Lipocalin-2 (24p3/Neutrophil Gelatinase-associated Lipocalin (NGAL)) Receptor Is Expressed in Distal Nephron and Mediates Protein Endocytosis*

Background: Localization and function of the lipocalin-2/NGAL/24p3 receptor (24p3R) in the kidney are unknown. Results: 24p3R is expressed in apical plasma membranes of the distal nephron and mediates high-affinity protein endocytosis in renal cells. Conclusion: 24p3R contributes to protein endocytosis and nephrotoxicity in distal nephron segments. Significance: This is the first study to investigate localization and function of 24p3R in relevant epithelia. In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+7-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+7-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123 ± 50 nm was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+7-MT toxicity in distal nephron segments.

toxicity affects the DT as well, both in experimental animals (20) and in Cd 2ϩ -exposed workers (21), although the mechanism of distal tubule damage has remained unresolved.
Recently, urinary neutrophil gelatinase-associated lipocalin (NGAL, rodent 24p3/human lipocalin-2) has emerged as a novel high-sensitivity biomarker of kidney damage that is upregulated by kidney injuries (22)(23)(24)(25). NGAL binds iron through association with bacterial (26) as well as mammalian siderophores (27,28), thereby affecting iron homeostasis of target cells and hence their survival and proliferation. During the course of renal insults, NGAL is secreted by the thick ascending limb of Henle loop, DT, and CD and excreted into the urine (25). It has been postulated that uptake of NGAL-siderophoreiron complexes by damaged nephron segments limits injury and promotes regeneration of damaged epithelia (29), but the underlying mechanisms are still unclear. Previously, Devireddy et al. (30) cloned a receptor for murine 24p3 whose affinity for 24p3/NGAL is almost 1000-fold higher (K D ϳ92 pM) (31) than that of megalin (K D ϳ60 nM) (32). The 24p3 receptor (24p3R) protein is expressed in the kidney and Madin-Darby canine kidney cells (30). Surprisingly, its localization and function in the nephron have not yet been investigated.
We have generated specific antibodies against rodent 24p3R and investigated its localization in rodent kidney as well as its function in transiently transfected Chinese hamster ovary (CHO) and renal DT cells. Here we demonstrate that 24p3R is expressed in the DT and medullary CD and is responsible for high-affinity RME of the low molecular weight protein MT as well as high molecular weight proteins, transferrin, and albumin in cultured cells. Furthermore, 24pR mediates Cd 2ϩ 7 -MT toxicity in cultured cells.
Immunofluorescence and Peroxidase Light Microscopy of Mouse Kidney-Experiments on mice were carried out in strict accordance with state health and ethical regulations. Care of animals was in accordance with institutional guidelines. The protocol for sacrifice of animals was approved by the Committee on the Ethics of Animal Experiments of the University of Freiburg (permit number X-07/27A). C57/BL6 male adult mice were anesthetized with ketamine (90 mg/kg of body weight) and xylazine (12 mg/kg of body weight) and fixed with 4% PFA by perfusion through the heart. Kidneys were excised, postfixed in 4% PFA overnight at 4°C, dehydrated, and embedded in paraffin. Staining was performed as described previously (33) Sections were incubated with ␣-CT-24p3R (1 g/ml) and with Cy3 (1:600) or HRP (1:50) anti-rabbit IgG. For double immunofluorescence, fixed ␣-CT-24p3R was incubated together with anti-calbindin antibody. Sections were viewed with a Zeiss Axioplan2 fluorescence microscope with ApoTome module (Jena, Germany). Control experiments were performed by substituting the primary antibody with preimmune serum (supplemental Fig. 1A).
Immunohistochemistry of Rat Kidney-Animal housing conditions and experiments on male Wistar rats (Taconic Europe) were approved by the Danish Ministry of Justice. All procedures have been described in detail previously (34). ␣-CT-24p3R was used at 0.25 g/ml. For preincubation experiments, affinitypurified antibodies were preincubated at 4°C overnight with 10-fold molar excess of antigenic peptide. Images were taken using an HCX PL APO 63 oil objective lens on a Leica DMRE microscope (Leica Microsystems, Herlev, Denmark).
