Remote Ischemic Conditioning on Recipients of Deceased Renal Transplants Does Not Improve Early Graft Function: A Multicenter Randomized, Controlled Clinical Trial.

Delayed graft function is a frequent complication following deceased donor renal transplantation, and is closely related to ischemia-reperfusion injury. Experimental and clinical studies have shown protection by remote ischemic conditioning (RIC). We hypothesized that recipient RIC before kidney graft reperfusion reduces the time to graft recovery. This multicenter, blinded, randomized, controlled clinical trial included 225 adult recipients of renal transplants from deceased donors at four transplantation centers in Denmark, Sweden, and the Netherlands. Participants were randomized 1:1 to RIC or sham-RIC. RIC consisted of 4 × 5-min thigh occlusion by an inflatable tourniquet each followed by 5-min deflation, performed during surgery prior to graft reperfusion. The tourniquet remained deflated for sham-RIC. The primary endpoint was the estimated time to a 50% decrease in baseline plasma creatinine (tCr50) calculated from plasma creatinine measurements 30 days posttransplant or 30 days after the last, posttransplant dialysis. No significant differences were observed between RIC and sham-RIC-treated patients in the primary outcome median tCr50 (122 h [95% confidence interval [CI] 98-151] vs. 112 h [95% CI 91-139], p = 0.58), or the number of patients receiving dialysis in the first posttransplant week (33% vs. 35%, p = 0.71). Recipient RIC does not reduce the time to graft recovery in kidney transplantation from deceased donors. ClinicalTrials.gov: NCT01395719.

Repeated Contrast Administration Is Associated With Low Risk of Postcontrast Acute Kidney Injury and Long-Term Complications in Patients With Severe Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) frequently require radiographic examinations. We investigated the impact of repeated contrast administrations on short- and long-term kidney function and mortality in kidney transplantation candidates. In a prospective study, 81 predialysis transplantation candidates underwent computed tomography angiography (CTA) and invasive coronary angiography (ICA) as part of a pretransplant cardiovascular evaluation. Postcontrast plasma creatinine (P-creatinine) changes were compared with a precontrast control period. We identified postcontrast acute kidney injury (AKI) in 10 patients (13%) after CTA and in two patients (3%) after ICA. Compared with the control period, relative changes in P-creatinine were significantly higher after CTA (p < 0.001) and ICA (p < 0.01). Diabetic kidney failure (p < 0.05) and contrast dose >0.8 mL/kg (p < 0.001) were associated with increases in P-creatinine. All cases of postcontrast AKI were reversible, and we found no differences between the progression rates of the kidney failure during 12 months before and after contrast exposure (p = 0.56). In a Cox regression analysis, creatinine changes after CTA or ICA were not associated with increased need for dialysis treatment or mortality. Contrast exposure and transient postcontrast AKI did not increase the risk of accelerated CKD progression or the time to initiation of dialysis or death.

Controversies in nephrology: renal albumin handling, facts, and artifacts!

In this article, we discuss and contradict a recent publication by Russo et al., which suggests that the filtration of large amounts of albumin followed by transtubular transport of intact albumin is a physiological phenomenon.

Characterization of proteinuria and tubular protein uptake in a new model of oral L-lysine administration in rats.

Intravenous infusion of basic amino acids is used experimentally and pharmacologically to prevent renal proximal tubular uptake of filtered proteins. Intravenously injected L-lysine is rapidly cleared from plasma and the effect on tubular protein reabsorption is transient. To obtain a more sustained effect, we developed a model of oral L-lysine administration and characterized this model by analyzing urinary protein excretion and proximal tubule uptake of filtered proteins. Rats placed in metabolic cages were treated with 20 mmol/kg/6 h of L-lysine, glycine, or water. Urines were analyzed for proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and radioimmunoassay. Proximal tubule uptake of proteins and expression of apical membrane receptors were investigated by immunocytochemistry. In vitro uptake and receptor expression were studied using a yolk sac cell line. L-lysine administration produced increased urinary excretion of a large number of proteins while the effect on tubular accumulation of selected proteins was variable. L-lysine treatment induced changes in the localization of two receptors responsible for tubular endocytosis of filtered proteins. In conclusion, oral L-lysine treatment induced proteinuria, in particular albuminuria, as efficiently as previous reports on intravenous infusion. The effect on tubular protein accumulation was variable suggesting differential effects on tubular reabsorption and degradation of filtered proteins. Changes in tubular protein handling were accompanied by changes in the localization of the endocytic receptors, megalin, and cubilin. In vitro experiments supported the in vivo observations. The findings suggest that L-lysine may affect receptor trafficking in addition to possible effects on the direct binding of ligands to the receptors.

Renal albumin absorption in physiology and pathology.

