Proximal Tubule Uptake Research Articles

Lrpap1 (RAP) Inhibits Proximal Tubule Clathrin Mediated and Clathrin Independent Endocytosis, Ameliorating Renal Aminoglycoside Nephrotoxicity.

Proximal tubules are exposed to many exogenous and endogenous nephrotoxins that pass through the glomerular filter. This includes many small molecules such as aminoglycoside and myeloma light chains. These filtered molecules are rapidly endocytosed by the proximal tubules and lead to nephrotoxicity. To investigate whether inhibition of proximal tubule uptake of filtered toxins can reduce toxicity we evaluated the ability of Lrpap1 or RAP to prevent proximal tubule endocytosis. Munich Wistar Frömter rats were used since both glomerular filtration and proximal tubule uptake can be quantified. The injury model chosen was the well-established gentamicin induced toxicity which leads to significant reductions in GFR and serum creatinine increases. Chronic kidney disease was induced with a right uninephrectomy and left 40 minute pedicle clamp. Rats had eight weeks to recover and to stabilize GFR and proteinuria. Multiphoton microscopy was used to evaluate endocytosis in vivo and serum creatinine and 24 hour creatinine clearances were used to evaluate kidney functional changes. Studies showed preadministration of RAP significantly inhibited both albumin and dextran endocytosis in outer cortical proximal tubules. Importantly, this inhibition was found to be rapidly reversible with time. RAP was also found to be an excellent inhibitor of proximal tubule gentamicin endocytosis. Lastly, gentamicin administration for 6 days resulted in significant elevation of serum creatinine in vehicle treated rats but not those receiving daily infusion of RAP prior to gentamicin. This study provides a model for the potential use of RAP to prevent, in a reversible manner, proximal tubule endocytosis of potential nephrotoxins thus protecting the kidney from damage.

Renal Megalin mRNA Downregulation Is Associated with CKD Progression in IgA Nephropathy

Introduction: Megalin plays an important role in proximal tubule uptake of filtered proteins. Downregulation and dysfunction of megalin were previously demonstrated in IgA nephropathy (IgAN); however, its relationship to IgAN progression remains unclear. Methods: We measured renal megalin mRNA and miR-148b, previously identified as a regulator of megalin, in a retrospective cohort of 417 IgAN patients at the time of biopsy, and evaluated their associations with chronic kidney disease (CKD) progression event, defined as end-stage renal disease or ≥40% decline in estimated glomerular filtration rate, using Cox proportional hazard models. Risk classification statistics were calculated for CKD progression. Results: During a median follow-up of 43 months, 121 (29.0%) patients reached the CKD progression event. Patients in the highest tertile of renal megalin mRNA had a lower risk for CKD progression than in the lowest tertile (hazard ratio (HR): 0.407, 95% confidence interval (CI) 0.231–0.719; p = 0.002). Log megalin mRNA was independent and negatively associated with CKD progression in IgAN (HR: 0.529, 95% CI 0.377–0.742; p < 0.001). The addition of renal megalin mRNA to a model with traditional risk factors improved risk prediction of disease progression (C statistic from 0.76 to 0.80; integrated discrimination index: 0.04 [95% CI: 0.02–0.07]). Moreover, patients in the highest tertile of renal miR-148b had a 2.3-fold higher risk for CKD progression compared with those in the lowest tertile. Conclusions: Lower renal megalin mRNA levels were associated with a greater risk of CKD progression in IgAN independent of clinical and pathological characteristics, suggesting that renal megalin could be an important prognostic factor for IgAN.

Modelling normal and nephrotic axial uptake of albumin and other filtered proteins along the proximal tubule.