Transient Transfections-The full-length clone of rat 24p3R (GenBank accession number NM_177421.3) (IRAKp961A-13231Q, I.M.A.G.E. clone 7312276) was obtained from imaGenes (Berlin, Germany) and cloned into the expression vector pcDNA3.1ϩ carrying neomycin (G418) and ampicillin resistance (Invitrogen). CHO cells were transiently transfected 24 after seed-ing using Lipofectamine 2000 in medium without antibiotics. After 6 h, medium was replaced, and cells were incubated for an additional 48 h. For isolation of plasma membranes, transfected cells were transferred to 75-cm 2 flasks 48 h after transfection. 24p3R-positive cells (24p3R-CHO) were selected in 500 g/ml G418 supplemented medium for 2 weeks with medium change every 48 h.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-cDNA was synthesized from 2 g of total RNA, and RT-PCR was carried out with Maxima TM Hot Start Green PCR master mix (Fermentas). Primers, cycling protocols, GenBank accession numbers, and product sizes are shown in supplemental Table 1.
Isolation of Plasma Membranes-Plasma membranes were obtained by differential ultracentrifugation at 4°C. Cells were homogenized by nitrogen pressure cavitation in a Parr Instruments 45-ml cell disruption vessel (Moline, IL) for 2 min at 350 p.s.i. To remove unbroken cells, nuclei, and large debris, homogenate was centrifuged at 300 ϫ g for 10 min. Mitochondria were pelleted at 4000 ϫ g for 10 min, and the resulting supernatant was centrifuged for 30 min at 35,000 ϫ g to yield a microsomal fraction in the pellet. Plasma membranes were isolated by discontinuous sucrose gradient ultracentrifugation exactly as described previously (37). The plasma membrane fraction was washed with 10ϫ volume of 5 mM Tris/HCl, pH 7.4, centrifuged for 30 min at 233,000 ϫ g, and resuspended in 5 mM Tris/HCl, pH 7.4, supplemented with protease inhibitor mixture. Plasma membrane enrichment was verified by immunoblotting and showed about 20-fold enrichment of Na ϩ ,K ϩ -ATPase (data not shown).
Cell Viability Assay-Cd 2ϩ 7 -MT was prepared from rabbit apo-MT (19), and cell viability was determined by the MTT test (36), both as described previously.
Live Immunofluorescence Staining of Cultured Cells-Experiments with non-permeabilized cells (see Fig. 3, B and D) were performed as described elsewhere (41) with the following modifications. Cells were blocked for 1 h with 1% bovine serum albumin and incubated with 5 g/ml ␣-NT-24p3R for 2 h followed by Alexa Fluor 488-conjugated secondary antibody (1:600) for 1 h before fixation with 4% PFA at room temperature for 30 min.
Quantification of 24p3R-mediated Internalization of Fluorescent Dye-labeled Proteins-MT was coupled to Alexa Fluor 546, as described previously (40). Concentration dependence of uptake of Alexa Fluor 546-coupled MT, Alexa Fluor 546-Tf, and FITC-albumin was estimated following a 24-h incubation period in serum-free medium. In addition, timedependent uptake of 1.4 M Alexa Fluor 546-coupled MT was determined after 6 -36 h in serum-free medium. For each experiment, 10 images of transfected and non-transfected cells were acquired and quantified as described earlier (39) or by counting the percentage of transfected cells with internalized fluorescent dye-labeled proteins (positive cells).
To determine 24p3R-specific uptake of fluorescent dye-labeled proteins, fluorescence values in the vector-transfected cells were subtracted from the fluorescence values in the corresponding 24p3R-transfected cells.
Microscale Thermophoresis-Binding interactions between 24p3R-CHO plasma membrane vesicles and the ligand Alexa Fluor 546-MT were measured using microscale thermophoresis, which determines differences in the thermophoretic properties of single molecules or ligands and ligand-receptor complexes in microliter volumes on a Monolith NT.015 (Nano-Temper Technologies GmbH, Munich, Germany). Thermophoresis is based on the thermodiffusive properties of biomolecules in liquids (42)(43)(44). Binding of a fluorescent ligand to its receptor changes the hydration shell of the receptor and thereby its entropy. An infrared (IR) laser diode creates a hot spot, which induces movement of the complex along the temperature gradient and depends on the concentration of the ligand-receptor complex. For each plasma membrane preparation, specific binding of Alexa Fluor 546-MT (0.7 M) to 24p3R membrane vesicles was compared with nonspecific binding to vector membrane vesicles by serial vesicle dilutions in 5 mM Tris/HCl, pH 7.4. The vesicle concentration showing the greatest "signal-to-noise" ratio was used for determining EC 50 values. Briefly, a titration series with constant 24p3R and varying ligand Alexa Fluor 546-MT was preincubated for 30 min at 37°C in the dark to achieve ligand-receptor binding equilibrium. All measurements were performed in hydrophobic capillaries at room temperature. Plotting normalized fluorescence F norm at a given time t against the Alexa Fluor 546-MT concentration resulted in a binding curve, and EC 50 values were obtained from fitting the binding curves with the Hill equation assuming a Hill coefficient of 1.0.
