Publications by authors named "Jakub Gburek"

24 Publications

  • Page 1 of 1

Renal Handling of Albumin-From Early Findings to Current Concepts.

Int J Mol Sci 2021 May 28;22(11). Epub 2021 May 28.

Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Borowska 211A, 50-556 Wrocław, Poland.

Albumin is the main protein of blood plasma, lymph, cerebrospinal and interstitial fluid. The protein participates in a variety of important biological functions, such as maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver, as well as in the kidney tubular epithelium. Long-lasting investigation in this area has delineated the principal route of its catabolism involving glomerular filtration, tubular endocytic uptake via the multiligand scavenger receptor tandem-megalin and cubilin-amnionless complex, as well as lysosomal degradation to amino acids. However, the research of the last few decades indicates that also additional mechanisms may operate in this process to some extent. Direct uptake of albumin in glomerular podocytes via receptor for crystallizable region of immunoglobulins (neonatal FC receptor) was demonstrated. Additionally, luminal recycling of short peptides into the bloodstream and/or back into tubular lumen or transcytosis of whole molecules was suggested. The article discusses the molecular aspects of these processes and presents the major findings and controversies arising in the light of the research concerning the last decade. Their better characterization is essential for further research into pathophysiology of proteinuric renal failure and development of effective therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22115809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199105PMC
May 2021

The effects of chicken egg white cystatin and proteinase inhibitor on cysteine peptidase-like activity in the sera of patients with breast cancer.

Adv Clin Exp Med 2021 03;30(3):323-330

Department of Pathophysiology, Wroclaw Medical University, Poland.

Background: The activity of autogenic proteolytic enzymes is regulated in vivo by autogenic inhibitors. They play important roles in maintaining a balance in many processes in the human body. In pathological conditions, enzymes are overexpressed and the balance is disturbed. Such uncontrolled changes may lead to the development of local or systemic cancer.

Objectives: To evaluate the effects of specific inhibitors, i.e., chicken egg white cystatin (CEWC) and proteinase inhibitor (E-64) on autogenic cysteine peptidases (CPs) in the sera of patients reporting for subsequent stages of treatment after being diagnosed with breast cancer. Cysteine peptidases play a vital role in the basic processes that are associated with cancer progression.

Material And Methods: We selected serum samples from 108 patients with a diagnosis of breast cancer (stages IIA-IIIA) who had received no previous treatment. The blood samples were centrifuged, and the resulting serum was placed in liquid nitrogen and stored at -80°C. The biochemical tests were performed at the laboratory of the Department of Physical Chemistry and Microbiology.

Results: For CEWC, we found an inhibitory effect in 37 out of 108 samples; for E-64, 14 out of 22 samples displayed an inhibitory effect. In the remaining blood samples, these inhibitors caused an increase in fluorescence. In a parallel test, we added pure cathepsin B to 9 serum samples, and then used CEWC to inhibit the activity of autogenic CPs. Chicken egg white cystatin completely inhibited the cathepsin B that was added to the serum without changing its effect on the autogenic CPs.

Conclusions: The results suggest that there may be a potential difference between the commercially available cathepsin B and its autogenic analogues found in the serum of cancer patients. The increase in fluorescence induced in the reaction between the inhibitors and autogenic CPs is still unexplained. There was no relationship between the observed inhibition/activation of CPs and any of the available indicators of the health of the patients examined.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.17219/acem/126288DOI Listing
March 2021

Iron Complexes of Flavonoids-Antioxidant Capacity and Beyond.

Int J Mol Sci 2021 Jan 11;22(2). Epub 2021 Jan 11.

Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, CZ-121 08 Prague, Czech Republic.

Flavonoids are common plant natural products able to suppress ROS-related damage and alleviate oxidative stress. One of key mechanisms, involved in this phenomenon is chelation of transition metal ions. From a physiological perspective, iron is the most significant transition metal, because of its abundance in living organisms and ubiquitous involvement in redox processes. The chemical, pharmaceutical, and biological properties of flavonoids can be significantly affected by their interaction with transition metal ions, mainly iron. In this review, we explain the interaction of various flavonoid structures with Fe(II) and Fe(III) ions and critically discuss the influence of chelated ions on the flavonoid biochemical properties. In addition, specific biological effects of their iron metallocomplexes, such as the inhibition of iron-containing enzymes, have been included in this review.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22020646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827006PMC
January 2021

Differences in cysteine peptidases-like activity in sera of patients with breast cancer.

