Abrogation of Protein Uptake through Megalin-Deficient Proximal Tubules Does Not Safeguard against Tubulointerstitial Injury
Franziska Theilig*,
Wilhelm Kriz,
Timo Jerichow*,
Petra Schrade*,
Brunhilde Hähnel,
Thomas Willnow,
Michel Le Hir and
Sebastian Bachmann*
* Institute of Anatomy, Charité Universitätsmedizin, and Max-Delbrück-Center for Molecular Medicine, Berlin, and Zentrum für Medizinische Forschung, Mannheim, Germany; and Institute of Anatomy, University of Zurich, Zurich, Switzerland
Address correspondence to: Prof. Sebastian Bachmann, Charité, Universitätsmedizin Berlin, Institut für Vegetative Anatomie, Philippstrasse 12, 10115 Berlin. Phone: +49-30-450-528-001; Fax: +49-30-450-528-922; E-mail: sbachm{at}charite.de
Received for publication November 22, 2006.
Accepted for publication March 12, 2007.
Sustained proteinuria and tubulointerstitial damage have beenclosely linked with progressive renal failure. Upon excess proteinendocytosis, tubular epithelial cells are thought to producemediators that promote inflammation, tubular degeneration, andfibrosis. This concept was tested in a transgenic mouse modelwith megalin deficiency. Application of an anti–glomerularbasement membrane serum to transgenic megalin-deficient mice[Cre(+)/GN] and megalin-positive littermates [Cre(–)/GN]produced the typical glomerulonephritis (GN) with heavy proteinuriain both groups. Tubulointerstitial damages correlated closelywith glomerular damages in pooled Cre(+)/GN and Cre(–)/GNmice. Owing to a mosaic pattern of megalin expression in themutant mice, Cre(+)/GN kidneys permitted side-by-side analysisof megalin-deficient and megalin-positive tubules in the samekidney. Protein endocytosis was found only in megalin-positivecells. TGF-, intercellular adhesion molecule, vascular cellularadhesion molecule, endothelin-1, and cell proliferation werehigh in megalin-positive cells, whereas apoptosis, heat-shockprotein 25, and osteopontin were enhanced in megalin-deficientcells. No fibrotic changes were associated with either phenotype.Tubular degeneration with interstitial inflammation was foundonly in nephrons with extensive crescentic lesions at the glomerulotubularjunction. In sum, enhanced protein endocytosis indeed led toan upregulation of profibrotic mediators in a megalin-dependentway; however, there was no evidence that endocytosis playeda pathogenetic role in the development of the tubulointerstitialdisease.
ESRD is defined by an irreversible decline of renal function.In most cases, the disease starts in the glomerulus and is thentransferred to the tubulointerstitium. Concerning this transfer,two major hypotheses, although not necessarily mutually exclusive,are under discussion. One is focused on the glomerular damageas the presumptive central event, causing obstruction of theinitial nephron segment and consecutive degeneration of thetubule ("glomerular hypothesis" [1]). The other, commonly assumedhypothesis refers to the loss of glomerular permselectivityand consequent protein leakage into the tubular fluid, followedby excessive protein reabsorption by the proximal tubule. Underthese conditions, proximal tubule cells are thought to producea variety of inflammatory mediators that induce peritubularinflammation, tubular damage, and fibrosis ("tubular hypothesis"[2–6]).
Protein reabsorption by the proximal tubule involves bindingto megalin, a 600-kD transmembrane glycoprotein that belongsto the LDL receptor gene family. Megalin has been introducedas the key molecule for the uptake of all major proteins thatare increased in proteinuria (7,8).
Studies in animal models and cell culture systems have shownthat protein overload on renal tubules leads to the expressionof inflammatory and profibrotic products (9–12). However,both approaches are hampered by common drawbacks, such as thedifficulty in animal models to identify links of causality betweenthe various altered parameters and the reductionist nature ofthe isolated cell system. We have made use of a transgenic mousemodel with kidney-specific megalin deficiency to study the roleof tubular endocytosis in degenerative renal damage (13). Megalin-deficientkidneys exhibit massive reduction in proximal tubular endocytosis,resulting in low molecular weight proteinuria. A key featureof this model, the mosaic remnant expression of megalin in asubset of proximal nephrons (20 to 40% [14]), first seemed tobe an obstacle for the interpretation of our results but actuallyturned out to be a major advantage that allowed us to comparethe responses to protein overload in neighboring megalin-positiveand megalin-deficient cells.