Statistics-Unless otherwise indicated, the experiments were repeated at least three times. Analyses by unpaired Student's t test or for more than two groups, one-way analysis of variance assuming equality of variance with Levene's test, and Tukey's post hoc test were carried out with GraphPad Prism 5.0. Results with p Ͻ 0.05 were considered to be statistically significant.

RESULTS
24p3R Is Expressed Apically in Rodent DCT and IMCD Cells-The 24p3R has been previously found by immunoblotting to be expressed in mouse kidney tissue and Madin-Darby canine kidney cells, a DT cell line (30). Using the specific antibody against the C-terminal sequence of 24p3R (␣-CT-24p3R), immunolabeling was present in both renal cortex and medulla in mouse kidney (Fig. 1, A and B). Medullary outer stripe and medullary CD revealed strong 24p3R immunostaining, which appeared sharp and partly localized to apical cell membranes. In renal cortex, prominent 24p3R apical and intracellular immunostaining was observed in the distal convoluted tubules (DCT) (Fig. 1, C and D, arrows). In contrast, in glomeruli and PT 24p3R, expression was not detectable. Double labeling for 24p3R and calbindin, a marker for late DCT and connecting tubule (calbindin is expressed in some cortical collecting duct cells in the rat, but its abundance is drastically reduced when compared with late DCT and connecting tubule) (45,46), showed a complex cellular labeling pattern (Fig. 1E).The majority of DCT cells demonstrated co-localization of the two proteins, 24p3R apically and calbindin intracellularly. Other cells, however, were 24p3R-positive but calbindin-negative, suggest- ing that 24p3R is expressed in both early and late DCT. When sections were incubated with preimmune serum, a diffuse, weak, nonspecific labeling throughout the renal tissue could be observed, demonstrating specificity of the labeling obtained with ␣-CT-24p3R (supplemental Fig. 1A).
In rat kidney, 24p3R was also predominantly expressed in the DCT cells, both apically ( Fig. 2A, arrows) and intracellularly ( Fig. 2A). Glomeruli and other cortical tubular segments (including PT) were not stained ( Fig. 2A, asterisks). Another location of strong 24p3R expression was inner medullary collecting ducts (IMCD) (Fig. 2, B-D). At higher magnification, 24p3R was clearly localized luminally (Fig. 2, C and D, arrows) and laterally (Fig. 2, C and D, arrowheads). Intracellular punctate 24p3R staining was also detected (Fig. 2, C and D), suggesting intracellular vesicles. No staining was observed after preabsorption of the peptide with the immunizing peptide (not shown). Overall, the distribution of 24p3R was comparable in mouse and rat kidney. Similar results were obtained with ␣-NT-24p3R, although the antibody showed more nonspecific background.
24p3R Is Expressed in the Rodent Distal Tubule Cell Line mDCT209-The expression of 24p3R was further investigated in immortalized DCT (mDCT209 (35)) and PT (WKPT-0293 Cl.2 (36)) cell lines. As shown in Fig. 3A, strong staining of 24p3R was detected with the specific antibody against the NH 2terminal sequence of 24p3R (␣-NT-24p3R) in mDCT209 cells by laser scanning confocal microscopy, which extended to cellular borders, whereas no major staining was detected in WKPT-0293 Cl.2. This is in agreement with the distribution of 24p3R in the corresponding nephron segments in rodent kidneys ( Figs. 1 and 2). Plasma membrane localization of 24p3R in mDCT209 cells was confirmed by live staining with ␣-NT-24p3R using non-permeabilized cells (Fig. 3B, upper panel,  arrows). Both rat and murine full-length cDNAs of 24p3R contain a single open reading frame of 520 amino acids (30), and an ϳ60-kDa polypeptide should be detected. No signal was detectable for 24p3R in both homogenate and plasma membranes (PM) of WKPT-0293 Cl.2 cells (Fig. 3B). In contrast, a double band at ϳ60 kDa was clearly detectable in PM of mDCT209 cells (Fig. 3B, lower panels, arrows), but not in homogenate, indicating an enrichment of 24p3R. Furthermore, when ␣-CT-24p3R was preabsorbed with a 10-fold excess of the antigenic peptide, the double band at ϳ60 kDa vanished, confirming specificity of the signal (Fig.  3B). An additional signal at ϳ140 kDa was also detected in mDCT209 cells, which disappeared by co-incubation with antigenic peptide (Fig. 3B) and could represent a 24p3R dimer. These data were complemented by RT-PCR for 24p3R in mDCT209 cells, which yielded only one band (Fig. 3C).
