Roberto Parrilla

Modification of kidney barrier function by the urokinase receptor

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

1H NMR detection of cerebral myo‐inositol

A previously unassigned group of prominent multiplets of the 360 MHz 1H NMR spectrum of acid stable metabolite extracts from rat brain is shown to arise from free myo‐inositol. This conclusion is derived from a systematic analysis of the high‐resolution 1H NMR spectra of brain acid extracts, in which appropriate conditions and optimal proton signals have been selected for the quantitative analysis of up to 15 metabolites. Developmental variations in the cerebral content of myo‐inositol could be readily detected using this approach, which provides a novel alternative to study myo‐inositol metabolism under physiological or pathological conditions.

Control of hepatic gluconeogenesis: role of fatty acid oxidation

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the Vmax even at the lowest (25 microM) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate]0.5 as a function of octanoate concentration. The latter occurs at low (less than 0.1 mM), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (greater than 0.5 mM), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

Enhanced Proliferation of Lymphoblasts from Patients with Alzheimer Dementia Associated with Calmodulin-Dependent Activation of the Na+/H+ Exchanger

We have recently reported that lymphoblasts from late onset Alzheimers disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.

Expression of podocalyxin enhances the adherence, migration, and intercellular communication of cells

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin(GFP) (PODXL(GFP)) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Delta(451)) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Delta(429)), by RGD peptides, or by inhibitors of the vitronectin receptor (alphavbeta3). CHO-PODXL(GFP) also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with beta1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking beta1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.

Cellular redistribution of metabolites during glucagon and insulin control of gluconeogenesis in the isolated perfused rat liver.

Abstract Livers isolated from fasted rats were perfused in a blood-free recirculating system using alanine (10 m m ) as the carbon source. Glucagon at a concentration of 2.1 × 10 −9 m enhanced gluconeogenesis, ureogenesis, and ketogenesis. The proportion of alanine utilized to glucose formed remained rather constant in all the situations studied, suggesting that the contribution of glycogen breakdown to the total glucose output was negligible. The glucagon stimulation of gluconeogenesis was accompanied by a decrease in the [ATP]/[ADP]ratio and a rise in the reduction state of the cytosolic and mitochondrial NAD systems. The calculation of the intracellular distribution of metabolites indicates that glucagon increases the intramitochondrial oxaloacetate concentration. This finding seems to support the hypothesis of pyruvate carboxylation, the first nonequilibrium enzymic step in the gluconeogenic sequence, as one of the main sites of glucagon action. The rise in the mitochondrial:cytosolic concentration gradient of malate suggests that glucagon may also act by facilitating the transfer of three-carbon units from the mitochondria to the cytosol. The fact that insulin reversed virtually all the glucagon-induced changes strongly suggests that both hormones act on common steps. It is remarkable that these insulin effects occur at glucagon/insulin ratios similar to those normally found in the portal vein of the intact animal.

Apolipoprotein E genotype in Spanish patients of Alzheimer's or Parkinson's disease

Blood donors of the Madrid area show a 6% frequency of apolipoprotein E genotype carrying allele epsilon 4. This frequency is smaller than other populations of Caucasian origin. This proportion decreases to 4% in a selected sample of healthy individuals of ages > 60 years. The frequency (34%) of the allele epsilon 4 was significantly increased in patients of late onset Alzheimers disease, similarly to other populations. An earlier age of onset of the dementia is observed in the patients of late-onset Alzheimers disease carrying the allele epsilon 4. No increased frequency in allele epsilon 4 frequency was found in patients of early-onset Alzheimers disease. Patients of Parkinsons disease do not show any differences in the frequency of the alleles of apolipoprotein E when compared with healthy individuals.

Podocalyxin enhances the adherence of cells to platelets

Abstract.Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.

The effect of starvation in the rat on metabolite concentrations in blood, liver and skeletal muscle

The effects of starvation on the concentration of blood and tissue metabolites was determined at different times in rats starved up to 6 days.The concentration of the hepatic gluconeogenic intermediates: pyruvate, phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate, decreased by 50% by the third day and then gradually increased to control levels by the sixth day of starvation. The change in the concentration of these metabolites correlates inversely with the plasma free fatty acid levels and the β-hydroxybutyrate: acetoacetate (B/A) ratios, suggesting that the increased generation of reducing equivalents from free fatty acid oxidation stimulate glyceraldehyde-phosphate dehydrogenase. However, this apparent stimulation does not appear to control glyconeogenic flux. The concentration of hepatic oxaloacetate increased gradually during starvation and correlated closely with the increase in hepatic gluconeogenic flux suggesting that the hepatic concentration of oxaloacetate may be important in controlling the rate of hepatic gluconeogenesis.The calculated equilibrium expression: [alanine] [α-ketoglutarate]/[pyruvate] [glutamate] and [aspartate] [α-ketoglutarate]/[oxdaloacetate] [glutamate] for alanine aminotransferase and aspartate amino transferase respectively, remained fairly constant in all the tissues studied throughout the starvation period, despite large changes in the concentrations of the individual reactants, indicating that these reactions are near equilibrium.The β-hydroxybutyrate: acetoacetate (B/A) ratios, taken to reflect the mitochondrial redox state, increased more than 4-fold in liver and muscle after the first day of starvation. It decreased after the third day of starvation and returned close to prefasting levels by the sixth day. The change in B/A ratios in blood was similar to those observed in the organs, except that the magnitude of the change was less. The changes observed in the mitochondrial redox state during starvation correlate closely with the changes in plasma free fatty acid concentrations and with the rate of fatty acid oxidation.