We induced glomerulonephritis (GN) in these mice and controlsto test the hypothesis that excessive protein uptake by proximaltubule cells may lead to tubulointerstitial damage. Morphologicchanges and the expression of inflammatory and cell-cycle parameterswere comparatively analyzed in megalin-expressing cells withintensive protein endocytosis and in megalin-deficient cellswith compromised endocytosis. Our results provide importantnew information with respect to the validation of the "glomerularhypothesis" as compared with the "tubular hypothesis."
Animals and Treatments
Experiments were performed in adult female, kidney-specific,Cre-positive megalin knockout mice (megalin lox/lox; apoE Cre),here termed Cre(+), and in Cre-negative control mice (megalinlox/lox), termed Cre(–) (13,14). The extent of megalingene deletion in the proximal epithelia of Cre(+) naturallyvaried to substantial amounts, thereby determining variabilityin the degree of basal proteinuria. For preselection of suitablemice, the degree of remnant megalin expression in Cre(+) wastherefore estimated by sampling of urinary levels of vitaminD–binding protein (13). For further assessment of themegalin status in mice that were selected for histologic analysis,a rate of 60 to 80% of megalin-deficient proximal tubule profiles(PTP) was standardized immunohistochemically. A total of 24pooled littermates [Cre(+) and Cre(–) mice] from severallitters were used. Twelve Cre(+) and 12 Cre(–) mice wereimmunologically primed by subcutaneous injection of rabbit IgGin complete Freund's adjuvant. GN was induced 6 d later by theintravenous injection of an anti-mouse glomerular basement membrane(GBM) serum (15) in eight Cre(+) and eight Cre(–) mice,whereas four mice of each genotype received an injection ofvehicle (0.9% NaCl). During treatment, mice were allowed freeaccess to standard diet and tap water. Another 18 d later, allmice were killed. For urinalysis, mice were individually placedin metabolic cages for 24 h between day 0 and day 6 and on thelast day before being killed. Urine protein and creatinine concentrationwere determined (kits from Roche Diagnostics, Mannheim, Germany).Urine was electrophoresed using BSA as a standard. The experimentswere conducted in accordance to the German law for the protectionof animals (Registered under G 0178/03).
Morphologic and Cytochemical Preparations
Mice were anesthetized by an intraperitoneal injection of sodiumpentobarbital (0.06 mg/g body wt), and the kidneys were perfusion-fixedusing 3% paraformaldehyde and prepared for light microscopyand electron microscopy analysis (14,16).
Periodic acid-Schiff–stained paraffin sections were usedfor scoring analyses. Semithin plastic sections (1 µm)were serially sectioned and stained with Richardson solution;several series from tissue blocks of Cre(+) and Cre(–)were cut, and the relevant glomeruli were selected in the midstof a series and traced toward both sides. Ultrathin sectionswere contrasted with uranyl acetate and lead citrate. Immunolabelingwas performed on cryostat or paraffin sections or on semithinLR white-plastic sections. As primary antibodies, sheep anti-megalin(gift from P. Verroust, INSERM U538, Paris, France), guineapig anti-megalin (generated against the c-terminus of rat megalin),rat anti–intercellular adhesion molecule-1 (anti–ICAM-1;clone KAT-1; ImmunoKontact, Abingdon, UK), rat anti–vascularcellular adhesion molecule-1 (anti–VCAM-1; BD Transduction,Heidelberg, Germany), rabbit anti–heat shock protein 25(anti–HSP25; Calbiochem, Schwalbach, Germany), rabbitanti–collagen I (Research Diagnostics Natu-Tec, Frankfurt,Germany), rat anti-CD68 (Serotec, Düsseldorf, Germany),rabbit anti–-smooth muscle actin (Abcam, Cambridge, UK),goat anti-vimentin (Sigma, Taufkirchen, Gernamy), rabbit anti–monocytechemoattractant protein-1 (anti–MCP-1; Santa Cruz Biotechnology,Santa Cruz, CA), rabbit anti-PCNA (Santa Cruz Biotechnology),rat anti–Ki-67 (Dako, Hamburg, Germany), or rabbit anti-S100A4(Dako) were used. Suitable cy2- or cy3-coupled (Dianova, Hamburg,Germany) or horseradish peroxidase–coupled (Dako) secondaryantibodies were applied. Horseradish peroxidase signal was generatedwith diaminobenzidine added with H2O2. For megalin immunostainingof semithin sections, 12 nm of colloidal gold–coupledanti-sheep antibody was used in combination with IntenSE-silverenhancement system (Amersham, Freiberg, Germany). For doubleimmunostaining, various primary antibodies were administeredconsecutively. Specificity controls were done as described previously(14).