24p3R Mediates Endocytosis of MT, Tf, and Albumin in 24p3R-CHO and mDCT209 Cells-In contrast to HEK293 cells in which expressed 24p3R was trapped intracellularly (supplemental Figs. 1B and 2), 4 CHO cells expressed 24p3R in the PM, thus allowing study of putative functions of the receptor. Transient transfection of 24p3R induced overexpression of the gene product (Fig. 3, D-F, supplemental Fig. 1C) that could be detected in the PM using both ␣-NT-24p3R (Fig. 3D, arrows) and ␣-CT-24p3R (Fig. 3E, supplemental Fig. 1D, arrows). Moreover, live staining of non-permeabilized cells clearly demonstrated surface staining of 24p3R using ␣-NT-24p3R (Fig.  3D, arrowheads).
Next we investigated the internalization of the filtered low molecular weight protein MT as well as of the high molecular weight proteins albumin and Tf, which are all ligands of the megalin-cubilin-amnionless complex in the PT ( (18,19); reviewed in Refs. 4 -6). 24p3R-and vector-CHO cells were exposed to 0. 35  . Immunohistochemical localization of 24p3R in the rat kidney. A, in the renal cortex, using ␣-CT-24p3R 24p3R localizes to tubules that morphologically resemble the DCT. Diffuse staining of the DCT cells is observed, but some labeling is associated with the apical plasma membrane (arrows). PT are not labeled (asterisks). G ϭ glomerulus. B, 24p3R is detected throughout the IMCD. C and D, at higher magnification, 24p3R labeling is clearly associated with both the apical (arrows) and the lateral cell membrane (arrowheads). 546-Tf, or FITC-albumin for 24 h. The magnitude of ligand uptake in 24p3R-transfected cells was about 2-3-fold that of vector-transfected cells and showed high-affinity saturating at ϳ1 M of the ligand with the exception of MT, which showed the greatest magnitude of uptake (Fig. 4, A and B). Furthermore, co-localization of internalized Alexa Fluor 546-MT (1.4 M for 18 h) with the lysosomal marker cathepsin-L was demonstrated in 24p3R-CHO cells, thus proving 24p3R-mediated endocytosis and subsequent trafficking to lysosomes (Fig. 4C). Similarly, Alexa Fluor 546-MT (2.8 M for 24 h) was internalized by mDCT209 cells, which express 24p3R (Fig. 4D).
24p3R Mediates Cd 2ϩ 7 -MT Toxicity in 24p3R-CHO and mDCT209 Cells-In previous studies (39,47), we demonstrated that in PT cells, the divalent metal transporter DMT1 is expressed in lysosomal membranes where it mediates efflux of Cd 2ϩ from endocytosed Cd 2ϩ 7 -MT into the cytosol and subsequent mitochondrial apoptosis (reviewed in Refs. 48 and 49). Here we tested whether this mechanism is also operative in 24p3R-CHO cells as another approach to monitor the function of 24p3R. Hence, we measured cell viability with and without Cd 2ϩ 7 -MT as an "end point" of 24p3R-mediated RME. As shown in Fig. 5A, cell viability, as measured by MTT assay, significantly decreased as a function of the Cd 2ϩ 7 -MT concentration (up to 2.8 M MT saturated by 20 M Cd 2ϩ for 24 h), which clearly mirrored the concentration dependence of Alexa Fluor 546-MT internalization (Fig. 5A). A similar relationship between cell viability and Alexa Fluor 546-MT internalization was observed in the kinetics of a fixed Cd 2ϩ 7 -MT concentration (1.4 M MT and 9.8 M Cd 2ϩ ) for 0 -36 h (Fig. 5B). Similarly, mDCT209 cells, which express 24p3R, showed a concentration-dependent decrease of cell viability induced by Cd 2ϩ 7 -MT (Fig. 5C). To prove that both Alexa Fluor 546-MT internalization and Cd 2ϩ 7 -MT cell death were caused by 24p3R in 24p3R-CHO cells, MT binding was competed with a saturating concentration of 500 pM 24p3 (K D for 24p3R ϳ92 pM (31)). As shown in h) were significantly reduced (but not abolished, possibly due to concomitant 24p3R-independent endocytosis) by co-incubation with 500 pM 24p3 (Fig. 5, G and H).

