Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques.

We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 µm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.

Albumin upregulates eNOS mRNA through ETRA/B in human proximal tubular epithelial cells.

BACKGROUND: Proximal tubular cells respond to proteinuria by expressing several cytokines and inflammatory molecules that induce interstitial fibrosis. Increased attention has been drawn toward the systems of endothelin (ET) and nitric oxide (NO). This work contributes to the elucidation of the interplay between these two systems in proximal tubular epithelial cells (PTECs) after exposure in proteinuric conditions. METHODS: HK-2 cells, a human PTEC line, were incubated with albumin, simulating proteinuric conditions. Cells were then lysed and either total RNA was isolated or whole cell extracts were prepared. PreproET-1, ET receptors (ETRA and ETRB) and NO synthases (eNOS, iNOS) mRNA accumulation was estimated by RT-PCR, and proteins by Western blot analysis. NO production was assessed using Griess reaction. Furthermore, we treated HK-2 cells with NO donor sodium nitroprusside, NO inhibitor L-NAME, ETRA inhibitor BQ123, ETRB inhibitor BQ788 and purified ET-1, and investigated the potential interplay between albumin-induced stimulation of NO or ET-1 systems. RESULTS: We found that albumin upregulates preproET-1, ETRA, ETRB, eNOS and iNOS mRNA as well as protein and stimulates NO production. Additionally, we recorded an ETRA/B dependent regulation of albumin-induced eNOS expression. CONCLUSIONS: For the first time an in vitro albumin-induced ET-1 and NO interplay was revealed.

Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17.

RATIONALE: The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. OBJECTIVE: Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. METHODS AND RESULTS: Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. CONCLUSIONS: Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.

PKB/Akt partners with Dab2 in albumin endocytosis.

Albumin in the glomerular filtrate is normally retrieved by concerted efforts of clathrin, LDL-type receptor megalin- and clathrin-associated sorting proteins. In glomerular diseases, albumin overload triggers a proapoptotic and inflammatory response contributing to tubulointerstitial fibrosis and tubular atrophy. The relationship between albumin overload-induced proximal tubule injury and albumin endocytosis remains to be discovered. We investigated presence of a possible overlap between endocytosis and cell survival. We showed a novel interaction between prosurvival protein, protein kinase B (PKB/Akt), and adaptor protein, disabled 2 (Dab2), with coimmunoprecipitation. Further delineation of this interaction by GST pull-down experiments utilizing different Dab2 constructs identified proline-rich domain as the interacting partner. Expression of Dab2 and PKB/Akt was downregulated at high concentrations of albumin associated with apoptosis. We then examined the physiological relevance of this interaction with functional studies. Overexpression of PKB/Akt increased albumin uptake in human proximal tubule cells. Conversely, inhibition of PKB/Akt with a nonselective Akt/PKB signaling inhibitor-2 and a dominant negative construct of PKB/Akt resulted in a decrease in albumin uptake. Inhibition of Dab2 by silencing RNA abolished PKB/Akt-induced albumin uptake demonstrating the physiological importance of this novel interaction. We concluded that PKB/Akt is part of an endocytic machinery and it mediates albumin uptake through its interaction with Dab2. The role that PKB/Akt plays in the endocytic cascade may dictate its decreased expression in proteinuric states in an attempt to limit albumin endocytosis that may tilt the balance between cell survival and apoptosis toward cell death.

Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1.

Activated neutrophils interacting with the vessel wall can alter vascular permeability to macromolecules such as albumin via release of various secretion products that induce changes in the endothelial monolayer. In the current work we used cremaster microvessels of anesthetized mice to show that, in addition to this paracrine mechanism, leukocyte ligation of endothelial ICAM-1 directly activates endothelial cell (EC) signaling, altering EC permeability to albumin [i.e., solute permeability (P(s))]. We show that antibody cross-linking of surface ICAM-1 in intact microvessels is sufficient to increase P(s) even in the absence of interacting leukocytes. Unstimulated arterioles do not support leukocyte-EC interactions, but despite this, antibody ligation of ICAM-1 in these vessels induced a twofold increase in P(s). Similarly, in venules that were depleted of interacting neutrophils, P(s) was decreased to below resting levels and was restored by ligation of ICAM-1. Use of function-blocking antibodies to separately block leukocyte rolling or adhesion under unstimulated or TNF-α-activated conditions established that both rolling and adhered leukocytes contribute to P(s) regulation in situ. Both rolling and adhesion activated EC-dependent signaling mechanisms that increased P(s). ICAM-1 ligation with primary antibody alone or primary followed by secondary antibodies showed that regulation of P(s) is directly dependent on the degree of ICAM-1 clustering. Under physiological versus inflamed conditions, respectively, this ICAM-1 clustering-dependent regulation of P(s) switches from PKC dependent and Src independent to Src dependent and PKC independent. This study thus identifies a new mechanism by which antiadhesion treatment may constitute a potential therapy for tissue edema.