Albumin is the most abundant plasmaprotein serving multiple functions as a carrier of metabolites, hormones, vitamins, and drugs, as an acid/base buffer, as antioxidant and by supporting the oncotic pressure and volume of the blood. The presence of albumin in urine is considered to be the result of the balance between glomerular filtration and tubular reabsorption. Albuminuria has been accepted as an independent risk factor and a marker for renal as well as cardiovascular disease, and during the past decade, evidence has suggested that albumin itself may cause progression of renal disease. Thus, the reduction of proteinuria and, in particular, albuminuria has become a target in itself to prevent deterioration of renal function. Studies have shown albumin and its ligands to induce expression of inflammatory and fibrogenic mediators, and it has been hypothesized that increased filtration of albumin causes excessive tubular reabsorption, resulting in inflammation and fibrosis, resulting in the loss of renal function. In addition, it is known that tubular dysfunction in itself may cause albuminuria owing to decreased reabsorption of filtered albumin, and, recently, it has been suggested that significant amounts of albumin fragments are excreted in the urine as a result of tubular degradation. Thus, although both tubular and glomerular dysfunction influences renal handling of albumin, it appears that tubular reabsorption plays a central role in mediating the effects of albumin on renal function. The present paper will review the mechanisms for tubular albumin uptake and the possible implications for the development of renal disease.

Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule.

The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and beta(2)- and alpha(1)-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.

BACKGROUND: Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. METHODS: In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. RESULTS: OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. CONCLUSION: The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.

Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show that previously identified low-molecular-weight renal albumin binding proteins are fragments of cubilin. In addition, we find that mice lacking the endocytic receptor megalin show altered urinary excretion, and reduced tubular reabsorption, of albumin. Because cubilin has been shown to colocalize and interact with megalin, we propose a mechanism of albumin reabsorption mediated by both of these proteins. This process may prove important for understanding interstitial renal inflammation and fibrosis caused by proximal tubule uptake of an increased load of filtered albumin.

High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites: radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity.

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.

Characterization of a kidney proximal tubule cell line, LLC-PK1, expressing endocytotic active megalin.

Reabsorption and cellular handling of glomerular filtered vitamins, peptides, and hormones in the proximal tubule are essential, but thus far, poorly elucidated processes. The multiligand receptor megalin, initially described as a Heymann nephritis antigen and later identified as a member of the LDL receptor gene family, mediates reabsorption of several molecules, such as transcobalamin-vitamin B12 and albumin, in the proximal tubule. Consequently, a differentiated cell line of proximal tubular origin expressing megalin is an important requisite for examination of the above-mentioned processes. This study shows, using electron microscopy, that the cell line LLC-PK1, originating from the proximal tubule, maintained differentiated morphology and had a well developed endocytotic apparatus. Furthermore, by immunoblotting and immunohisto- and cytochemistry, megalin was identified in the endocytotic compartments of these cells. Megalin was situated mainly in the endosomes and in the dense apical tubules, but it was also identified in coated pits and in the brush border. The ability of megalin to mediate internalization and degradation of labeled receptor-associated protein (RAP) in a RAP-inhibitable manner was demonstrated. By autoradiography, the endocytosed, iodinated RAP was located in endosomes and lysosomes in the apical part of the cells. Moreover, the LLC-PK1 cells assembled in a monolayer with a hindrance toward diffusion of labeled mannitol, inulin, and dextran at a satisfactory level for the study of proximal tubule handling of smaller proteins. This study reveals a proximal tubule cell line expressing megalin in a functional manner well suited for binding, uptake, and transcellular transport studies.

Megalin-mediated endocytosis in renal proximal tubule.

Megalin, a 600 kDa membrane protein belonging to the IDL receptor family is highly expressed in the endocytic pathway of renal proximal tubules. In addition, this receptor is found in several other epithelia facing transcellular fluids but is also expressed in the parathyroid glands. Recent studies have established this protein as probably the most important receptor for endocytosis of macromolecules filtered in the renal glomeruli. The ligands reported to bind to megalin consist of a variety of different substances including albumin, vitamin-carrier complexes, proteinases and proteinase-inhibitor complexes, lipoprotein particles, receptor associated protein (RAP), different drugs and calcium.

Membrane receptors for endocytosis in the renal proximal tubule.