DISCUSSION
We investigated the distribution of 24p3R in rodent kidney, which was identically distributed in mouse and rat kidney ( Figs. 1 and 2). In the cortex, it was mainly found in the DCT, but not in the PT (Figs. 1, C-E, and 2A), and in the medulla, strong levels of expression were detected in IMCD (Figs. 1, A and B, and 2, B-D), both intracellularly and in plasma membranes. This differential 24p3R expression was also observed in cell lines derived from proximal and distal

Renal NGAL/24p3/Lipocalin-2 Receptor and Protein Endocytosis
nephron segments (Fig. 3). Interestingly, 24p3R expression was also detected in primary cultures of rat collecting duct cells. 5 Besides its established function as a receptor for lipocalin-2/ NGAL/24p3 (30,31), a putative role of 24p3R in RME of (metalbinding) proteins was tested with a focus on the putative ligands MT, Tf, and albumin based on the following rationale. (i) These proteins bind metals, such as iron, zinc, or cadmium, but also calcium (17, 50 -52); (ii) they are filtered by the kidney glomer-uli (17,53); and (iii) they are ligands of the megalin-cubilinamnionless receptor complex in the PT (4).
In CHO cells overexpressing 24p3R, the magnitude of uptake of all three proteins was increased by maximally 2-3-fold (Fig.  4B). Putative ligands were internalized at sub-to low micromolar concentrations (Fig. 4B), indicating high-affinity binding to the receptor. From microscale thermophoresis experiments, an EC 50 of MT binding to 24p3R was calculated to 123 Ϯ 50 nM, which indicates a Ͼ500-fold higher affinity than its affinity to the megalin receptor (18). Moreover, uptake of MT and Tf was competed by picomolar concentrations of 24p3 (Fig. 5, D, F, and   5 B. Edemir, C. Langelueddecke, and F. Thévenod, unpublished results. G), demonstrating high-affinity binding of 24p3 to its receptor and proving specificity of binding. The significant uptake of all three proteins in vector-transfected CHO cells indicates that additional uptake pathways are present in CHO cells (e.g. Tf receptor or fluid-phase endocytosis), and this could also account for the incomplete block of Alexa Fluor 546-MT uptake and Cd 2ϩ 7 -MT toxicity by 500 pM 24p3 in mDCT209 cells (Fig. 5, G and H). Using embryonic collecting duct or Madin-Darby canine kidney cells, Yang et al. (54) demonstrated that iron bound to Tf or 24p3 is taken up by two independent pathways with no overlapping specificity. This work, which assumes mutually exclusive uptake mechanisms of Tf and 24p3 in renal cells, apparently contradicts our observations of 24p3R-specific Tf binding and endocytosis in CHO cells that is prevented by 500 pM 24p3 (Fig. 5F). The work of Yang et al. (54) showed fluorescent images of Alexa Flour 488-Tf uptake in the presence of 24p3. Unfortunately, uptake of Alexa Fluor 488-Tf in the absence of 24p3 was not shown, and quantitative analysis of Alexa Fluor 488-Tf uptake was not performed, which would have been helpful to distinguish between 24p3R-and Tf receptor-mediated Tf uptake. Here we carried out quantitative analysis of the fluorescence data to clarify the competition of Tf uptake by 24p3 in vector-and 24p3R-transfected CHO cells (Figs. 4B and 5). The observed Tf uptake in the presence of 24p3 in the study by Yang et al. (54) and in our study most likely represents uptake via a Tf receptor (55). Assuming a K D of Tf for the Tf receptor of about 5 nM (56) and a K D of Tf for the 24p3R in the same magnitude as the EC 50 of metallothionein (ϳ100 nM), a significant proportion of Tf should be taken up by 24p3R in the concentration range tested (0.35-2.8 M Tf).
