Urinary TCP1-eta: A Cortical Damage Marker for the Pathophysiological Diagnosis and Prognosis of Acute Kidney Injury.

Acute kidney injury (AKI) is a serious syndrome with increasing incidence and health consequences, and high mortality rate among critically ill patients. Acute kidney injury lacks a unified definition, has ambiguous semantic boundaries, and relies on defective diagnosis. This, in part, is due to the absence of biomarkers substratifying AKI patients into pathophysiological categories based on which prognosis can be assigned and clinical treatment differentiated. For instance, AKI involving acute tubular necrosis (ATN) is expected to have a worse prognosis than prerenal, purely hemodynamic AKI. However, no biomarker has been unambiguously associated with tubular cell death or is able to provide etiological distinction. We used a cell-based system to identify TCP1-eta in the culture medium as a noninvasive marker of damaged renal tubular cells. In rat models of AKI, TCP1-eta was increased in the urine co-relating with renal cortical tubule damage. When kidneys from ATN rats were perfused in situ with Krebs-dextran solution, a portion of the urinary TCP1-eta protein content excreted into urine disappeared, and another portion remained within the urine. These results indicated that TCP1-eta was secreted by tubule cells and was not fully reabsorbed by the damaged tubules, both effects contributing to the increased urinary excretion. Urinary TCP1-eta is found in many etiologically heterogeneous AKI patients, and is statistically higher in patients partially recovered from severe AKI. In conclusion, urinary TCP1-eta poses a potential, substratifying biomarker of renal cortical damage associated with bad prognosis.

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: role of the Akt/mTOR survival pathway and Bcl-2 family proteins.

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.

Aquaporins, anti-aquaporin-4 autoantibodies and neuromyelitis optica.

The classification, distribution and functions of the different molecules of aquaporins (AQPs), including aquaporins, aquaglyceroporins and superaquaporins are reviewed together with their potential diagnostic and therapeutic uses. We analyzed the pathogenic importance of anti-AQP4 autoantibodies in neuromyelitis optica and related syndromes, as well as their diagnostic and predictive potential, prognosis, and monitoring of the disease. Finally, the analytical methods and current recommendations for testing anti-AQP4 autoantibodies in clinical practice are described.

Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines.

Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.

Urinary levels of regenerating islet-derived protein III ß and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats.

A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicin's nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine.

Biobancos, laboratorios clínicos e investigación biomédica / Biobanks, clinical laboratorios and biomedical research

Los biobancos son instituciones públicas o privadas sin ánimo de lucro dedicadas a la recogida, el procesamiento, el almacenamiento y la distribución de especímenes biológicos humanos, junto a los datos asociados con esas muestras. El Instituto de Salud Carlos III ha creado y financiado una red de biobancos hospitalarios cuya finalidad es el almacenamiento de muestras para investigación. Esta red de biobancos pretende integrarse en la Red Europea de Infraestructuras en Biobancos y Recursos Moleculares (BBMR), cuyo objetivo es favorecer la realización de estudios en gran escala. Se revisan los principales aspectos, tanto organizativos, como operativos de los biobancos y su relación con los laboratorios clínicos y la investigación biomédica (AU)

Biobanks are non-profit public or private institutions for the collection, processing, storage and distribution of human biological specimens, linked to associated data of those samples. The Instituto de Salud Carlos III has set up and funded a hospital biobank network focused on the storage of samples for research. This network is expected to integrate into the Biobanking and Biomolecular Resources Research Infrastructure (BBMR) whose objective is to favour large-scale studies. The main organisational and operational aspects of biobanks are reviewed as well as their relationship with clinical laboratories and biomedical research (AU)

Sub-nephrotoxic doses of gentamicin predispose animals to developing acute kidney injury and to excrete ganglioside M2 activator protein.

We studied whether nephrotoxic drug administration sensitizes to acute renal failure (ARF) by administering a sub-nephrotoxic dose of gentamicin. This pre-treatment sensitized animals with no sign of renal injury to develop ARF when exposed to a second potential nephrotoxic drug, also given at sub-nephrotoxic doses that would be otherwise harmless to non-sensitized animals. We identified urinary ganglioside M2 activator protein (GM2AP) as a biomarker of an enhanced sensitivity to suffer ARF following sub-nephrotoxic treatment with gentamicin. Sub-nephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by reverse transcriptase-PCR, western blot, and immunostaining, nor was its serum level modified. The origin of increased GM2AP in the urine is thought to be a defective tubular handling of this protein as a consequence of gentamicin action. Hence, markers of acquired sensitivity may improve the prevention of ARF by enhancing our capacity to monitor for this condition, in a preemptive manner.