TGF-1, TGF-3, osteopontin, and endothelin-1 (ET-1) mRNA expressionwas studied by in situ hybridization using digoxigenin-labeledriboprobes (Roche). Sense and antisense probes were generatedby in vitro transcription of a 500-bp TGF-1, a 500-bp TGF-3,an 1100-bp osteopontin, and a 300-bp ET-1 cDNA template. Insitu hybridization was performed on paraffin sections and combinedwith immunohistochemistry as described previously (16). Fordetection of DNA fragmentation, terminal deoxynucleotidyl transferase–mediateddigoxigenin-deoxyuridine nick-end labeling (TUNEL) stainingwas performed using an In Situ Cell Death Detection Kit (Roche).A Leica DMRB fluorescence microscope equipped with a digitizedcamera system and MetaView software (Visitron, Puchheim, Germany)and a Zeiss EM 906 were used for evaluation.
Quantifying Assessments
A semiquantitative score of the glomerular and tubulointerstitialdamage was established as described previously (17). For assessmentof inflammatory parameters, stress proteins, and apoptosis,sections that were double immunostained with megalin and ICAM-1,TGF-1, TGF-3, ET-1, osteopontin, MCP-1, or HSP25 were evaluatedby counting of megalin-positive and/or megalin-deficient PTP,respectively, for the simultaneous presence or absence of theseparameters. Megalin half-positive versus half-negative PTP portionswere separately assigned. VCAM-1, PCNA, Ki-67, and TUNEL signalswere evaluated by single cell counts. Five mice per group wereevaluated throughout. Ultrastructural parameters of proximaltubules were estimated from 100 electron micrographs per kidney(n = 4 mice per group). Megalin-positive cells were distinguishedfrom megalin-deficient cells using structural criteria (14).Cell height, microvillar length, and tubular basement membranethickness were determined by calculation of the means of threeindividual measurements per cell performed at random. The mitochondrialand lysosomal contents of tubular cells were estimated by stereology(18) using the MetaVue analyzing software (MetaVue, München,Germany).
Statistical Analyses
Data were analyzed using SPSS statistic program (SPSS, Chicago,IL). Quantitative data are presented as means ± SD. Forstatistical comparison, the Mann-Whitney rank sum test was used.For the assessment of correlation, the Pearson test was used.P < 0.05 was considered statistically significant.
GN and Megalin-Deficient Mice
Application of the nephritogenic antibody to Cre(–) andCre(+) mice induced GN with prominent proteinuria as establishedpreviously (15,19). Immunohistochemical detection of the nephritogenicantibody that bound to the GBM demonstrated specificity of theGN (Figure 1A). The entire kidneys of either genotype were affected,albeit with higher damage and proteinuria occurring in Cre(+)compared with Cre(–) mice (Table 1). This was confirmedby urine electrophoresis showing elevated baseline protein levelsin Cre(+) mice owing to the expected low molecular weight proteinuria(Figure 1B) (13) and disproportionate enhancement of proteinuriain Cre(+) mice with GN. Most of the urine protein in GN wasconcentrated at the albumin level (Figure 1B).
Figure 1. Characterization of Cre(–) and Cre(+) mice 18 d after the induction of glomerulonephritis (GN). (A) Immunostaining of glomerular basement membrane (GBM) deposits with horseradish peroxidase–coupled anti-rabbit IgG. (B) Coomassie staining of urinary proteins. M is the marker for protein size; BSA serves as positive control. Compared with Cre(–) mice, Cre(+) mice show a low molecular weight proteinuria with major bands at the BSA level. Cre(+) mice with GN [Cre(+)/GN] show markedly elevated proteinuria compared with Cre(–) mice with GN [Cre(–)/GN]. (C) Immunogold-silver staining for megalin in an overview of a Cre(+) control kidney. The black silver deposit at the basis of the microvilli identifies cells with remnant megalin expression; semithin section stained with Richardson's blue. Bars indicate borders between megalin-positive and megalin-deficient cells. (D) Cre(+)/GN kidney, staining as in C; intracellular blue dots reveal massive uptake of filtered proteins, which is visible only in megalin-positive cells. (E) Double immunostaining of mouse IgG (green fluorescence) and megalin (red fluorescence) in Cre(+)/GN cryosection. IgG uptake is strictly co-localized with megalin. Bar indicates the border between a megalin-positive and a megalin-deficient epithelial portion. Magnifications: x250 in A; x200 in C; x800 in D; x300 in E.