Recent studies indicate that filtered albumin is retrieved in the proximal tubule (PT) via three pathways: receptor-mediated endocytosis via cubilin (high affinity) and megalin (low affinity), and fluid-phase uptake. Expression of megalin is required to maintain all three pathways, making it challenging to determine their respective contributions. Moreover, uptake of filtered molecules varies between the sub-segments (S1, S2 and S3) that make up the PT. Here we used new and published data to develop a mathematical model that predicts the rates of albumin uptake in mouse PT sub-segments in normal and nephrotic states, and partially accounts for competition by β2 -microglobulin (β2m) and immunoglobulin G (IgG). Our simulations indicate that receptor-mediated, rather than fluid-phase, uptake accounts for the vast majority of ligand recovery. Our model predicts that ∼75% of normally filtered albumin is reabsorbed via cubilin; however, megalin-mediated uptake predominates under nephrotic conditions. Our results also suggest that ∼80% of albumin is normally recovered in S1, whereas nephrotic conditions or knockout of cubilin shifts the bulk of albumin uptake to S2. The model predicts β2m and IgG axial recovery profiles qualitatively similar to those of albumin under normal conditions. In contrast with albumin, however, the bulk of IgG and β2m uptake still occurs in S1 under nephrotic conditions. Overall, our model provides a kinetic rationale for why tubular proteinuria can occur even though a large excess in potential PT uptake capacity exists, and suggests testable predictions to expand our understanding of the recovery profile of filtered proteins along the PT. KEY POINTS: We used new and published data to develop a mathematical model that predicts the profile of albumin uptake in the mouse proximal tubule in normal and nephrotic states, and partially accounts for competitive inhibition of uptake by normally filtered and pathological ligands. Three pathways, consisting of high-affinity uptake by cubilin receptors, low-affinity uptake by megalin receptors and fluid phase uptake, contribute to the overall retrieval of filtered proteins. The axial profile and efficiency of protein uptake depend on the initial filtrate composition and the individual protein affinities for megalin and cubilin. Under normal conditions, the majority of albumin is retrieved in sub-segment S1 but shifts to sub-segment S2 under nephrotic conditions. Other proteins exhibit different uptake profiles. Our model explains how tubular proteinuria can occur despite a large excess in potential proximal tubule uptake capacity.

Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Terminal complement complex C5b-9 reduced megalin and cubilin-mediated tubule proteins uptake in a mouse model of trichloroethylene hypersensitivity syndrome

Trichloroethylene (TCE), a commonly used industrial solvent and degreasing agent, is known to cause trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including skin, liver and kidney. Clinical evidence have shown that the kidney injury occurs in THS and our previous studies suggested that the terminal complement complex C5b-9 deposited in impaired renal tubules induced by TCE with unclear mechanisms. In the present study, we questioned whether activation of the complement system with renal deposition of C5b-9 contributes to TCE-induced kidney injury in THS. We established a BALB/c mouse model of TCE sensitization with or without pretreatment of exogenous CD59, a C5b-9 inhibitory protein. H&E staining, PAS staining, and biochemical detection of urinary proteins were performed to assess renal function. Deposition of C5b-9 and expression of CD59 were evaluated by immunohistochemistry. Sub-lytic effects of C5b-9 in tubular epithelial cells were assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Expression of endocytosis receptors megalin and cubilin on proximal tubules were assessed by immunofluorescence and qRT-PCR. We found that TCE sensitization induced structural and functional changes of renal tubules in mice, associated with the deposition of sub-lytic C5b-9 on proximal tubular epithelial cells. TCE sensitization decreased proximal tubule uptake of filtered proteins and renal expression of megalin and cubilin, phenotypes that were attenuated by pretreatment with exogenous CD59. Overall, our findings reveal a novel mechanism underlying sub-lytic C5b-9 acting on megalin and cubilin, contributes to the renal tubules damage by TCE exposure.