Role of vitamin C transporters and biliverdin reductase in the dual pro-oxidant and anti-oxidant effect of biliary compounds on the placental-fetal unit in cholestasis during pregnancy.

Maternal cholestasis causes oxidative damage to the placental-fetal unit that may challenge the outcome of pregnancy. This has been associated with the accumulation of biliary compounds able to induce oxidative stress. However, other cholephilic compounds such as ursodeoxycholic acid (UDCA) and bilirubin have direct anti-oxidant properties. In the present study we investigated whether these compounds exert a protective effect on cholestasis-induced oxidative stress in placenta as compared to maternal and fetal livers, and whether this is due in part to the activation of anti-oxidant mechanisms involving vitamin C uptake and biliverdin/bilirubin recycling. In human placenta (JAr) and liver (HepG2) cells, deoxycholic acid (DCA) similar rates of free radical generation. In JAr (not HepG2), the mitochondrial membrane potential and cell viability were impaired by low DCA concentrations; this was partly prevented by bilirubin and UDCA. In HepG2, taurocholic acid (TCA) and UDCA up-regulated biliverdin-IX alpha reductase (BVR alpha) and the vitamin C transporter SVCT2 (not SVCT1), whereas bilirubin up-regulated both SVCT1 and SVCT2. In JAr, TCA and UDCA up-regulated BVR alpha, SVCT1 and SVCT2, whereas bilirubin up-regulated only SVCT2. A differential response to these compounds of nuclear receptor expression (SXR, CAR, FXR and SHP) was found in both cell types. When cholestasis was induced in pregnant rats, BVR alpha, SVCT1 and SVCT2 expression in maternal and fetal livers was stimulated, and this was further enhanced by UDCA treatment. In placenta, only BVR alpha was up-regulated. In conclusion, bilirubin accumulation and UDCA administration may directly and indirectly protect the placental-fetal unit from maternal cholestasis-induced oxidative stress.

Multiple protective effects of melatonin against maternal cholestasis-induced oxidative stress and apoptosis in the rat fetal liver-placenta-maternal liver trio.

Maternal cholestasis is usually a benign condition for the mother but induces profound placental damage and may be lethal for the fetus. The aim of this study was to investigate the protective effects in rat maternal and fetal livers as also the placenta of melatonin or silymarin against the oxidative stress and apoptosis induced by maternal obstructive cholestasis during the last third of pregnancy (OCP). Melatonin or silymarin administration (i.e. 5 mg/100 g bw/day after ligation of the maternal common bile duct on day 14 of pregnancy) reduced OCP-induced lipid peroxidation, and prevented decreases in total glutathione levels. However, the protective effect on OCP-induced impairment in the GSH/GSSG ratio was mild in the placenta and fetal liver, while absent in maternal liver. Melatonin or silymarin also reduced OCP-induced signs of apoptosis (increased caspase-3 activity and Bax-alpha upregulation) in all the organs assayed. Moreover, melatonin (but not silymarin) upregulated several proteins involved in the cellular protection against the oxidative stress in rats with OCP. These included, biliverdin-IX alpha reductase and the sodium-dependent vitamin C transport proteins SVCT1 and SVCT2, whose expression levels were enhanced in maternal and fetal liver by melatonin treatment. In contrast, in placenta only biliverdin-IX alpha reductase and SVCT2 were upregulated. These results indicate that whereas the treatment of cholestatic pregnant rats with melatonin or silymarin affords a direct protective antioxidant activity, only melatonin has dual beneficial effects against OCP-induced oxidative challenge in that it stimulates the expression of some components of the endogenous cellular antioxidant defense.

Urinary proteomics.

Protein measurement in urine has been used for many years for the diagnosis and monitoring of renal disease. The pattern of urinary protein excretion can be used to identify the cause of the disease and to classify proteinuria. In recent years, proteomics has proven to be a powerful tool in investigation and clinical medicine. Proteomics employs a protein separation method and the identification of proteins using mass spectrometry. One of the objectives of clinical proteomics is the identification of biological markers of disease. To accomplish this, it is necessary to have a normal proteome of the medium in question, which in our case is urine. Comparison of the normal urinary proteome with the urinary proteome from patients with a defined disease can detect proteins expressed differentially from one another. The aim of this review is to present the situation of urinary proteomics, putting special emphasis on its application in the diagnosis of glomerular diseases, renal allograft rejection, urological cancers and urolithiasis.