Megalin deficiency in Cre(+) mice was incomplete. Previous studiesusing urinary excretion of vitamin D–binding protein asan indicator for the degree of megalin deficiency showed thatknockout among PTP ranged between 60 and 80% (13). In our mice,an average of 69.8 ± 5.5% of all PTP were megalin-deficientas revealed by immunohistochemical quantification (Figure 1C).The presence or absence of megalin immunoreactivity in the brushborder membrane (BBM) and underlying endocytic compartment wasclearcut (Figure 1C). Uptake of filtered proteins in PTP orsingle cells with remnant megalin expression was visualizedimmunohistochemically (Figure 1, D and E). Numerically, 79.1± 13.2% of megalin-positive PTP showed uptake, whereasall megalin-deficient PTP were negative.
Histopathology of the Kidneys under Partial Megalin Deficiency and Proteinuria
Both treated groups developed crescentic GN with tubulointerstitialdisease 18 d after injection of the anti-GBM serum. The histopathologicchanges were qualitatively identical in both groups but quantitativelymore pronounced in Cre(+) than in Cre(–) mice (Figure 2,A through D). Glomeruli exhibited severe tuft alterations andcellular crescent formation (Figures 3, A and B, and 4). Tubulointerstitialdamage presented with proximal tubular dilation and cast formation(Figure 2B) but also with atrophy, degeneration, and collapse(Figure 4, A and B). Vivid inflammation with peritubular hypercellularityand matrix deposition was encountered adjacent to the degeneratingtubules (Figure 4, B and D). The degree of sclerotic, inflammatory,and degenerative damage was substantially higher in Cre(+)/GNcompared with Cre(–)/GN (Figure 2, E and F). However,a strict, linear correlation (r = 0.992, P < 0.001) was foundbetween the glomerular and the tubulointerstitial damage scores(Figure 2G).
Figure 2. Histopathologic characterization. (A and B) Overview of kidney cortex of a representative Cre(–)/GN (A) versus Cre(+)/GN kidney (B). Inflammation, glomerulosclerosis, obstruction or dilation of tubules, and tubulointerstitial fibrosis have evolved in Cre(–)/GN tissue but are much more pronounced in Cre(+)/GN (semithin sections). (C and D) Glomerulosclerosis and inflammatory detail as revealed by periodic acid-Schiff staining in representative Cre(–)/GN (C) versus Cre(+)/GN mice (D) on paraffin sections as used for damage scoring. (E and F) Tissue damage, given as percentage changes of the glomeruli (E) and of the tubulointerstitium per optical field (F) according to the respective ranges of scores from Cre(–)/GN and Cre(+)/GN mice. Data are means ± SD. *P < 0.05. (G) Correlation between damage scores of the pooled groups. Linear regression line with r indicating linear correlation coefficient. Magnifications: x80 in A and B; x100 in C and D.
Figure 3. Electron microscopy. (A and B) Glomeruli from Cre(–)/GN (A) and Cre(+)/GN kidneys (B). Both glomeruli show inflammatory changes such as microvillous transformation (arrows) and crescent formation (star). In favor of demonstrating the initial tubular segment, in B, tuft adherence is not contained in this section plane but was identified by serial sectioning in this glomerulus as well. Both glomeruli reveal intact initial tubular segments. Enlarged inserts show the presence (A) or absence (B) of an endocytic apparatus, identifying this portion as megalin-positive (A) or megalin-deficient (B). (C and D) Proximal tubule cells derived either from megalin-positive (C) or megalin-deficient S1 segments (D) of a Cre(+)/GN kidney. Again, inserts illustrate presence (C) or absence (D) of the endocytic apparatus. Numerous primary and secondary lysosomes are present in C but not in D. Magnifications: x450 in A and B; x4600 in C and D.
Figure 4. Representative images from semithin serial sectioning of kidneys from Cre(–) (A) and Cre(+) mice (B through F) with GN. Glomeruli show extensive crescents, and the connections to the tubule are obstructed as traced by serial sectioning (A and B). In A, the proximal tubules that belong to the collapsed glomerulus are fully collapsed (stars) and the distal tubules are narrowed (*). Interstitial inflammation is moderate. In B, all tubule profiles that belong to the collapsed glomerulus are atrophied with their remnants surrounded by inflammation and widened capillaries. In both A and B, tubules from neighboring nephrons do not seem to be affected by the inflammatory process. (C) Detail shows an inflamed glomerulus with encroachment of the crescentic process onto the first segment of the proximal tubule (arrows), revealing intercellular clefts (arrowheads; a sign of early damage [19]). The adjacent tubulointerstitium is normal. (D) Inflammatory reaction surrounding an atrophied profile (stars) that belongs to a massively damaged glomerulus. It is surrounded by a severe inflammatory response. Note that the inflammatory response does not affect the adjacent structures. (E and F) Affected glomerulus with encroachment of the crescentic inflammation toward the tubule via the glomerulotubular junction (arrows in E). The epithelium of the initial tubular segment is atrophied. Its lumen is patent, whereas downstream segments (stars; continuity shown in F) are collapsed as part of local, severe tubulointerstitial inflammation. Magnification, x300.