Moreover, proteins internalized via 24p3R were trafficked to lysosomes, as shown for MT (Fig. 4C), suggesting lysosomal degradation of endocytosed proteins. This is important because this observation is in line with the data by Yang et al. (54), who , the temperature jump after applying the IR laser beam (movement of unbound ligand, which is a very rapid process), the thermophoresis (movement of ligand-receptor complexes, which usually lasts ϳ30 s), and the re-equilibration of all reagents after switching off the laser (with two components, a rapid one for the unbound ligand and a slow phase due to back diffusion of complexes). The IR laser is switched on at t ϭ 5 s, the fluorescence decreases as the temperature increases, and the labeled MT moves away from the heated spot because of thermophoresis. When the IR laser is switched off, the molecules diffuse back. The thermophoretic depletion of unbound Alexa Fluor 546-MT is about four times that of Alexa Fluor 546-MT bound to 24p3R.
showed co-localization of endocytosed 24p3 in renal cells with acidic intracellular compartments expressing DMT1. We (39,47) and others (57) have previously demonstrated expression of DMT1 in lysosomes of PT cells and hepatocytes. Therefore we used the property of MT as a high-affinity Cd 2ϩ -binding protein to further characterize RME via 24p3R. In PT cells, endocytosed Cd 2ϩ 7 -MT is degraded in lysosomes and free Cd 2ϩ transported via DMT-1 into the cytosol, which induces cell death (39,58). Indeed, a similar mechanism was operative in 24p3R-transfected CHO cells and mDCT209 cells and Cd 2ϩ 7 -MT-induced cell death could be prevented by co-incubation with 24p3 (Fig. 5, E and H). Overall, these data strongly support the notion that the whole machinery for RME of proteins via 24p3R is present in the distal nephron where it may operate under certain physiological and pathophysiological circumstances.
Although bulk protein reabsorption takes place in the PT, experimental evidence has demonstrated that a small but significant proportion of filtered proteins is reabsorbed by the distal nephron (3,(7)(8)(9)(10)(11)(12). Considering that megalin-cubilin-amnionless operates as a high-capacity, low-affinity receptor complex for endocytotic reabsorption of filtered proteins (4, 6), a certain proportion of these proteins should bypass reabsorption in the PT, either as the consequence of low affinity (e.g. MT with a K D of ϳ100 M (18)) or due to exhaustion of the reabsorptive capacity of the system (e.g. following glomerular or PT damage and ensuing proteinuria) (6,13,14,53). A high-affinity protein reabsorption system in the distal nephron, as suggested by the present study, could contribute to exhaustive protein reabsorption by the nephron under physiological conditions, as deduced from previous micropuncture studies, or limit protein losses associated with renal diseases, including various forms of inherited or acquired Fanconi syndrome (53).
Chronic cadmium toxicity targets the kidney PT (48), largely due to RME of filtered Cd 2ϩ 7 -MT via megalin (18,19). However, other segments of the nephron are also affected, although the literature on the subject is scarce (20,21). It is conceivable that some filtered MT/Cd 2ϩ 7 -MT escapes PT reabsorption and reaches distal nephron segments (see above). MT is excreted into the urine of both non-exposed and cadmiumexposed humans (for a review, see Ref. 17). In cadmium-exposed humans, urinary MT originates from the damaged PT epithelium. However, in non-exposed healthy individuals, this indicates that filtered MT indeed escapes PT reabsorption (59). Accordingly, Sabolic et al. (17) observed MT droplets in the PT lumen of adult rats, which vanished in the distal nephron, suggesting reabsorption in these nephron segments.
Lipocalin-2/NGAL/24p3 plays a role as an iron-sequestering protein in the antibacterial innate immune response (60), and lipocalin-2-deficient mice have increased susceptibility to bacterial infections in the urinary tract (24). In this process, 24p3R in the distal nephron is thought to mediate iron sequestration into epithelia and thereby promote bacteriostasis. Hence, the predominant localization of 24p3R in IMCD could participate in a line of defense against ascending infections and is compatible with the described function of 24p3 in bacteriostasis (29). In addition, lipocalin-2/NGAL/24p3 is induced by distal nephron segments and released into the urine following renal damage, suggesting its involvement in autocrine/paracrine modulation of renal epithelial defense and repair (25,29). Other postulated non-antibacterial functions of 24p3 include regulation of cell proliferation (61) or apoptosis (62) in the context of pathological conditions. However, the presence of a receptor for 24p3 in distal nephron segments suggests additional unknown functions of 24p3R and its endogenous ligand(s) in the kidney, such as adaptive response to cell stress and antiapoptosis, which are the aim of ongoing studies.