Abstract 353: Aldehyde Scavenging Reduces Renal Injury Role of IsoLG-modified apoAI

Reactive aldehydes, including isolevuglandins (IsoLG) are lipoxidation products that can modify lipoproteins and promote cellular dysfunction. Previously, we showed that proteinuric injury increases urinary apoAI and causes renal lymphangiogenesis. We now investigate the role of IsoLG and effects of IsoLG scavenging on renal injury and lymphatics. Nphs1-hCD25 mice (NEP25) expressing podocyte-specific human CD25 become proteinuric after injection of immunotoxin (LMB2). NEP25 mice were treated with the aldehyde scavenger, PPM (1g/L) or vehicle from the onset of proteinuria until sacrifice (2 weeks). We assessed proteinuria [measured as albumin:creatinine ratio (ACR)], urinary apoAI, IsoLG, KIM-1 (marker of tubular injury), and the renal expression of lymphatic markers (LYVE-1 and podoplanin). In vitro , we assessed the effects of apoAI or modified apoAI (IsoLG-apoAI) +/- PPM on proximal tubular cells (PTC) and lymphatic endothelial cells (LEC). As expected, LMB2 significantly increased ACR and urinary KIM-1. Proteinuric injury also significantly increased urinary IsoLG (262%). Similar to our previous data, proteinuric kidneys increased expression of apoAI, VEGF-C, podoplanin and LYVE-1 compared to wild type mice. In vitro , PTC uptake of IsoLG-apoAI was significantly increased (39%), an effect that was abrogated by exposure to PPM (22%). In LECs, compared to apoAI, IsoLG-apoAI significantly increased viability (27%) and migration (13%), effects that were abrogated by exposure to PPM. In vivo , proteinuric NEP25 mice treated with PPM showed significantly reduced ACR (22%), urinary KIM-1 (30%) and IsoLG excretion (64%). We conclude that aldehyde scavenger lessens proteinuric renal damage through mechanisms that include reducing proximal tubule uptake of IsoLG-modified apoAI and preserving lymphatic endothelial cell functionality.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

MicroRNA-148b regulates megalin expression and is associated with receptor downregulation in mice with unilateral ureteral obstruction.

Megalin is a multiligand, endocytic receptor that is important for the normal, proximal tubule reabsorption of filtered proteins, hormones, enzymes, essential nutrients, and nephrotoxins. Megalin dysfunction has been associated with acute, as well as chronic kidney diseases. Tubular proteinuria has been observed following unilateral ureteral obstruction (UUO), suggesting megalin dysfunction; however, the pathophysiological mechanism has not been determined. To identify potential regulators of megalin expression, we examined renal microRNAs (miRNAs) expression and observed an upregulation of microRNA-148b (miR-148b) in obstructed mouse kidneys 7 days after UUO, which was associated with a significant reduction in proximal tubule megalin expression and accumulation of megalin ligands. By in silico miRNA target prediction analysis, we identified megalin messenger RNA (mRNA) as a potential target of miR-148b and confirmed using a dual-luciferase reporter assay that miR-148b targeted the 3'-untranslated region of the megalin gene. Transfection of LLC-PK1 cells with miR-148b mimic reduced endogenous megalin mRNA and protein levels in a concentration-dependent manner, while transfection with miR-148b inhibitor resulted in an increase. Our findings suggest that miR-148b directly downregulates megalin expression and that miR-148b negatively regulates megalin expression in UUO-induced kidney injury. Furthermore, the identification of a miRNA regulating megalin expression may allow for targeted interventions to modulate megalin function and proximal tubule uptake of proteins, as well as other ligands.

Abstract P118: The Complexity of the Albuminuria Development in Salt-sensitive Hypertension

Hypertension is one of the most prevalent diseases worldwide, and is a major risk factor for developing albuminuria, renal failure and cardiovascular diseases. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a canonical rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate complex changes in renal function that occur during salt-induced hypertension. Following a high salt (HS) diet, SS rats exhibited elevated blood pressure, accompanied by increased glomerular sieving of albumin (GSC alb =0.0686) and decreased serum albumin (-Δ0.54 g/dL), which corresponded to increased daily filtered albumin up to 3.7 vs 0.8 g at normal diet. Pathologically, hypertensive animals had significant tubular damage as indicated by increased prevalence of granular casts (+Δ2.20 casts/image), dilation/expansion and necrosis of PT epithelial cells, increased vascular injury (+Δ0.61 leakage/image), and progressive inflammation. HS diet significantly reduced total PT uptake of albumin, which also coincided with reduced transcellular transport of albumin back into circulation. Collectively, these results indicate that both the glomerulus and the proximal tubule contribute to albuminuria and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level.