Details of the glomerular changes and related consequences fortubule morphology were analyzed in serial sections tracing theglomerulotubular junctions and adjacent tubular profiles ofaffected glomeruli in Cre(+) and Cre(–) mice with GN.In GN, the initial proximal tubule was intact as long as therewas no major encroachment of crescents from affected glomeruli(Figure 3, A and B). An entry of crescents into glomerulotubulartransition, however, resulted in tubular degeneration, as detailedpreviously (19,20) (Figure 4, C through F). Further encroachmentof the crescents was associated with collapse and obstructionof the tubule. Peritubular inflammation was restricted to thevicinity of collapsed segments, whereas unaffected nephronsthat extended into areas of tubulointerstitial damage had maintainedan intact structure (Figure 4D).
Ultrastructural Features of Proximal Tubular Changes Relative to Megalin Expression
Tubules from glomeruli that revealed crescent formation butnot obstruction of the glomerulotubular junction were analyzed.Both glomeruli and tubules ranged within intermediate damagescores (>25 and <75%; Figure 2, E and F). The respectivetubular cells, however, lacked destructive changes such as cellularatrophy, dedifferentiation, and autophagy, which could haveresulted from the exaggerated protein load (Figure 3, C andD). Apart from the general, drastic reduction of the endosomalapparatus in megalin-deficient cells, resulting in their moderatelydecreased cell height, specific GN-related differences wereregistered among groups in megalin-positive versus megalin-deficientcells with respect to total cell and brush border height, mitochondrialvolume, and tubular basement membrane thickness. Predictably,lysosomes displayed major changes selectively in the megalin-positivecells (Table 2).
Table 2. Numerical evaluation of structural parameters, proximal tubulea
Histochemical Evaluation of Parameters Related to Inflammation and Cell Cycle
The expression of proteins whose upregulation was shown earlierin tubulointerstitial inflammation and of parameters relatedto the cell cycle were quantitatively compared in megalin-deficientversus megalin-expressing PTP of Cre(+)/GN mice. Control Cre(+)and Cre(–) mice as well as Cre(–)/GN mice were comparedon a qualitative level.
ICAM-1 and VCAM-1.
In control tissues, ICAM-1 was only faintly expressed in theBBM of a few single proximal tubule cells and in capillary endothelia,whereas VCAM-1 was not detectable. In Cre(+)/GN, ICAM-1 wasupregulated predominantly in the BBM of megalin-positive PTP;with a similar distribution, VCAM-1 was upregulated in the basolateralcell aspect (Figures 5, A and B, and 8A). These changes werealso found in Cre(–)/GN. Data suggest that an upregulationof the inflammatory adhesion molecules ICAM-1 and VCAM-1 isassociated with enhanced protein endocytosis.
Figure 5. Histochemistry of inflammatory markers in Cre(+) mice with GN. (A) Double labeling of intercellular adhesion molecule-1 (ICAM-1; green) and megalin (red). ICAM-1–positive expression in brush border is mostly co-localized with megalin. White bars indicate the border between megalin-positive and megalin-deficient cells. (B) Double labeling of vascular cellular adhesion molecule-1 (VCAM-1; green immunofluorescence) and megalin (red immunofluorescence). Basolateral VCAM-1 expression mostly co-localizes with megalin (#), whereas there is no VCAM-1 staining in megalin-deficient proximal profiles (identified by interference contrast; *). (C and D) Double labeling of TGF-1 mRNA expression (in situ hybridization; C) and megalin (red immunofluorescence; D). TGF-1 mRNA was expressed in profiles where megalin was co-localized (#) but absent from megalin-deficient profiles (*). Similarly, TGF-3 mRNA and megalin (E and F) and endothelin-1 mRNA and megalin (G and H) were mostly co-localized. Magnifications: x200 in A; x150 in B through H.
Figure 8. Quantification of histochemical data. Numerical evaluation of proximal tubular profiles is presented except for VCAM-1 immunoreactivity, proliferation, and apoptosis, where single VCAM-1–, PCNA-, Ki-67–, or TUNEL-positive cells located in megalin-positive or in megalin-deficient proximal tubule profiles have been counted. Data are means ± SD. *P < 0.05.