Multiplexed testing in the autoimmunity laboratory.

A variety of technologies for autoantibody profiling have been developed. The main techniques are line-blot immunoassays, bar-coded nanoparticle immunoassays, and bead-based assays with flow cytometry detection and antigen microarrays. Some of these technologies are only able to measure a limited number of autoantibodies, while others can detect elevated numbers. Assays for antinuclear antibody specificities using line-blot immunoassays and bead-based assays with flow cytometry detection are already commercialised. Antigen microarrays for autoantibody measurement are only in the development phase, although in the not too distant future these assays will probably appear on the market. Multiplexed testing in the autoimmunity laboratory appears to have a promising future, since this technique permits a reduction in analytical time, with a shorter turnaround time.

Maternal ethanol consumption during pregnancy enhances bile acid-induced oxidative stress and apoptosis in fetal rat liver.

Ethanol is able to cross the placenta, which may cause teratogenicity. Here we investigated whether ethanol consumption during pregnancy (ECDP), even at doses unable to cause malformation, might increase the susceptibility of fetal rat liver to oxidative insults. Since cholestasis is a common condition in alcoholic liver disease and pregnancy, exposure to glycochenodeoxycholic acid (GCDCA) has been used here as the oxidative insult. The mothers received drinking water without or with ethanol from 4 weeks before mating until term, when placenta, maternal liver, and fetal liver were used. Ethanol induced a decreased GSH/GSSG ratio in these organs, together with enhanced gamma-glutamylcysteine synthetase and glutathione reductase activities in both placenta and fetal liver. Lipid peroxidation in placenta and fetal liver was enhanced by ethanol, although it had no effect on caspase-3 activity. Although the basal production of reactive oxygen species (ROS) was higher by fetal (FHs) than by maternal (AHs) hepatocytes in short-term cultures, the production of ROS in response to the presence of varying GCDCA concentrations was higher in AHs and was further increased by ECDP, which was associated to a more marked impairment in mitochondrial function. Moreover, GCDCA-induced apoptosis was increased by ECDP, as revealed by enhanced Bax-alpha/Bcl-2 ratio (both in AHs and FHs) and the activity of caspase-8 (only in AHs) and caspase-3. In sum, our results indicate that although AHs are more prone than FHs to producing ROS, at doses unable to cause maternal liver damage ethanol consumption causes oxidative stress and apoptosis in fetal liver.

Present and future of the autoimmunity laboratory.

At present, autoimmunity laboratories are very dynamic owing to the constant and increasing availability of new tests, mainly due to the detection of new autoantibodies. The main characteristic of the autoimmunity laboratory and the one that differentiates it from other laboratories that use immunoassays as basic techniques is that it determines antibodies (autoantibodies) and not antigens. For this reason, immunoassay techniques must employ antigens as reagents. Indirect immunofluorescence has and continues to be a basic technique in autoimmunity studies. However, over the last few years, a significant trend at autoimmunity laboratories has been the gradual replacement of immunofluorescence microscopy by immunoassay. Of the several different forms of immunoassay, the enzyme-linked immunosorbent assay (ELISA) format is the one most used in autoimmunity laboratories. Recombinant DNA technology has allowed the production of large quantities of antigens for autoantibody analysis. Flow cytometry for the analysis of microsphere-based immunoassays allows the simultaneous measurement of several autoantibodies. Likewise, autoantigen microarrays provide a practical means to analyse biological fluids in the search for a high number of autoantibodies. We are now at the beginning of an era of multiplexed analysis, with a high capacity of autoantibody specificities. Future trends in this field include immunoassays with greater analytical sensitivity, simultaneous multiplexed capability, the use of protein microarrays, and the use of other technologies such as microfluidics.

Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid.