[Resident cardiac stem cells].

The search for sources of stem/progenitor cells the use of which has a potential to affect course of ischemic heart disease and chronic heart failure is conducted nowadays in many countries. Resident cardiac stem cells (CSC) were revealed during recent years on the basis of expression of c-kit, sca-1, MDR1, and islet-1 markers. In vitro experiments demonstrated possibility of their differentiation into cardiomyocytes, smooth muscle cell and endothelial cells. Introduction of CSC in injured myocardium in animals facilitated its partial repair and short term improvement of cardiac function. This holds promise for the use of these cells in the future. In the review we have attempted to summarize literature data on resident CSC and their application for the treatment of heart diseases.

Structural and mutagenic approach to create human serum albumin-based oxygen carrier and photosensitizer.

SUMMARY: Human serum albumin (HSA) is a versatile protein found at high concentration in blood plasma and binds a range of insoluble endogenous and exogenous compounds. We have shown that complexation of functional molecules into HSA creates unique proteins never seen in nature. Complexing an iron-protoporphyrin IX into a genetically engineered heme pocket of recombinant HSA (rHSA) generates an artificial hemoprotein, which binds O(2) reversibly in much the same way as hemoglobin. A pair of site-specific mutations, (i) introduction of a proximal histidine at the Ile-142 position and (ii) substitution of Tyr-161 with Phe or Leu, allows the heme to bind O(2). Additional modification on the distal side of the heme pocket provides rHSA(triple mutant)-heme complexes with a variety of O(2) binding affinity. Complexing a carboxy-C(60)-fullerene (CF) into HSA generates a protein photosensitizer for photodynamic cancer therapy. Energy transfer occurs from a photoexcited triplet-state of HSA-CF (HSA-(3)CF(*)) to O(2), forming singlet oxygen ((1)O(2)). This protein does not show dark cytotoxicity, but induceds cell death under visible light irradiation.

Getting to the meat of the matter: beyond protein supplementation in maintenance dialysis.

Until recently, patients on dialysis with low serum albumin levels were characterized as suffering from protein malnutrition suggesting that the cause of this malady was due to an inadequate intake of protein. In fact, these patients tend to suffer from a wasting syndrome similar to cachexia commonly associated with inflammation in which there is loss of lean body mass and fat mass is underutilized. The term protein energy wasting has been used to characterize this syndrome and suggests that the simple addition of protein supplements to the dietary regimen of hemodialysis patients will not cure this malady. Correction of the underlying inflammatory disorder which drives losses of body protein and fuel reserves is far more important and is the single most effective therapy. Protein supplements which may promote albumin synthesis and synthesis of liver-related proteins tend to increase muscle catabolism. Muscle growth is not fostered by increasing dietary protein above recommended goals for dialysis patients, but can be promoted by the addition of protein of high biological value that is rich in leucine and other essential amino acids in tandem with repetitive exercises. Ultimately, correction of PEW hinges on the diagnosis and treatment of co-morbid conditions in combination with strategies to replenish caloric and protein stores. A supplementary exercise program would allow recovery of lean body mass. Given the multiple co-morbidities that exist in this population, therapy would have to be individualized.

Statins but not thiazolidinediones attenuate albumin-mediated chemokine production by proximal tubular cells independently of endocytosis.

