Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

<div><p>The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an <i>EC</i>_<i>50</i> of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.</p> </div

NGAL-R mediates apical-to-basolateral transcytosis of PC3, MT and Tf in confluent Caco-2 BBE cell monolayers.

<p>Transcytosis of fluorescence-labelled ligands through confluent monolayers of Caco-2 BBE cells plated on Transwell culture inserts. Fluorescent ligands of hNGAL-R were added to the apical compartment at a final concentration of 700 nM and the apical decrease as well as the basolateral increase of the concentration of the fluorescent ligands was recorded over a period of 8 h (<b>A</b>, <b>C</b> and <b>E</b>). Confluent colon-like Caco-2 BBE monolayers display rapid NGAL-R dependent apical-to-basolateral transcytosis of A488-PC3 (<b>A</b>), A546-MT (<b>C</b>) and A546-Tf (<b>E</b>), which saturates at 4-8 h. Means ± SEM of 9-14 experiments are shown. Data are fitted to a one-phase exponential decay function. For further details, see Experimental Procedures. Experiments with or without 500 pM hNGAL in the apical compartment were also performed to determine the contribution of hNGAL-R to transcytosis (<b>B</b>, <b>D</b>, <b>F</b> and <b>G</b>). To obtain a quantitative estimate of the apical decrease and basolateral delivery of the fluorescent ligands due to transcytosis and to determine the impact of hNGAL on this process experimental data were integrated over a period of 8 h and expressed as “area under the curve” (AUC) (<b>B</b>, <b>D</b> and <b>F</b>). hNGAL significantly reduces the apical decrease of fluorescent ligand concentration. Means ± SEM of 9-14 experiments are shown; * <i>P</i><0.05; ** <i>P</i><0.01. A plot of the ratio of basolateral to apical AUC of fluorescent PC₃, MT and Tf, as shown in B, D and <b>F</b>, provides an estimate of transcytosis efficiency as well as the proportion of intracellular ligand trapping in the absence or presence of 500 pM hNGAL (<b>G</b>). In controls, intracellular A546-MT trapping is significantly increased compared to A488-PC₃ or A546-Tf, but not with hNGAL. The data are expressed as a percentage of apical AUC. Means ± SEM of 9-14 experiments are shown; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Expression of hNGAL-R in Caco-2 BBE cells.

<p>RT-PCR for hNGAL-R and GAPDH in colon-like Caco-2 BBE cells (<b>A</b>). A PCR product of 296 bp is amplified from colon-like Caco-2 BBE cell cDNA using specific primers for human NGAL-R and reverse transcriptase (+RT), but not in the control reaction without reverse transcriptase (-RT). The housekeeping gene human GAPDH is used as a control. A 326 bp PCR product is only amplified in the presence of reverse transcriptase (+RT). Immunoblotting of colon-like Caco-2 BBE cell homogenate (Ho) and plasma membranes (PM) (<b>B</b>). Specific signals are detected in PM of colon-like Caco-2 BBE cells with antibodies against hNGAL-R (α-CT-24p3-R; 1:500) and the α1-subunit of Na⁺,K⁺-ATPase (1:500). Live immunofluorescence staining of non-permeabilized colon- and duodenum-like Caco-2 BBE cells (<b>C</b> and <b>D</b>). Immunofluorescence staining with α-NT-24p3-R (1:100) reveals hNGAL-R expression (red fluorescence) at apical (asterisks) and lateral plasma membranes (arrows) of colon-like Caco-2 BBE cells (<b>C</b>). No staining for hNGAL-R is detected in duodenum-like Caco-2 BBE cells (<b>D</b>).</p

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

<div><p>Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.</p></div

LC MS/MS analysis of proteins pulled down by H95 preferentially in samples from aldosterone treated rats but not detected in IP without beads or without antibody.

<p>Ctrl: control, Aldo: aldosterone treated, n.d.: not detected.</p

Immuno-gold electron micrographs of anti-RPL22 stained cryo-sections.

<p>Top left panel is a cellular overview of a distal renal tubule and the red box indicates the area magnified in left bottom panel. Here, the red box corresponds to the highest magnification electron micrograph (right panel). Arrows point to gold particles.</p

Identification of the tubular segment and cell structures displaying aldosterone-induced punctate immunoreactivity.

<p>Example of double immunofluorescence staining with RPL22 (green) and antibodies against tubule markers (all in red): NKCC2 (A), NCC (B), calbindin-D_28K (C), H⁺-ATPase (D), and AQP2 (E & F) in kidney cortex from aldosterone treated rats. Fluorescence signals are overlaid on the corresponding DIC image.</p