TGF-.
TGF-1 and TGF-3 were localized by in situ hybridization. NoTGF- signal was detected in control tissues. GN caused elevatedexpression of both cytokines in the glomeruli, proximal, anddistal tubules of Cre(–) and Cre(+) kidneys. In Cre(+)/GNkidneys, expression of TGF-1 and TGF-3 was distinctly higherin megalin-positive than in megalin-deficient PTP (Figures 5,C through F, and 8), suggesting a link to endocytosis as well.
ET-1.
The mRNA of this proinflammatory peptide was constitutivelyexpressed in the distal tubule of control kidneys, whereas inGN, glomerular, interstitial, and proximal and distal tubularexpression was induced. In Cre(+)/GN kidneys, megalin-positivePTP more frequently expressed ET-1, than megalin-deficient PTP(Figures 5, G and H, and 8).
Osteopontin and HSP25.
The stress-related cytokine osteopontin was mainly expressedin medullary collecting ducts of control kidneys. In GN, osteopontinmRNA signal was strong in glomeruli and proximal and distaltubules; in contrast to the findings listed previously, however,in Cre(+)/GN, megalin-deficient PTP were more frequently osteopontinmRNA-positive than megalin-positive PTP (Figures 6, A and B,and 8). The stress protein HSP25 was strongly induced in glomeruliand proximal nephron segments and, to a lesser extent, in thedistal tubules of GN kidneys. As for osteopontin, in Cre(+)/GN,a major share of the megalin-deficient PTP was strongly positivefor HSP25 compared with the megalin-positive PTP (Figures 6Cand 8).
Figure 6. Histochemistry of osteopontin (A and B) and heat shock protein 25 (HSP25) expression (C) in Cre(+) mice with GN. (A and B) Osteopontin mRNA (in situ hybridization; A) and megalin (#; brown immunoperoxidase staining; B) in consecutive sections mostly show mutually exclusive labeling with osteopontin mRNA expressed in megalin-deficient proximal tubules (*). Black bars indicate border between megalin-positive, osteopontin-negative and megalin-deficient, osteopontin-positive portions within the same tubule. (C) Double labeling of HSP25 (green immunofluorescence) and megalin (red immunofluorescence). Proximal tubule portions mostly show mutually exclusive staining for HSP25 (*) or megalin (#). White bars indicate border between megalin-positive, HSP25-negative and megalin-deficient, HSP25-positive portions within the same tubule. Magnification, x150.
MCP-1.
This major inflammatory chemokine of tubular cells was apicallypresent in proximal and distal tubules of Cre(+)/GN kidneysirrespective of megalin expression. It seemed that MCP-1 expressionpositively correlated with the individual nephron damage butwas unrelated to cellular megalin expression (data not shown).
Apoptosis/Regeneration.
For testing whether increased protein endocytosis had influencedepithelial cell cycle, proliferation (Ki-67 and PCNA) and apoptosismarkers (TUNEL) were applied and the results were quantifiedin Cre(+) control and Cre(+)/GN mice. Baseline levels in controlsshowed an upregulation of all parameters in megalin-deficientcells (Table 3). Parameters were further increased substantiallyin GN, with more pronounced proliferation levels in the megalin-positivecells and higher apoptosis levels in the megalin-deficient cells(Figures 7 and 8, Table 3).
Figure 7. Histochemical markers for cell proliferation (Ki-67; green) and apoptosis (terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling [TUNEL]; green) in control Cre(+) (A and C) and Cre(+)/GN (B and D). Sections are double stained for megalin (red). (A) Baseline Ki-67 signal in a megalin-deficient proximal tubule profile. (B) Enhanced Ki-67 signal is localized preferentially in megalin-positive proximal tubule profiles. (C and D) TUNEL signals in both conditions are mainly found in megalin-deficient profiles; note the increased signal in GN (D). Magnification, x150.
Peritubular Interstitium.
Collagen I–and CD68-positive fibroblasts were diffuselyenhanced in GN mice, with accumulations in the fibrotic interstitiumsurrounding degenerating tubule profiles whose epithelia werenot principally free of remnant megalin expression. Searchingacross coronary kidney sections for an immediate spatial vicinityof megalin-positive versus megalin-deficient tubule portionsand underlying positive fibroblasts, we could not detect a preferentialassociation of the labeled fibroblasts with one or the otherphenotype (Figure 9). Thus, there was no indication for a histotopographicassociation between protein endocytosis and inflammation orfibrosis.