BACKGROUND: Proximal tubular epithelial cells (PTEC) secrete chemokines under proteinuric conditions. Both statins and thiazolidinediones (TZDs) possess pleiotropic anti-inflammatory effects. This study examined the ability of statins and TZDs and the natural peroxisome proliferator activated receptor-gamma (PPARgamma) agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) to attenuate the proteinuria-induced pro-inflammatory phenotype of PTEC. METHODS: Mouse PTEC were treated with statins, TZDs and PGJ(2 )and effects on uptake and binding of FITC-albumin determined. PTEC were incubated with fatty acid free bovine serum albumin with or without statins/TZDs/PGJ(2), and the release of MCP-1 and RANTES measured. RESULTS: Statins and TZDs significantly inhibited PTEC albumin endocytosis. PGJ(2 )had no effect. Incubation of PTEC with albumin significantly stimulated production of MCP-1 and RANTES. Co-treatment with statins and PGJ(2) significantly reduced albumin-stimulated chemokine production, an effect reversed by the addition of mevalonate and geranylgeranyl pyrophosphate. In contrast, TZDs had no effect on albumin-mediated chemokine production. CONCLUSION: Statins and PGJ(2), but not TZDs, prevent the development of a PTEC pro-inflammatory phenotype in response to albumin. Albumin endocytosis is not a prerequisite for PTEC chemokine production, and inhibition of albumin endocytosis alone is insufficient to attenuate chemokine production. These studies suggest a therapeutic role for statins and some PPARgamma ligands in proteinuric renal disease.

Is beta2-microglobulin-related amyloidosis of hemodialysis patients a multifactorial disease? A new pathogenetic approach.

PURPOSE: Beta2-microglobulin amyloidosis (Abeta(2)M) is one of the main long-term complications of dialysis treatment. The incidence and the onset of Abeta(2)M has been related to membrane composition and/or dialysis technique, with non-homogeneous results. This study was carried out to detect: i) the incidence of bone cysts and CTS from Abeta(2)M; ii) the difference in Abeta(2)M onset between cellulosic and synthetic membranes; iii) other risk factors besides the membrane. METHODS: 480 HD patients were selected between 1986 to 2005 and grouped according to the 4 types of membranes used (cellulose, synthetically modified cellulose, synthetic low-flux, synthetic high-flux). The patients were analyzed before and after 1995, when the reverse osmosis treatment for dialysis water was started at our center, and the incidence of Abeta(2)M was compared between the two periods. Routine plain radiography, computer tomography (CT) and nuclear magnetic resonance imaging (MRI) as well as electromyography were used to investigate the clinical symptoms. RESULTS: Bone cysts occurred in 29.2% of patients before 1995 vs. 12.2% after 1995 (p<0.0001). CTS occurred in 24% of patients before 1995 vs. 7.1% after 1995 (p<0.0001). Bone cysts and CTS occurred in older patients, who began dialysis at a late age, with high CRP, low albumin, low residual GFR, and low Hb. Cox regression analysis showed that the risk factor for bone cysts was high CRP (RR 1.3, p<0.01), while albumin (RR 0.14, p<0.0001) and residual GFR (RR 0.81, p<0.0001) were revealed to be protective factors. Cox analysis for CTS confirmed CRP as a risk factor (RR 1.2, p<0.01), and albumin (RR 0.59, p<0.0001) and residual GFR (RR 0.75, p<0.0001) as protective factors. The comparison obtained between membranes did not suggest any protective effect on Abeta(2)M. CONCLUSIONS: The findings that the inflammatory status as well as low albumin and the residual GFR of the uremic patient are predictive of Abeta(2)M lesions suggests that Abeta(2)M has a multifactorial origin rather than being solely a membrane- or technique-related side effect.

