Access to Grafts in a Liver Transplant Center: Does It Rely on the Severity of the Waiting List Population?

BACKGROUND: The number of transplants performed relies, partially, on recipients' variables on the waiting list. The goal of this study was to compare recipients from a high-volume liver center in Argentina with the rest of the country. METHODS: This study was a retrospective analysis of liver transplant recipients nationally between January 2013 and April 2017. It involved extracting data from the open database CRESI-SINTRA (the Argentinian database of the National Procurement Organization, an equivalent to the United Network for Organ Sharing); expressing results by percentages, medians, and interquartile ranges (IQRs); and comparing the national population with the population transplanted at Hospital El Cruce (HEC). The Mann-Whitney U test was used for analysis. RESULTS: Nationally, 1434 liver transplants were performed. A total of 177 (12.34%) were emergency status; 811 (56.6%) were by the Model for End-Stage Liver Disease (MELD) (n = 759)/PELD (Pediatric End-Stage Liver Disease) (n = 52), with a median graft assignment position of 5 (IQR, 3-10) in 57.2 days (IQR, 11-217). Median MELD access was 29 points (IQR, 24-33). A total of 446 (31.1%) had MELD exceptions; 249 (55.8%) of these were due to Milan hepatocellular carcinoma. At the HEC, 167 liver transplantations were performed; 26 (15.6%) were emergency status and 97 (58.1%) by MELD (none PELD). Their median graft assignment position was 4 (IQR, 4-16) in 19.1 days (IQR, 4-90); median MELD access was 28 points (IQR, 24-31). Forty-five patients (26.9%) had MELD exceptions; 31 (68.9%) were due to hepatocellular carcinoma. CONCLUSIONS: Our center has a larger proportion of recipients transplanted by emergency status and MELD, similar MELD access, and less waiting list time, reflecting our wide policy of liver graft acceptance.

Controversies in nephrology: response to 'renal albumin handling, facts, and artifacts'.

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.

Two-photon microscopy: visualization of kidney dynamics.

The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.

The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.

Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion.

Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK(10) cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK(10) cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogeneous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK(10) cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells.

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK(1) cells.

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK(1)) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for approximately 20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.

The homeodomain coordinates nuclear entry of the Lhx3 neuroendocrine transcription factor and association with the nuclear matrix.

LIM homeodomain transcription factors regulate development in complex organisms. To characterize the molecular signals required for the nuclear localization of these proteins, we examined the Lhx3 factor. Lhx3 is essential for pituitary organogenesis and motor neuron specification. By using functional fluorescent derivatives, we demonstrate that Lhx3 is found in both the nucleoplasm and nuclear matrix. Three nuclear localization signals were mapped within the homeodomain, and one was located in the carboxyl terminus. The homeodomain also serves as the nuclear matrix targeting sequence. No individual signal is alone required for nuclear localization of Lhx3; the signals work in combinatorial fashion. Specific combinations of these signals transferred nuclear localization to cytoplasmic proteins. Mutation of nuclear localization signals within the homeodomain inhibited Lhx3 transcriptional function. By contrast, mutation of the carboxyl-terminal signal activated Lhx3, indicating that this region is critical to transcriptional activity and may be a target of regulatory pathways. The pattern of conservation of the nuclear localization and nuclear matrix targeting signals suggests that the LIM homeodomain factors use similar mechanisms for subcellular localization. Furthermore, upon nuclear entry, association of Lhx3 with the nuclear matrix may contribute to LIM homeodomain factor interaction with other classes of transcription factors.

Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations.

Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.

[Mortality among the workers of the Instituto Mexicano del Seguro Social from 1983 to 1987]. / La mortalidad de los trabajadores del Instituto Mexicano del Seguro Social de 1983 a 1987.

A study on mortality of 2,268 workers of the Mexican Social Security Institute was done during 1983-1987 in order to obtain accurate information to specifically determine to those, activities which promote the health and improve the life conditions of the workers of the aforementioned institution. This information relates to the first step of the study, so it doesn't lead us to value judgements, because it would fall into speculations. However, it creates an important data to subsequently explore, by means of further studies, in the casualty of the existing results.