Figure 9. Histochemistry of interstitial parameters in Cre(+) mice with GN. (A) Double labeling of collagen I (green) and megalin (red immunofluorescence). Collagen I is predominantly expressed in areas of glomerular and tubular degeneration. Of note, collagen I was mostly in close proximity to megalin-deficient (*) rather than to megalin-positive proximal tubule profiles (#). (B) Double labeling of macrophage marker CD68 (green/yellow immunofluorescence; arrows) and megalin (red). CD68-positive macrophages (individual cells or clusters of cells) are localized to fibrotic areas and to the vicinity of megalin-deficient (*) rather than to megalin-positive proximal tubule profiles (#). Magnification, x90.
We used transgenic Cre(+) mice with kidney-specific megalindeficiency and control Cre(–) littermates to evaluatethe effect of experimental GN and ensuing tubular protein overloadon the development of tubulointerstitial disease. Symptoms ofGN had developed in either genotype as previously establishedin rodent models (12,15,19–21). The Cre(+)/GN mice, however,unexpectedly presented a higher degree of glomerular as wellas tubulointerstitial damage than the Cre(–)/GN mice.
The reason that Cre(+) mice reacted stronger to the experimentalprotocol than Cre(–) mice is not clear. However, poolingall Cre(+)/GN and Cre(–)/GN mice resulted in a strongpositive correlation between glomerular and tubulointerstitialdamage. This suggests that protein reabsorption has only a minorimpact on the development of tubulointerstitial injury. However,the primary differences in disease manifestation among groupsinterfered with the interpretation of some of the data raisedin Cre(+) as compared with Cre(–) mice. Therefore, themain emphasis in our study was put on intragroup comparisonsbetween megalin-positive and megalin-deficient cells in theCre(+)/GN group based on the mosaic remnant expression of megalin(14).
Structurally, changes in GN in both lines supported the previouslyestablished view that tubulointerstitial injury develops subsequentto the encroachment of a glomerular crescent onto the initialpart of the proximal tubule (1,19). Tracing of the glomerulotubularjunction in serial sections of Cre(+) and Cre(–) kidneysrevealed that the affection of the initial proximal tubule inGN depended on the degree of the encroachment of the glomerulardamage rather than on the presence or absence of megalin. Theinitial portion of the proximal tubule was structurally normaldespite obvious protein leakage, as long as the glomerulotubularjunction was patent. Conversely, more advanced encroachmentof the crescents toward the tubule or obstruction were associatedwith degeneration and collapse of the tubule. Notably, thiswas independent of the expression of megalin as assessed bythe occurrence or absence of an endocytic machinery in the tubularcells. This fits with previous experimental studies in ratsand mice (19,20,22) as well as with findings in human disease,such as congenital nephrotic syndrome of the Finnish type (23),showing that heavy proteinuria does not necessarily induce tubularlesions.
As the second major finding, our results confirm that GN elicitedan upregulation of the expression of ICAM-1, VCAM-1, TGF-1,TGF-3, ET-1, MCP-1, HSP25, and osteopontin, parameters thatare known to be associated with proteinuria. In addition, therates of cell proliferation and of apoptosis increased. Boththe megalin-positive and the megalin-deficient segments wereaffected. Comparable changes were described previously (fora review, see reference [6]). However, because the developmentof tubulointerstitial disease in Cre(+)/GN mice with a majorityof megalin-deficient proximal tubules obviously followed thesame pattern as in Cre(–)/GN mice with normal proximaltubules, the role of the protein overload hypothesis may bequestioned. Because the inflammatory parameters did not coincidewith the tubulointerstitial injury, their commonly assumed pathogeneticrole could in fact be different. This raises the question asto the relevance of their upregulation.
It is known that ICAM-1, VCAM-1, and ET-1 have several potentialbinding sites for NF-B in their promoter region and that bindingof NF-B may be relevant for their activation (24–28).Substantial protein uptake in cultured cells also induced signalingvia NF-B (for review, see reference [29]). It is therefore reasonableto assume that in GN, the expression of ICAM-1, VCAM-1, andET-1 within tubular cells reflects the activation of NF-B. Inrenal pathophysiology, NF-B–mediated responses are oftenconsidered pathogenic (29). However, there is also good evidencethat NF-B may mediate protective cellular adaptation to stressand also prevent apoptosis (30,31).
