rRNAs. Finally, a positive correlation between the expression of CNBP and TCOF1 in mesenchymal cells from both control and TCS subjects was found. Based on this, we suggest CNBP as an additional target for new alternative therapeutic treatments to reduce craniofacial defects not only in TCS but also in other neurocristopathies.

Ischaemia/reperfusion in rat renal cortex: vesicle leakiness and Na+, K+-ATPase activity in membrane preparations

BACKGROUND Despite the central role of Na(+), K(+)-ATPase (NKA) in ischaemic renal injury (IRI), cortical NKA activity values during renal ischaemia remain controversial. In this study, we explore why cortical NKA activity shows such behaviour during ischaemia in rats. METHODS Ischaemia was induced by unilateral renal artery clamping (40 min, I) followed or not by reperfusion (60 min, IR). NKA alpha- and beta-subunit abundance was analysed by western blot. We studied the NKA detergent sodium dodecyl sulphate (SDS) enzymatic activation in isolated membrane preparations from control and ischaemic kidneys. RESULTS NKA activity was diminished in I cortical homogenates (C = 9.3 +/- 1.1, I = 4.7 +/- 1.1* micromol Pi/h mg Prot, n = 4-6, *P < 0.05 versus C). This was rapidly recovered after reperfusion (IR = 9.9 +/- 1.2 micromol Pi/h mg Prot). alpha-subunit levels were increased, while beta-subunit was unchanged. At SDS 0.9 mg/ml (maximal detergent activation), the activities were indistinguishable (C = 90.5 +/- 2.2, I = 91.4 +/- 15.1 micromol Pi/h mg Prot). The analysis of detergent activation of NKA activity is widely used to estimate membrane leakiness in plasma membrane preparations. Our results suggest a higher population of sealed impermeable vesicles in preparations from ischaemic renal tissue. CONCLUSION The well-known effect of ischaemia on renal cell cytoskeleton could explain the observed changes in the leakiness of membrane vesicles.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs.

Recently, we have provided evidence for the involvement of a cytosolic tyrosine-phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.