Cancer Biomark 2020 ;27(3):335-341

Department of Pathophysiology, Wroclaw Medical University, Wroclaw, Poland.

Background: The key role in carcinogenesis with destruction of the extracellular matrix is played by proteases released by invasive cancer cells. Cysteine peptidases, such as cathepsin B and L, take an important role in cancer progression and metastasis.

Objectives: Cysteine peptidase-like activity (CPA) in sera of patients with breast cancer at different stages of disease and the influence of genetic predisposition associated with BRCA-1 gene mutations were analysed.

Methods: CPA in serum was determined with the spectrofluorometric technique using Z-Phe-Arg-AMC as a substrate. Determination was carried out in 111 breast cancer patients in comparison to a control group of 50 healthy subjects.

Results: The highest CPA was found in breast cancer patients with a hereditary predisposition bearing BRCA1 gene mutations, and the lowest activity was found in patients who had a tumour surgically removed and before adjuvant therapy. The differences in the activities between control group and cancer groups were statistically significant (p< 0.05), except from group of cancer patients in complete remission (p< 0.52).

Conclusions: Serum CPA in patients with breast cancer differs depending on the cancer stage and treatment methods. Our study demonstrate the correlation between BRCA-1 gene mutations and the increased level of CPA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/CBM-190327DOI Listing
November 2020

Parameters of Oxidative and Inflammatory Status in a Three-Month Observation of Patients with Acute Myocardial Infarction Undergoing Coronary Angioplasty-A Preliminary Study.

Medicina (Kaunas) 2019 Sep 13;55(9). Epub 2019 Sep 13.

Department of Toxicology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

: Patients with acute myocardial infarction (MI) are usually treated with percutaneous transluminal coronary angioplasty (PTCA), which is burdened with a risk of postoperative complications, often accompanied by biochemical disturbances. The aim of our study was to evaluate a set of selected parameters of oxidative and inflammatory status, which could be useful in the management of post-procedural care in MI patients after PTCA. : In this preliminary study, ischemia modified albumin (IMA), advanced oxidation protein products (AOPP), thiol groups (SH), total antioxidant status (TAS), insulin growth factor-1 (IGF-1), presepsin (PSP), and trimethylamine N-oxide (TMAO) were chosen as candidate biomarkers, and were determined in patients with MI who underwent PTCA at two time points: During cardiac episodes (at admission to the hospital, T0) and 3 months later (T3). : Most of the examined parameters were significantly different between patients and control subjects (except for IMA and TAS), but only hsCRP changed significantly during the time of observation (T0 vs. T3). Discriminant analysis created a model composed of AOPP, hsCRP, PSP, and TMAO, which differentiated male subjects into a group with MI and a control (without cardiovascular diseases). : This set of parameters seems useful in evaluating inflammatory and oxidative status in MI patients after PTCA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/medicina55090585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780791PMC
September 2019

Beneficial effect of ovocystatin on the cognitive decline in APP/PS1 transgenic mice.

Adv Med Sci 2019 Mar 30;64(1):65-71. Epub 2018 Nov 30.

Department of Psychiatry, Wroclaw Medical University, Wroclaw, Poland.

Purpose: Cystatin C plays an important role in the course of neurodegenerative diseases and has a beneficial effect through inhibiting cysteine proteases and amyloid-β aggregation. It also induces proliferation and autophagy. Cystatin isolated from chicken egg white, called ovocystatin, has been widely used in the medical and pharmaceutical research due to its structural and biological similarities to human cystatin C. The aim of this study was to assess the effect of administering ovocystatin on the development of dementia-specific cognitive deficits in APP/PS1 transgenic mice.