Similarly, TGF- is generally considered to play a pivotal rolein renal pathology regarding fibrogenesis and scarring (32),yet increased collagen expression in our model was not specificallyassociated with the upregulated TGF- transcripts. TGF- isoformsare considered to be multifunctional, and recent work emphasizedthe dosage dependence of their effects; in glomerular epithelialcells, a critical TGF- concentration threshold that specifiesa molecular switch from growth arrest/differentiation to proapoptoticsignaling and apoptosis was found (33). In line with this, lowerdosages of TGF- promoted neurogenesis within ganglia, whereasslightly higher dosages induced apoptosis (34). Therefore, itmay be suggested that the TGF-–mRNA increases in responseto enhanced protein uptake were insufficient to initiate deleteriouseffects. Rather, we speculate that the increases representeda protective response to stress, apparently including diminishedsusceptibility to proapoptotic signals in Cre(+)/GN.
Consequently, we assume that in GN, increased protein uptakeby megalin-positive cells induces an anti-stress response thatincludes augmented expression of ICAM-1, VCAM-1, TGF-1, TGF-3,and ET-1, which along with selectively enhanced cell proliferationmay serve protective rather than pathologic functions. Regardingthe increased rate of apoptosis in megalin-deficient comparedwith megalin-positive cells, we therefore suggest that megalin-deficientcells, which lack the protective stress response that is elicitedby protein uptake, may have an increased susceptibility to undefinedstimuli of disease progression in our model. A study by Erkanet al. (35) supports the view that apoptosis might be causedby stimuli other than protein overload, because there was enhancedapoptosis in kidneys with FSGS and membranoproliferative GNbut not in kidneys with minimal-change disease. Reactive oxygenspecies may as well menace the intactness of the proximal tubule(36), but this issue has also been discussed controversially(37–39).
It may also be considered that altered intracellular supplyof potentially vital small molecules to the proximal tubule,caused by the disrupted endocytosis, could have proapoptoticinfluence to contribute to the selective increase of TUNEL signalof megalin-deficient cells in GN. As yet, there is no basisfor a speculation about the possible role of such moleculesin GN, but a recent article suggested another pathway for regulationof stress responses via megalin (40). It was found that megalinbinds to and activates protein kinase B (Akt). Activated Aktpromotes cell survival, partly via NF-B, and inhibits apoptosis.
An enhanced expression of the stress-related products osteopontinand HSP25 was also found preferentially in megalin-deficientPTP in GN. Because upregulation of both products was furtherobserved in distal tubules, displaying only minor protein uptake(37), changes likely did not depend on protein overload; notably,their upregulation correlated with the increased apoptosis ratein megalin-deficient cells (r = 0.925 for osteopontin and r= 0.975 for HSP25).
Our data suggest that excessive uptake of filtered proteinsby proximal tubules has no major impact on the development ofthe tubulointerstitial disease in GN. Upregulation of inflammatoryproteins in response to excessive protein uptake may representa stress-protective function rather than a disease-inducingmechanism. Instead, our results support the view that expandingdamages at the glomerulotubular junction represent the crucialmoment in the transmission of the disease from the glomerulusto the tubule.
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Top
Abstract
Introduction
, intercellular adhesion molecule, vascular cellular adhesion molecule, endothelin-1, and cell proliferation were high in megalin-positive cells, whereas apoptosis, heat-shock protein 25, and osteopontin were enhanced in megalin-deficient cells. No fibrotic changes were associated with either phenotype. Tubular degeneration with interstitial inflammation was found only in nephrons with extensive crescentic lesions at the glomerulotubular junction. In sum, enhanced protein endocytosis indeed led to an upregulation of profibrotic mediators in a megalin-dependent way; however, there was no evidence that endocytosis played a pathogenetic role in the development of the tubulointerstitial disease. 
-smooth muscle actin (Abcam, Cambridge, UK), goat anti-vimentin (Sigma, Taufkirchen, Gernamy), rabbit anti–monocyte chemoattractant protein-1 (anti–MCP-1; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PCNA (Santa Cruz Biotechnology), rat anti–Ki-67 (Dako, Hamburg, Germany), or rabbit anti-S100A4 (Dako) were used. Suitable cy2- or cy3-coupled (Dianova, Hamburg, Germany) or horseradish peroxidase–coupled (Dako) secondary antibodies were applied. Horseradish peroxidase signal was generated with diaminobenzidine added with H2O2. For megalin immunostaining of semithin sections, 12 nm of colloidal gold–coupled anti-sheep antibody was used in combination with IntenSE-silver enhancement system (Amersham, Freiberg, Germany). For double immunostaining, various primary antibodies were administered consecutively. Specificity controls were done as described previously (14). 
B in their promoter region and that binding of NF-
