Jakub Gburek

Hemoglobin binding sites on renal brush-border membranes

Prolonged exposure of renal tubules to hemoglobin markedly reduces kidney function and eventually leads to acute renal failure called pigment nephropathy. Intracellular hemoglobin toxicity is one of main pathomechanisms involved in the disease development. However, the process in which hemoglobin is taken up by renal tubular epithelium has not been characterized so far. Isolated renal brush-border membranes of the rat and radioiodinated rat and human hemoglobins were used. Binding properties were examined by the use of rapid filtration technique. Partial isolation of hemoglobin binding proteins was achieved by affinity chromatography. Our experiments showed that both human and rat hemoglobins can be specifically bound to renal brush-border membranes by one class of low affinity (Kd, 7.7 microM) and high capacity (Bmax, 0.18 nmol/mg protein) binding sites. The sites were relatively selective for hemoglobin. Albumin did not compete with hemoglobin. Cationic molecules cytochrome C and lysine exhibited some competition while strong competition of myoglobin was observed. The binding was affected by EGTA indicating a Ca2+ requirement for the interaction. There was a rise in binding in pH 5.4. Fall in binding activity after preincubation of the membranes with peptidases suggested the proteinaceous nature of the binding sites. Affinity chromatography of membrane proteins extract yielded heterogeneous preparation consisting of proteins with molecular masses of 110, 72, 38 and 27 kDa respectively. The existence of binding sites for hemoglobin in renal brush-border membranes strongly suggests that uptake of the protein by tubular epithelia occurs via adsorptive endocytosis. Increased binding of hemoglobin to the membranes under acidic conditions may explain exacerbation of hemoglobinuric acute renal failure in aciduric states.

Clearance of chicken cystatin from the rat circulation

Chicken cystatin (mixed form) was prepared from egg white. Radioactively labeled preparations were administered intravenously to rats. 125I-cystatin disappeared from the rat circulation with a half-life of approximately 73 min. The radiolabeled inhibitor was rapidly taken up by the kidneys. Percoll density gradient analysis showed that it was incorporated into lysosomes. Within 24 hr after the injection of 125I-cystatin, 25% of the administered radioactivity was recovered in the urine, but only 2% was in the protein-bound form.

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

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.

Immunohistochemical localization of haemoglobin binding sites in the distal tubule of the rat kidney

Although it is well established that haemoglobin can be taken up by kidney tubular epithelium, the exact mechanism of the process has not been elucidated so far. We have undertaken a study to determine whether any specific binding sites for haemoglobin are present on the membranes of renal tubular cells. Paraffin sections of rat kidney cortex were incubated with haemoglobin, and the bound molecules were detected by means of a combined avidin–peroxidase and ImmunoMax method. Haemoglobin binding sites were observed in the apical membrane of distal tubules. Binding occurred for both rat haemoglobin and swine and human haemoglobins, and the proteins could compete with each other. Competition experiments with other proteins showed that the binding is specific for haemoglobin and that the net charge of the protein is not critical for the interaction. We failed to detect the binding sites in proximal tubules, where most of the filtered proteins are reabsorbed. The role of the binding sites in the distal nephron is unclear. Our findings may be essential for the further understanding of the pathomechanism of haemoglobin-induced acute renal failure.

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

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.

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

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.

Binding of glycated ovocystatin to rat renal brush border membranes

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.