Materials/methods: The study was conducted on transgenic B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax mice. Ovocystatin was administered to four-month-old transgenic (AD) and wild type (NCAR) mice in drinking water for 24 weeks (at a dose of 40 and 4 μg/ mouse). The locomotor activity and cognitive functions were determined using an actimeter and the Morris water maze test, respectively.

Results: The results of the study indicate that ovocystatin has a beneficial effect on the cognitive functions in APP/PS1 transgenic mice. The strongest effects of ovocystatin were found in the group of AD mice, where ovocystatin was administered in drinking water at a dose of 40 μg/mouse (p < 0.05). Mice from the AD group swam statistically significantly further in the target zone during the trial in the Morris water maze compared to the AD (vehiculum) group (p < 0.05).

Conclusions: The obtained results encourage further research into the protective effect, which may be used as an adjuvant in the treatment of deteriorating cognitive functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.advms.2018.08.002DOI Listing
March 2019

Inhibition of Advanced Glycation End-Product Formation and Antioxidant Activity by Extracts and Polyphenols from Scutellaria alpina L. and S. altissima L.

Molecules 2016 Jun 14;21(6). Epub 2016 Jun 14.

Department of Biology and Pharmaceutical Botany, Wroclaw Medical University, ul. Borowska 211, Wroclaw 50-556, Poland.

Methanolic extracts from the aerial parts and roots of two Scutellaria species, S. alpina and S. altissima, and five polyphenols from these plants demonstrated a significant ability to inhibit the formation of advanced glycation end-products (AGE) in vitro. S. alpina, which is richer in polyphenolic compounds, had strong antiglycation properties. These extracts demonstrated also high activity in the FRAP (ferric-reducing antioxidant power), antiradical (DPPH) and lipid peroxidation inhibition assays. Among the pure compounds, baicalin was the strongest glycation inhibitor (90.4% inhibition at 100 μg/mL), followed by luteolin (85.4%). Two other flavone glycosides had about half of this activity. Verbascoside was similar to the reference drug aminoguanidine (71.2% and 75.9%, respectively). The strong correlation observed between AGE inhibition and total flavonoid content indicated that flavonoids contribute significantly to antiglycation properties. A positive correlation was also observed between antiglycative and antioxidant activities. The studied skullcap species can be considered as a potential source of therapeutic agents for hyperglycemia-related disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules21060739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273165PMC
June 2016

[Clathrin-independent endocytosis - role in disease processes and pharmaceutical aspects].

Postepy Hig Med Dosw (Online) 2015 Jul 7;69:763-76. Epub 2015 Jul 7.

Katedra i Zakład Biochemii Farmaceutycznej, Uniwersytet Medyczny im. Piastów Śląskich we Wrocławiu.

Clathrin-independent endocytosis (CIE) is the process of cellular uptake of various particles, including pathogens, without the coat protein clathrin. It occurs commonly in mammalian cells and is regulated by protein-lipid composition of the cell membranes. Understanding of different routes of CIE allowed the identification of novel molecular mechanisms involved in uptake of molecules and cell signaling and explained their role in pathological processes. In this paper we characterize diseases associated with genetic defects of proteins involved in CIE and the relationship between expression of these proteins and pathology of atherosclerosis, hypercholesterolemia, diabetes and neoplasia. The role of CIE in bacterial, viral, fungal, and protozoal infections is also presented. In the second part we describe the plausible use of clathrin-independent endocytosis in increasing drug absorption, their penetration through biological membranes, and the design of specific nanocarriers for selective cell uptake.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5604/17322693.1160614DOI Listing
July 2015

Antipapain activity in the serum of patients with breast cancer.

Cancer Biomark 2015 ;15(1):99-102

Department of Basic Sciences, Wrocław Medical University, Wrocław, Poland.

Background: Complement information about the share the role of antipapain activity in serum people with breast cancer.

Objective: We measured the activity of cysteine peptidase inhibitors in the sera of 150 patients with breast cancer. Patients were divided into four groups depending on the cancer type and treatment method. We also analysed the control group. The activity of cysteine peptidase inhibitors was defined as a 'defensiveness' marker.