Albúmina en el paciente crítico: ¿mito o realidad terapéutica?: [revisión] / Albumin in the critically ill patient: myth or real therapeutics?: [review]

Under normal conditions, the plasmatic oncotic pressure is determined mainly by albumin. Numerous trials in critically ill patients have showed that hypoalbuminemia is associated to poor outcome. So, the administration of exogenous albumin is an attractive therapeutic strategy, widely spread in different clinical scenes. Nevertheless, its use has been questioned in the last period and up to date there is no clear evidence of the real effectiveness and/or utility. This article reviews the physiological and pathophysiological concepts that would justify the use of synthetic albumin. According to current literature, discussion about the rationality of its use in different pathological situations exists, trying to outline those clinical conditions that could or could not benefit with its administration. Certainly, clinical guidelines with recommendations about the benefits and indications of this therapy are required. Hypoalbuminemia in the critically ill patient is produced principally by redistribution, secondary to changes in capillary permeability: "transcapillary leakage". The crucial interrelation between osmotic plasmatic pressure and albumin concentration in healthy individuals is lost in several critical conditions. Agreements on indications for use of albumin have not been achieved, since in different clinical context (resuscitation, sepsis, post-surgical, burns, nephrotic syndrome, ARDS) there are no significant advantages in morbidity and mortality of critically ill patients, compared to other cristalloids or synthetic colloids used. It is extremely important to develop clinical guidelines with recommendations on benefits and indications for the use of albumin in critically ill patients.

La albúmina es la principal determinante de la presión oncótica plasmática. La reducción de sus niveles séricos se asociaría a malos resultados clínicos, fundamentalmente, en la población de pacientes críticos, por lo cual su administración exógena resulta una estrategia terapéutica atractiva y ampliamente difundida. Su uso, sin embargo, ha sido cuestionado en el último tiempo, no existiendo a la fecha una clara evidencia de su real eficacia y/o utilidad. Objetivo: Revisar los conceptos fisiológicos y fisiopatológicos que subyacen al uso de albúmina sintética y evaluar la racionalidad de su utilización en distintas situaciones patológicas, intentando perfilar las condiciones clínicas que pudieran o no beneficiarse de su administración. La hipoalbuminemia en el paciente crítico está dada principalmente por un fenómeno de redistribución, secundario a cambios en la permeabilidad capilar (escape transcapilar), y la correlación entre presión osmótica plasmática y concentración de albúmina en individuos sanos, se pierde en condiciones críticas. A pesar de la literatura existente, no se han logrado acuerdos sobre las indicaciones para el uso de albúmina, ya que en los distintos contextos clínicos revisados, (resucitación, sepsis, post quirúrgicos, quemados, síndrome nefrótico, SDRA), no aparecen ventajas significativas en la morbimortalidad al compararla con el uso de cristaloides u otros coloides sintéticos, sin dejar de mencionar además el costo económico que representa su uso. Se requieren guías clínicas de consenso, basadas en la evidencia, que establezcan recomendaciones acerca de los beneficios e indicaciones de esta herramienta terapéutica, que por ahora aparece con indicaciones muy limitadas en los pacientes críticos.

Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing myeloablative stem cell transplantation.

Iron overload could be a significant contributor to treatment-related mortality (TRM) for patients with hematologic malignancies undergoing hematopoietic stem cell transplantation (HSCT). We studied 590 patients who underwent myeloablative allogeneic HSCT at our institution, and on whom a pretransplantation serum ferritin was available. An elevated pretransplantation serum ferritin level was strongly associated with lower overall and disease-free survival. Subgroup multivariable analyses demonstrated that this association was restricted to patients with acute leukemia or myelodysplastic syndrome (MDS); in the latter group, the inferior survival was attributable to a significant increase in TRM. There was also a trend toward an increased risk of veno-occlusive disease in patients with high ferritin. Our results argue that iron overload plays an important role in transplantation outcome for patients with acute leukemia or MDS, as it does in thalassemia. They also suggest future prospective trials to examine the potential benefit of chelation therapy in this setting.

PKB and megalin determine the survival or death of renal proximal tubule cells.

Renal proximal tubule cells have a remarkable ability to reabsorb large quantities of albumin through megalin-mediated endocytosis. This is an essential process for overall body homeostasis. Overstressing this endocytic system with a prolonged excess of albumin is injurious to proximal tubule cells. How these cells function and protect themselves from injury is unknown. Here, we show that megalin is the sensor that determines whether cells will be protected or injured by albumin. Megalin, through a novel mechanism, binds PKB in a D-3-phosphorylated phospholipid-insensitive manner, anchoring PKB in the luminal plasma membrane. Whereas low doses of albumin are protective, an overload of albumin decreases megalin expression followed by a reduction of plasma membrane PKB, PKB activity, and Bad phosphorylation induced by PKB. The result is albumin-induced apoptosis. These results reveal a model for PKB distribution in the plasma membrane and elucidate mechanisms involved in both the protective and toxic effects of albumin on proximal tubule cells. In addition, our findings suggest a mechanism for the progression of chronic kidney disease to end-stage renal disease.