The renal proximal tubule exhibits a very extensive apical endocytic apparatus consisting of an elaborate network of coated pits and small coated and noncoated endosomes. In addition, the cells contain a large number of late endosomes/prelysosomes, lysosomes, and so-called dense apical tubules involved in receptor recycling from the endosomes to the apical plasma membrane. This endocytic apparatus is involved in the reabsorption of molecules filtered in the glomeruli. The process is very effective as demonstrated by the fact that although several grams of protein are filtered daily in the human glomeruli, human urine is virtually devoid of proteins under physiological conditions. Several key receptors appear to be involved in this function, which serves not only to conserve protein as such for the organism but also to reabsorb vital substances such as different vitamins in complex with their binding proteins. Recent research has established megalin, a 600-kDa protein belonging to the LDL receptor family, as probably the most important receptor in this process in the proximal tubule mediating endocytosis of a large variety of ligands and therefore classifying it as a scavenger receptor. More specific receptors like the folate receptor, IGF-II/Man-6-P receptor, and gp280/IFR, identical to the intrinsic factor receptor, are also functioning in the apical endocytic pathway of renal proximal tubules. A better understanding of these receptors will give us new insight into these very important processes for the organism.

Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein.

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.

Internalization and apical-to-basolateral transport of folate in rat kidney proximal tubule.

Folate derivatives are filtered in the glomeruli and reabsorbed within the nephron. The amount filtered largely exceeds the minimum daily requirements. Thus folate reabsorbed within the kidney must be returned to the circulation. To establish whether renal proximal tubule can accomplish this by transport, of [3H]folate across the cell, microperfusion of rabbit, proximal tubule with [3H]folate and [14C]inulin was performed. Transtubular transport of [3H]folate was 5 +/- 1% (0.25 +/- 0.07 fmol/min) of perfused amount/mm tubule and remained constant during a 2-h perfusion period. An accumulation of 15 +/- 4% (0.8 +/- 0.3 fmol/min) of perfused amount/mm tubule was observed during the same period. Furthermore, to determine whether endocytosis may be involved in the initial process of folate uptake in proximal tubule cells, we performed light microscopy autoradiography on cryosections of rat kidney cortex incubated with [3H]folate. Folate binding sites were located apically as well as intracellularly similar to the location of [3H]folate when injected into the abdominal aorta and visualized by light microscopy autoradiography. Thus folate binding sites as well as internalized folate is localized both apically and intracellularly. Micropuncture of rat proximal tubules with folate-coupled collodial gold particles showed significantly increased endocytosis of folate gold when evaluated quantitatively and compared with controls injected with noncoupled gold particles (0.22 +/- 0.08 vs. 0.03 +/- 0.01 gold particles/micron 2 tubule cell). The results show that kidney proximal tubule cells are capable of transcellular transport of [3H]folate with limited capacity. Folate gold particle uptake suggests that folate can be internalized by endocytosis.

Hormone, growth factor, and vitamin handling by proximal tubule cells.

The proximal tubule in the kidney is a main organ for the metabolic clearance of small peptide hormones, and is intensively involved in reabsorption, storage and homeostatic regulation of different vitamins. Recently, the endocytic membrane protein megalin has been shown to bind several smaller peptides, including insulin and epidermal growth factor, as well as to bind and mediate uptake of transcobalamin-B12, attracting new attention on the cellular mechanisms involved in these processes.

Fiber type composition of articular branches of the tibial nerve at the knee joint in man.

BACKGROUND: We examined the fiber type composition of the articular branches of the tibial nerve in human cadavers. Our primary motivation was to assess the suitability of these nerve branches for making neural recordings by using an interface, such as a nerve cuff electrode or a microelectrode array. Articular branches of the tibial nerve innervate primarily the posterior joint capsule of the knee (Gardner, 1948. Anat. Rec, 101:109-130); the main branch corresponds anatomically to the posterior articular nerve (PAN), which has been studied extensively in animals such as the cat. MATERIALS AND METHODS: By using light microscopy, we counted the numbers of myelinated fibers in articular branches of the tibial nerve removed from eight cadavers. Unmyelinated fibers were counted in the same specimens by using electron microscopy, and the percentage of unmyelinated fibers was calculated. RESULTS: We found on average 2,280 myelinated fibers in the main articular branch and 279 fibers in individual capsular ramifications. Myelinated fiber diameter histograms showed either bimodal (peaks at 3-4 and 9 microns) or unimodal (peak at 3-4 microns) distributions, depending on the specimen. These histograms were similar in appearance for both the individual capsular ramifications and the main articular branch of the tibial nerve. Numbers of unmyelinated fibers ranged from 4,176 to 5,200 in the main tibial nerve branch (average percentage of unmyelinated fibers = 69.6%) and from 750 to 2,250 in the individual capsular ramifications (average percentage of unmyelinated fibers = 78.5%). CONCLUSIONS: The percentage of unmyelinated fibers is comparable to that found in articular nerves in other species. We discovered that the main articular branch of the tibial nerve contains a branch projecting distal to the knee joint capsule; therefore, the best location for placement of a neural recording interface to record from capsular afferents appears to be the well-defined ramifications of the articular branch that penetrate the joint capsule. Branches that contain only these ramifications are 0.5-1.0 mm in diameter and, on average, have 658 myelinated axons, which should be a sufficient number from which to record.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2 water channels.