Methods: The activity of cysteine peptidase inhibitors was measured against papain using the colorimetric method and the BANA substrate.

Results: The highest activity of enzymes was found in the group of patients with BC and hereditary predisposition to it, and the lowest activity was found in patients after surgical treatment.

Conclusion: The activity of cysteine peptidase inhibitors in serum was measured against papain. We found that the activity levels were correlated with the cancer stage and treatment method. The lowest activity was found in patients after surgical treatment; the highest in women with active cancer and a hereditary predisposition to it.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/CBM-140435DOI Listing
August 2015

Evaluation of the antibacterial activity of cystatin against selected strains of Escherichia coli.

Folia Biol (Krakow) 2014 ;62(3):187-92

The aim of this study was to analyze the antibacterial activity of hen egg white cystatin against selected Escherichia coli strains. We used a monomeric solution of hen egg white cystatin in bovine serum albumin (BSA) with added phosphate buffered saline (PBS), and three test strains: Escherichia coli ATCC 23811, Escherichia coli ATCC 8739 and Escherichia coli ATCC 25922. The effect of cystatin against the tested strains was determined on the basis of minimal inhibitory concentration (MIC) and survival curves of the microorganisms in a cystatin-containing environment during incubation at various temperatures. Our study confirmed the activity of cystatin against the analyzed Escherichia coli strains. taining environment, as compared to control samples incubated in a ovocystatin-deficient medium. Depending on the incubation temperature (20 degrees C or 37 degrees C) the reduction persisted up to 12 hours after incubation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3409/fb62_3.187DOI Listing
January 2015

Ovocystatin affects actin cytoskeleton organization and induces proapoptotic activity.

Acta Biochim Pol 2014 3;61(4):753-8. Epub 2014 Sep 3.

Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, Poland.

Ovocystatin is a chicken egg white protein, generally known for its inhibitory activity against cysteine proteases. However, biological activity of ovocystatin does not seem to be well recognized in respect to other possible cellular effects. Our attention has been focused on ovocystatin cytotoxic effects in relation to its influence on actin cytoskeleton organization and apoptosis induction. In vitro studies with human melanoma A375, human cervix HeLa cancer cells and normal human fibroblasts - NHDF were done. Cytotoxic activity of ovocystatin was seen in respect to apoptosis induction - manifested by cell shape changes, phosphatydylserine translocation and actin cytoskeleton reorganization. Normal human fibroblasts have shown lower sensitivity to ovocystatin as compared with human melanoma A375 and human cervix HeLa cancer cells. In conclusion, ovocystatin affects actin cytoskeleton organization and displays proapoptotic activity towards applied cell lines. This implicates its application as a potential anticancer drug. However, its adverse effects on normal cells should be taken into consideration.
View Article and Find Full Text PDF

Download full-text PDF

Source
September 2015

Influence of aminoglycoside antibiotics on chicken cystatin binding to renal brush-border membranes.

J Pharm Pharmacol 2013 Jul 19;65(7):988-94. Epub 2013 Mar 19.

Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Wrocław, Poland.

Objectives: Drug-induced kidney injury is a serious adverse event which needs to be monitored during aminoglycoside therapy. Urine cystatin C is considered an early and sensitive marker of nephrotoxicity. Cystatin C, a low-molecular-weight serum protein, and basic drugs have a common transport system expressed in the apical membrane of renal proximal tubular cells. The aim of this study was to investigate whether aminoglycoside antibiotics influenced cystatin C binding to the renal brush-border membrane.

Methods: The binding study was performed using a rapid filtration technique and affinity column displacement method.

Key Findings: Concentration-dependent inhibition of chicken cystatin binding to brush-border membranes by gentamicin was observed. The gentamicin interaction with brush-border membranes was of relatively low affinity (Ki = 32 μm) in comparison with the chicken cystatin affinity to the binding sites (Kd = 3.6 μm). Amikacin and gentamicin were only able to displace chicken cystatin from the chromatographic affinity column in concentrations several times higher than normally found in the tubular fluid during standard aminoglycoside therapy.