F344 rat liver nonparenchymal cell transplantation can increase the number of albumin-positive hepatocytes in the liver following hematopoietic reconstitution in irradiated analbuminemic rats.

The adult liver contains hematopoietic stem cells that can reconstitute the bone marrow. We tested whether bone marrow cells (BMCs) derived from liver nonparenchymal cells (LNPCs) can increase the number of hepatocytes within livers. LNPCs from Fischer 344 rats (F344) were infused into the penile veins of F344 congenic Nagase's analbuminenic rats (F344alb) immediately after whole-body irradiation, and the recipients were sacrificed 8 weeks later. Eleven of 15 (73.3%) F344alb that received the LNPC transplantation after irradiation survived, while only 1 of 8 (12.5%) F344alb that received irradiation alone was alive after 8 weeks. Normal albumin gene sequences were detected by PCR in BMCs of the recipient F344alb that received LNPC transplantation after irradiation, indicating that F344alb bone marrow was reconstituted by F344 LNPCs. Although single or pairs of albumin-positive (Alb+) hepatocytes were seen in the liver of untreated F344alb and those with irradiation or LNPC transplantation alone, clusters consisting of >3 Alb+ hepatocytes were detected in the livers of F344alb with the LNPC or BMC transplantation after irradiation together with single or double Alb+ cells. Normal albumin gene sequences were detected by PCR in the DNA isolated from such Alb+ hepatocyte clusters microdissected from the immunostained sections. The data indicate that BMCs derived from F344 LNPCs could increase the number Alb+ hepatocytes within the F344alb liver.

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells.

Albumin induces oxidative stress and cytokine production in proximal tubular cells (PTECs). Albumin-bound fatty acids (FAs) enhance tubulopathic effects of albumin in vivo. We proposed that FA aggravation of albumin-induced oxidative stress in PTECs might be involved. We hypothesized that mitochondria could be a source of such stress. Using a fluorescent probe, we compared reactive oxygen species (ROS) production after exposure of PTECs to bovine serum albumin (BSA) alone or loaded with oleic acid (OA-BSA) (3-30 g/l for 2 h). There was no difference in cellular albumin uptake, but OA-BSA dose-dependently induced more ROS than BSA alone (P<0.001). OA-BSA-induced ROS was significantly alleviated by mitochondrial inhibition, but not by inhibitors of nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) oxidase, xanthine oxidase, or nitric oxide synthase. Gene expression analysis showed that neither the NADPH oxidase component p22phox nor xanthine oxidase was induced by BSA or OA-BSA. OA-BSA, in contrast to BSA, failed to induce mitochondrial manganese superoxide dismutase 2 (SOD2) expression. OA-BSA showed a greater capacity than BSA to downregulate heme oxygenase-1 mRNA expression and accentuate inflammatory cytokine mRNA and protein. Supplementation of SOD activity with EUK-8 reduced ROS, and interleukin-6 protein expression was suppressed by both mitochondrial inhibition and SOD augmentation. Thus, in PTECs, FAs accentuate albumin-induced oxidative stress and inflammatory cytokine expression via increased mitochondrial ROS, while frustrating protective antioxidant responses.

Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion.

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the alpha2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against alphaIIbbeta3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase alphaIIbbeta3-mediated platelet adhesion to albumin, dependent on alpha2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.

Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation.

Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II increases albumin endocytosis by approximately 100% at 10(-9) M. This effect is completely reversed by 10(-9) M PD123319, a specific AT(2) receptor antagonist, but not by losartan, a specific AT(1) receptor antagonist, at concentrations up to 10(-7) M. The Ang II effect on albumin endocytosis is also reversed by: phosphoinositide 3-kinase inhibitors LY294002 (2.5 x 10(-6) M) or wortmannin (10(-7) M), the protein kinase B inhibitor (2 x 10(-5) M), and staurosporine (2 x 10(-6) M), an inhibitor of 3'-phosphoinositide-dependent kinase 1. Ang II induced the selective phosphorylation of protein kinase B (PKB) at the Thr-308 residue without a change in Ser-473 phosphorylation, a combination that leads to an increase in PKB activity. These effects were completely abolished by 3 x 10(-6) M staurosporine or 10(-8) M PD123319. Our experiments also showed that PKB is present in the membrane fraction in overnight-starved LLC-PK1 cells. Taken together, these data show that Ang II increases albumin endocytosis through an AT(2) receptor mediated by activation of PKB in the plasma membrane, which depends on the basal activity of the phosphatidyl-inositol 3-kinase.

Albumin therapy in clinical practice.

Albumin is the predominant product of hepatic protein synthesis and one of the more abundant plasma proteins. Among its multiple physiologic roles, it plays an essential part in the generation of colloid-oncotic pressure. In the United States, the indications for which albumin therapy are considered include hypovolemia or shock, burns, hypoalbuminemia, surgery or trauma, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, and sequestration of protein-rich fluids. The use of this relatively expensive therapy accounts for up to 30% of the total pharmacy budget in certain hospitals. The use of albumin therapy in different clinical situations and its influence in morbidity and mortality have been reviewed in multiple randomized controlled trials and meta-analyses. Despite frequent reviews, the use of albumin remains controversial in several clinical situations. At the same time, these valuable reviews seem to have documented the advantages of albumin therapy in the management of ascites and clarified the use of albumin in volume resuscitation. More studies have been recommended to investigate the use of albumin in different doses and its role in hypoalbuminemia. This article will provide an overview of albumin metabolism, use of albumin for volume expansion, the potential therapeutic role of albumin in liver disease, and the role of albumin therapy in nutrition.

Apoptotic response to albumin overload: proximal vs. distal/collecting tubule cells.

End-stage renal disease due to proteinuric states has a great impact on the quality of life by necessitating renal replacement therapy. Understanding the pathophysiologic consequences of proteinuria is crucial in order to develop treatment strategies to halt the progression. We have previously reported that cultured porcine proximal tubule cells respond to albumin overload by undergoing apoptosis. In this study, we investigated the differential apoptotic response to albumin in HKC-8 (proximal tubule) and MDCK (collecting/distal tubule) cells under high concentrations of albumin simulating the nephrotic milieu. Our results are consistent with marked cytotoxicity and apoptosis within 24 h of albumin incubation in HKC-8 cells that was closely related to the fatty acid content of the albumin. In contrast, in MDCK cells, albumin stimulated cell turnover by stimulating proliferation and late onset apoptosis regardless of the fatty acid content. Another important result of this study is the direct demonstration of albumin uptake by MDCK cells mediated by endocytosis via clathrin-coated pits. A comparison of albumin uptake between proximal and distal/collecting tubule cells revealed faster uptake in proximal tubule cells within 15 min but almost 100% albumin uptake of both cell types in 1 h. In summary, our findings demonstrate that both proximal and distal nephron segments are affected in proteinuric states, but the degrees of susceptibility to albumin and associated lipid moieties are distinct in the different nephron segments.

Inhibition of albumin synthesis in chronic diseases: molecular mechanisms.

The albumin gene is expressed specifically in the liver after birth, and this expression is regulated predominantly at the transcriptional level. Regulatory proteins occupy specific DNA sequences within the promoter and enhancer of the albumin gene. The interaction between the CCAAT/enhancer binding protein (C/EBP)-beta and the albumin DNA is critical for albumin synthesis. Cachexia-induced hypoalbuminemia is mediated by tumor necrosis factor (TNF)-alpha. In turn, TNF-alpha stimulates oxidative stress, NO synthesis, and phosphorylation of C/EBP-beta within its nuclear localization signal (NLS). Consequently, C/EBP-beta is exported from the nucleus, preventing it to act as a transcriptional factor on the albumin gene. Antioxidants, NOS inhibitors. and dominant negative, nonphosphorylatable C/EBP-beta peptides block phosphorylation of C/EBP-beta within the NLS and its nuclear export as well as rescue the abnormal albumin gene expression, suggesting potential therapeutic interventions.