BACKGROUND/AIMS: The sensitivity of fetal rat liver to maternal obstructive cholestasis during pregnancy (OCP), and the effect of ursodeoxycholic acid (UDCA) were investigated. METHODS: UDCA was administered (i.g. 0.6 mg/kg b.wt./day) from day 14 to day 21 of pregnancy after maternal common bile duct ligation. RESULTS: Impairment in the activity of antioxidant enzymes, levels of total glutathione and GSH/GSSG ratio and the degrees of lipid peroxidation and protein carbonylation were similar in livers of OCP mothers and fetuses at term, despite hypercholanemia was milder in fetuses. Treatment of OCP rats with UDCA reduced maternal and fetal liver oxidative stress. Although maternal hypercholanemia was not corrected, fetal serum concentrations of major bile acids (except UDCA and beta-muricholic acid) were reduced. Fetal liver expression of key enzyme in bile acid synthesis, Cyp7a1, Cyp27 and Cyp8b1 was not affected by OCP or UDCA treatment. In OCP fetal livers, the relative expression of Bax-alpha and Bcl-2 and the activity of caspase-3, but not caspase-8, were increased. These changes were markedly reduced in fetuses of OCP animals treated with UDCA. CONCLUSIONS: OCP induced moderate fetal hypercholanemia but marked liver oxidative stress and apoptosis that were partly prevented by treatment of pregnant rats with UDCA.

Laboratory screening of connective tissue diseases by a new automated ENA screening assay (EliA Symphony) in clinically defined patients.

BACKGROUND: The measurement of antinuclear antibodies (ANA) is used in the autoimmune laboratory for the screening of connective tissue diseases (CTD). ANA measurements are mainly performed by indirect immunofluorescence (IIF) on HEp-2 cells or by enzyme immunoassay (EIA). The objective of this study was to clinically evaluate an automated EIA for extractable nuclear antigens (ENA) which lacks anti-dsDNA for the screening of CTD. METHODS: The study involved a total of 170 serum samples, 54 from patients with CTD, 26 from patients with other autoimmune diseases, and 90 from patients with non-autoimmune diseases. For all sera, ANA detection was performed by IIF and by EliA Symphony (Pharmacia Diagnostics, Freiburg, Germany), an ENA screening which detects the following autoantibodies: SSA/Ro, SSB/La, U1RNP (70 kDa, A, C), Scl-70, JO-1, centromere B and Sm. Also, anti-dsDNA (EliA dsDNA, Pharmacia Diagnostics, Freiburg, Germany) was measured on all samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), efficiency, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were calculated. RESULTS: Diagnostic efficiency was similar for IIF (82.6%) and EliA Symphony (82.3%), as well as PLR (6.5 for IIF, and 7.3 for Eli Symphony), and NLR (0.35 for IIF, and 0.41 for EliA Symphony). The combined measurement of EliA Symphony and dsDNA increased sensitivity but not PLR. Area under receiver operator characteristic (ROC) curve was similar for IIF (0.847) and EliA Symphony (0.823). CONCLUSIONS: The results of the study demonstrate that EliA Symphony solely or combined with anti-dsDNA detection has an efficiency similar to HEp-2 cells IIF with a cut-off of 1:160 for the diagnosis of CTD.

Anti-cyclic citrullinated peptide autoantibodies in IgM rheumatoid factor-positive patients.

BACKGROUND: Antibodies to citrullinated proteins have been described in patients with RA and these appear to be the most specific markers of the disease. The objective of this study was to analyse the improvement in diagnostic accuracy of anti-cyclic citrullinated peptide autoantibodies and IgA rheumatoid factor in patients with clinical suspicion of RA and who were IgM rheumatoid factor-positive. Anti-CCP antibodies were measured with three different second-generation enzyme immunoassays. METHODS: We chose 133 serum samples with IgM RF levels greater than 20 IU/mL sent to our Laboratory from Specialized Care Units. Subsequently, patients were classified according to their clinical records. Eighty-seven had rheumatoid arthritis and 46 had other diseases. In all samples anti-CCP and IgA RF were measured by the corresponding ELISAs. RESULTS: Comparison of the three anti-CCP second-generation ELISAs revealed differences between them. Likewise, clinical performances in terms of sensitivity, specificity, and positive and negative likelihood ratios were different. In patients with IgM RF higher than 20 IU/mL, anti-CCP antibodies increased the clinical efficiency of IgM RF and offered better performance as compared with IgA RF. CONCLUSIONS: The use of anti-CCP antibodies affords good clinical efficiency and modifies the pre-test probability of the occurrence of RA in patients with IgM rheumatoid factor higher than 20 IU/mL.