Conclusion: Cystatin reabsorption in the proximal tubule cannot be significantly affected by aminoglycoside antibiotics because of their relatively low affinity to common binding sites on the brush-border membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jphp.12058DOI Listing
July 2013

Binding of glycated ovocystatin to rat renal brush border membranes.

Anim Sci J 2013 Oct 18;84(10):702-7. Epub 2013 Apr 18.

Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

Glycated proteins are considered as one of the factors involved in the pathogenesis of diabetic complications, including nephropathy. These proteins are formed endogenously under conditions of hyperglycemia, as well as being provided with food containing sugars, which was subjected to high temperature. Examples are egg products. One of the proteins found in eggs in a relatively high concentration is chicken cystatin (ovocystatin). It is now believed that some proteins can passage the intestinal epithelium by transcytosis directly into the bloodstream. Thus, glycated protein present in food can be an additional source of glycotoxins. The aim of this study was to compare the affinity of native and glycated cystatin to the brush border membranes of rat kidney. Kinetic analysis was performed with surface plasmon resonance technique using sensor chip L1. Dissociation constants for native and glycated cystatin (Kd ) were 2.76 μmol/L and 3.82 μmol/L, respectively. The results of our study indicate that glycation only slightly affects binding of cystatin to brush border membranes. This suggests that glycated cystatin and other glycated proteins may also be efficiently taken up in the kidney proximal tubule. The observation may be important for understanding the mechanisms involved in the development of diabetic nephropathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/asj.12060DOI Listing
October 2013

[Renal catabolism of albumin - current views and controversies].

Postepy Hig Med Dosw (Online) 2011 Oct 27;65:668-77. Epub 2011 Oct 27.

Katedra i Zakład Biochemii Farmaceutycznej, Akademia Medyczna im. Piastów Śląskich we Wrocławiu.

Albumin is the main protein of blood plasma, lymph, cerebrospinal fluid and interstitial fluid. The protein assists in many important body functions, including maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver as well as in the kidney tubular epithelium. Renal catabolism of albumin consists of glomerular filtration and tubular reabsorption. The tubular processes include endocytosis via the multiligand scavenger receptor tandem megalin and cubilin-amnionless complex. Possible ways of further catabolism of this protein are lysosomal proteolysis to amino acids and short peptides, recycling of degradation products into the bloodstream and tubular lumen or transcytosis of whole molecules. The article discusses the molecular aspects of these processes and presents the controversies arising in the light of the last decade of research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5604/17322693.964329DOI Listing
October 2011

Cubilin is essential for albumin reabsorption in the renal proximal tubule.

J Am Soc Nephrol 2010 Nov 26;21(11):1859-67. Epub 2010 Aug 26.

INSERM UMR S968, Institut de la Vision, 17 rue Moreau, 75012 Paris, France.

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2010050492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014001PMC
November 2010

Characterization of chicken cystatin binding to rat renal brush-border membranes.

Comp Biochem Physiol B Biochem Mol Biol 2007 Apr 21;146(4):482-8. Epub 2006 Nov 21.

Department of Pharmaceutical Biochemistry, Wrocław Medical University, Szewska 38/39, 50-139 Wrocław, Poland.

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K(d)=3.67-4.07 microM) and high capacity (B(max)=2.32-2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K(i)=0.7 microM) among other megalin/cubilin ligands tested. The presence of Ca(+2) ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpb.2006.11.004DOI Listing
April 2007

Total and lipid-bound plasma sialic acid as diagnostic markers in colorectal cancer patients: correlation with cathepsin B expression in progression to Dukes stage.

J Exp Ther Oncol 2006 ;5(3):223-9

Department of Pathophysiology, Wroclaw Medical University, Marcinkowski 1 Street, Wroclaw, Poland.

Purpose: Serum cathepsin B (CB), Total Sialic acid (TSA), total sialic acid (TSA) and lipid bound sialic acid (TSA) concentrations more useful than the other markers investigated for detecting different malignancies. Our aim was to investigate the possible correlation between serum CB with TSA, LSA in colorectal carcinoma with pathological stages progressed of the disease.