Megalin in acute kidney injury: foe and friend

The kidney proximal tubule is a key target in many forms of acute kidney injury (AKI). The multiligand receptor megalin is responsible for the normal proximal tubule uptake of filtered molecules, including nephrotoxins, cytokines, and markers of AKI. By mediating the uptake of nephrotoxins, megalin plays an essential role in the development of some types of AKI. However, megalin also mediates the tubular uptake of molecules implicated in the protection against AKI, and changes in megalin expression have been demonstrated in AKI in animal models. Thus, modulation of megalin expression in response to AKI may be an important part of the tubule cell adaption to cellular protection and regeneration and should be further investigated as a potential target of intervention. This review explores current evidence linking megalin expression and function to the development, diagnosis, and progression of AKI as well as renal protection against AKI.

Circulating Micro–RNAs in Acute Kidney Injury

Acute kidney Injury (AKI) remains a diagnostic and therapeutic dilemma. Translation of excellent basic science information into therapeutic advances in AKI now depends on early diagnosis and drug delivery before elimination of the potential for this target to influence cell injury. For instance,

Neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury--where do we stand today?

Neutrophil gelatinase-associated lipocalin (NGAL) is the first member of a new generation of diagnostics called ‘renal biomarkers’ that may be just a few steps away from entering clinical practice. What does this new test measure? Is this the new ‘troponin for the kidney’? What are the most important conclusions from clinical studies on NGAL? What are their limitations? How will NGAL affect clinical practice? Nephrologists will soon encounter these questions more frequently. Hence, in this editorial, we will attempt to briefly summarize the current state of the NGAL field. NGAL, a small 25-kDa protein of the lipocalin family, was discovered through a genome-wide analysis of kidney genes that are induced in response to experimental acute kidney injury (AKI) in animals [1]. NGAL was among the top upregulated genes in damaged kidneys and fulfilled the criteria of a promising biomarker of tubular damage since it was a secreted tubular protein that entered both urine and serum rapidly after the onset of AKI. Markedly, increased levels of NGAL in blood and urine could be detected following ischaemia-reperfusion injury in mice [1]. The early preclinical studies prompted a large and rapidly expanding body of clinical trials that addressed the utility of NGAL in diagnosing AKI. Studies in patients undergoing cardiopulmonary bypass revealed marked increases of plasma and urinary NGAL levels as early as 2 h after surgery in patients that developed AKI [2,3]. This increase in NGAL preceded the increase in serum creatinine by 24–48 h. Similar increases in NGAL levels were observed in other clinical settings, including contrast-induced AKI, sepsisassociated AKI and AKI following kidney transplantation [4–7]. Furthermore, several studies suggested that elevated NGAL levels are predictive of poor clinical outcomes in AKI, including dialysis initiation and mortality, even when adjusted for conventional predictors, such as creatinine levels [2,8–10]. Based on findings in mice and patients, a model of NGAL trafficking along the nephron was developed (Figure 1) which supported the concept of urinary or serum NGAL levels as biosensors of kidney damage [11]. NGAL messenger RNA and protein expression are markedly induced in tubules of the damaged kidney, resulting in elevated urinary and plasma levels of NGAL. In addition, circulating NGAL is filtered in the glomerulus but undergoes rapid and effective proximal tubular reabsorption unless the proximal tubule uptake machinery is damaged [12]. As a

Expression of renal cubilin and its potential role in tubulointerstitial inflammation induced by albumin overload