Methods: The study was performed on 177 patients (109 patients with colon and 68 patients with rectal) and 50 healthy individuals comprised the control group. Serum CB activity was determined using fluorogenic substrate. Serum TSA and LSA Concentrations were measured according to the method described by Katopodis.

Results: Plasma CB and TSA levels in the tumor group were significantly increased in comparison with the controls group (P < or = 0.0001). No significant differences were observed in LSA level between the tumor group and the controls group. T/N ratios for CB, TSA elevated 2.3-fold, 2.5-fold respectively). LSA 1.8-fold. Serum CB activity, TSA concentrations values in plasma samples of patients were increased significantly with pathological stages progressed (P < or = 0.0001). CB is seen to correlate more strongly with TSA in tumor group (P < or = 0.0001, r= 0.7277) in comparison with controls group. These correlations became more significant as the stage of the disease progressed.

Conclusion: The present investigations indicate that CB activity, serum TSA, concentrations are sensitive markers for detecting and earliest diagnosis of colorectal cancer. These markers with other clinical and biochemical criteria may play important metabolic roles in cancer progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
May 2006

Cathepsin D expression in human colorectal cancer: relationship with tumour type and tissue differentiation grade.

J Exp Ther Oncol 2005 ;5(2):145-50

Department of Pathophysiology, Medical University of Wroclaw, Marcinkowski 1 Street, Wroclaw, Poland.

Objective: Cathepsin D (CD) is one of the main proteolytic enzymes contributing to the development of cancer. The aim of this study was to CD activity assay in the homogenates of tissues from the centre of the tumour (0) and tumour free area 2 cm, and 5 cm from the tumour border in human colorectal cancer. Activity in the centre of the tumour was compared with immunohistochemical expression CD.

Methods: CD activity was measured using acid denatured Hb as a substrate. For immunohistochemical staining peroxidase method was used.

Results: Activity of CD was significantly higher (15-fold) in tumour tissue homogenates in comparison to normal mucosa adjacent (control) (p < or = 0.0001) and raised parallel to the stage of tumour tissue differentiation grade. CD activity decreased significantly (p < or = 0.0001) with the distance from the tumour border 2 cm (12.7 fold) and 5 cm (5.7 fold) in comparison to the centre of the tumour. In immunohistochemical examinations CD was detected as diffuse cytoplasmic as well as fine granular staining of the cytoplasm, with occasional coarse cytoplasmic granules staining in the same cases that were positive for both. Positive staining was observed in 2 of 3 in well-differentiated (66%), 4 of 10 in moderately-differentiated (40%) and 4 of 5 in poorly-differentiated (80%), tubular adencarcinomas represented: 3 of 7 (42%) and 9 of 13 in invasive adencarcinoma (69%).

Conclusion: We have observed a wide range of cathepsin D and their antigen expressions patterns in colorectal tumours with the development the disease stage, this finding may be used as a daignostic tumor marker in colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
March 2006

Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes.

Am J Physiol Renal Physiol 2006 Jun 31;290(6):F1525-33. Epub 2006 Jan 31.

Department of Physiology and Pathophysiology, Faculty of Medicine, Univ. of Witten/Herdecke, Witten, Germany.

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00359.2005DOI Listing
June 2006

Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex.

J Am Soc Nephrol 2005 Aug 23;16(8):2330-7. Epub 2005 Jun 23.

Address correspondence to: Dr. Pierre J. Verroust, INSERM U538, Centre Hospitalier Universitaire Saint Antoine, 27 Rue Chaligny, 75012 Paris, France.

Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with cubilin. By expression in transfected polarized MDCK cells of AMN and several cubilin fragments, including a functional "mini" version of cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with cubilin and co-localized with cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the cubilin-AMN complex to the apical membrane: AMN interacts with the EGF domains of cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2004110925DOI Listing
August 2005

Plasma protein haptoglobin modulates renal iron loading.

Am J Pathol 2005 Apr;166(4):973-83

Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5bis, 10126 Turin, Italy.

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1602399PMC
http://dx.doi.org/10.1016/S0002-9440(10)62319-XDOI Listing
April 2005

Protein reabsorption in renal proximal tubule-function and dysfunction in kidney pathophysiology.

Pediatr Nephrol 2004 Jul 14;19(7):714-21. Epub 2004 May 14.

Department of Cell Biology, Institute of Anatomy, University of Aarhus, 8000 Aarhus C, Denmark.

The endocytic receptors megalin and cubilin are highly expressed in the early parts of the endocytic apparatus of the renal proximal tubule. The two receptors appear to be responsible for the tubular clearance of most proteins filtered in the glomeruli. Since cubilin is a peripheral membrane protein it has no endocytosis signaling sequence. Cubilin binds to megalin and it appears that megalin is responsible for internalization of cubilin and its ligands, in addition to internalizing its own ligands. The importance of the receptors is underscored by the proteinuria observed in megalin-deficient mice, in dogs lacking functional cubilin, and in patients with distinct mutations of the cubilin gene. In this review we focus on the role of megalin- and cubilin-mediated endocytosis in renal pathophysiology. Association between disorders characterized by tubular proteinuria, such as megaloblastic anemia type-1, Dent disease, cystinosis, and Fabry disease and the dysfunction of proximal tubular endocytosis is discussed. The correlation between the high capacity of endocytosis in the proximal tubule and progressive renal disease in overload proteinuria is considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00467-004-1494-0DOI Listing
July 2004

Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin.

Am J Physiol Renal Physiol 2003 Sep 29;285(3):F451-8. Epub 2003 Apr 29.

Dept. of Cell Biology, Institute of Anatomy, Aarhus University, Denmark.

Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kd values of 2.0 and 3 microM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced (Kd = 5 microM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes (Ki approximately 2 microM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00062.2003DOI Listing
September 2003

Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin.

J Am Soc Nephrol 2002 Feb;13(2):423-430

*Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, Poland; Department of Cell Biology, Institut of Anatomy, University of Aarhus, Aarhus, Denmark; Institut Nationale de la Santé et de la Recherche St.-Antoine, Paris, France; Max Delbrueck Centre for Molecular Medicine, Berlin, Germany; Department of Microbiology, Michigan State University, East Lansing, Michigan; Department of Veterinary Prevention and Immunology, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.

The kidney is the main site of hemoglobin clearance and degradation in conditions of severe hemolysis. Herein it is reported that megalin and cubilin, two epithelial endocytic receptors, mediate the uptake of hemoglobin in renal proximal tubules. Both receptors were purified by use of hemoglobin-Sepharose affinity chromatography of solubilized renal brush-border membranes. Apparent dissociation constants of 1.7 microM for megalin and 4.1 microM for cubilin were determined by surface plasmon resonance analysis. The binding was calcium dependent in both cases. Uptake of fluorescence-labeled hemoglobin by BN-16 cells was inhibited by anti-megalin and anti-cubilin antibodies as well as by receptor-associated protein, a chaperone for LDL-receptor family proteins. Partial inhibition by myoglobin was observed, whereas bovine serum albumin, intrinsic factor-cobalamin complexes, and beta2-microglobulin did not affect the uptake. By use of immunohistochemistry, it was demonstrated that uptake of hemoglobin in proximal tubules of rat, mouse, and dog kidneys occurs under physiologic conditions. Studies on normal and megalin knockout mouse kidney sections showed that megalin is responsible for physiologic clearance of hemoglobin. Labeling intensities in kidneys from normal and cubilin-malexpressing dogs were similar, which suggests that, in the normal state, the role of cubilin in uptake of hemoglobin is rather limited. However, cubilin is likely to assist hemoglobin endocytosis in settings of hemoglobinuria. In conclusion, the study provides a molecular explanation for long-standing observations of hemoglobin uptake in renal proximal tubules that involve the endocytic receptors megalin and cubilin. The findings may prove to be essential for further research on the pathophysiology of hemoglobinuric acute renal failure and proteinuria-associated tubulointerstitial nephritis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.V132423DOI Listing
February 2002
-->