Sustained proteinuria is an independent risk factor leading to kidney fibrosis and end-stage renal failure. Over-reabsorption of filtered proteins, notably albumin, has been proved to trigger interstitial inflammation and fibrosis in proteinuric renal disease. Cubilin, an endocytic receptor expressed on the renal tubular brush border, is responsible for albumin reabsorption in physiologic condition. However, little is known about whether it is required for activation of tubular cells induced by albumin overload. In this work, we investigated the change of cubilin expression and its potential role in albumin-induced up-regulation of chemokines synthesis in vivo and in vitro. Twenty-six patients with nephrotic syndrome were enrolled in this study. Proximal tubule uptake of albumin, expression of apical membrane cubilin and infiltrating cells in kidney interstitium were determined by immunocytochemistry. In vitro, the transcription of cubilin in HK2 cells after exposure to albumin was analyzed by real-time PCR. Endocytosis of albumin in HK2 cells was examined by fluorescent microscope. The influence of inhibition of cubilin on albumin-induced expressions of monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and secreted (RANTES) was investigated by Western blot. The intensity of luminal cubilin and tubular accumulation of albumin were significantly increased in nephrotic kidneys. The expression of MCP-1 and RANTES was up-regulated, and there were spatial relationships in localization between these chemokines and cubilin as well as intracellular albumin in kidney tissues. Infiltration of CD-3 and ED-1-positive cells was predominant in tubulointerstitial areas displaying signs of increases of cubilin expression and albumin accumulation. In vitro, the transcription of cubilin mRNA in HK2 cells was enhanced after 24 h exposure to albumin in a dose-dependent manner. Inhibition of endocytosis of albumin by antisense cubilin nucleotide markedly reduced expression of MCP-1 and RANTES. Cubilin was required for handling a greater amount of protein in nephrotic status and albumin-induced production of MCP-1 and RANTES by renal tubular cells, which further initiated tubulointerstitial inflammation in proteinuric disease.

The kidney in vitamin B12and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins

Over the past 10 years, animal studies have uncovered the molecular mechanisms for the renal tubular recovery of filtered vitamin and vitamin carrier proteins. Relatively few endocytic receptors are responsible for the proximal tubule uptake of a number of different vitamins, preventing urinary losses. In addition to vitamin conservation, tubular uptake by endocytosis is important to vitamin metabolism and homeostasis. The present review focuses on the receptors involved in renal tubular recovery of folate, vitamin B12, and their carrier proteins. The multiligand receptor megalin is important for the uptake and tubular accumulation of vitamin B12. During vitamin load, the kidney accumulates large amounts of free vitamin B12, suggesting a possible storage function. In addition, vitamin B12 is metabolized in the kidney, suggesting a role in vitamin homeostasis. The folate receptor is important for the conservation of folate, mediating endocytosis of the vitamin. Interaction between the structurally closely related, soluble folate-binding protein and megalin suggests that megalin plays an additional role in the uptake of folate bound to filtered folate-binding protein. A third endocytic receptor, the intrinsic factor-B12 receptor cubilin-amnionless complex, is essential to the renal tubular uptake of albumin, a carrier of folate. In conclusion, uptake is mediated by interaction with specific endocytic receptors also involved in the renal uptake of other vitamins and vitamin carriers. Little is known about the mechanisms regulating intracellular transport and release of vitamins, and whereas tubular uptake is a constitutive process, this may be regulated, e.g., by vitamin status.

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.

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.

Effect of low-molecular-weight dextran on proteolytic and nonproteolytic lysosomal enzymes in isolated segments of rat proximal tubule.

Dextrans undergo glomerular filtration and are taken up by endocytosis into lysosomes of proximal tubule cells. We measured the activities of the intralysosomal proteolytic enzymes cathepsin B and L and the nonproteolytic acid phosphatase in microdissected segments of rat proximal tubules by means of fluorescent ultramicroassays. Cathepsin activities were increased following dextran injection in a dose-dependent manner. Cathepsin stimulation was not dextran-specific since enhanced proximal albumin uptake also increased cathepsin activities. Acid phosphatase remained almost unchanged. We conclude that increased proximal tubule uptake of macromolecules is accompanied by a rather specific stimulation of proteolytic enzymes in the lysosomes. For proteins this constitutes an adequate response to increased protein uptake to maintain the steady state between uptake and catabolism of proteins in lysosomes. For the nondigestible dextran the cathepsin induction is an inadequate response. Since lysosomal cathepsins are involved in breakdown of intracellular proteins, it is conceivable that the increased activities reflect increased cellular autophagy leading to proximal tubule damage.