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Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

2008; Elsevier BV; Volume: 74; Issue: 10 Linguagem: Inglês

10.1038/ki.2008.405

1523-1755

Yaeko Motoyoshi, Taiji Matsusaka, Akihiko Saito, Ira Pastan, Thomas E. Willnow, Shuki Mizutani, Iekuni Ichikawa,

Ion Transport and Channel Regulation

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury. Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury. Many glomerular diseases accompany proteinuria, and structural damage develops along the downstream tubule. This tubule injury is closely correlated with progressive loss of kidney function.1.Bruzzi I. Benigni A. Remuzzi G. Role of increased glomerular protein traffic in the progression of renal failure.Kidney Int. 1997; 52: S29-S31Google Scholar,2.Birn H. Christensen E.I. Renal albumin absorption in physiology and pathology.Kidney Int. 2006; 69: 440-449Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar Clinical studies indicated that the level of proteinuria predicts the risk of developing into end-stage renal failure.3.Abbate M. Zoja C. Remuzzi G. How does proteinuria cause progressive renal damage?.J Am Soc Nephrol. 2006; 17: 2974-2984Crossref PubMed Scopus (544) Google Scholar,4.Ruggenenti P. Perna A. Remuzzi G. Retarding progression of chronic renal disease: the neglected issue of residual proteinuria.Kidney Int. 2003; 63: 2254-2261Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar In that, when compared to selective proteinuria, nonselective proteinuria has a significantly greater chance of tubulointerstitial injury and progression of renal diseases.5.Zandi-Nejad K. Eddy A.A. Glassock R.J. et al.Why is proteinuria an ominous biomarker of progressive kidney disease?.Kidney Int. 2004; 66: S76-S89Abstract Full Text Full Text PDF Scopus (131) Google Scholar,6.Bazzi C. Petrini C. Rizza V. et al.A modern approach to selectivity of proteinuria and tubulointerstitial damage in nephrotic syndrome.Kidney Int. 2000; 58: 1732-1741Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,7.Bakoush O. Grubb A. Rippe B. et al.Urine excretion of protein HC in proteinuric glomerular diseases correlates to urine IgG but not to albuminuria.Kidney Int. 2001; 60: 1904-1909Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Although, in the past, many in vitro studies have been designed and executed to demonstrate the causal role of proteinuria for progressive renal cell injury,8.Wang Y. Rangan G.K. Tay Y.C. et al.Induction of monocyte chemoattractant protein-1 by albumin is mediated by Nuclear Factor κB in proximal tubule cells.J Am Soc Nephrol. 1999; 10: 1204-1213PubMed Google Scholar,9.Erkan E. De Leon M. Devarajan P. Albumin overload induces apoptosis in LLC-PK(1) cells.Am J Physiol Renal Physiol. 2001; 280: F1107-F1114PubMed Google Scholar,10.Sengul S. Zwizinski C. Simon E.E. et al.Endocytosis of light chains induces cytokines through activation of NF-kappaB in human proximal tubule cells.Kidney Int. 2002; 62: 1977-1988Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Zandi-Nejad et al.5.Zandi-Nejad K. Eddy A.A. Glassock R.J. et al.Why is proteinuria an ominous biomarker of progressive kidney disease?.Kidney Int. 2004; 66: S76-S89Abstract Full Text Full Text PDF Scopus (131) Google Scholar pointed to uncertainties underlying those studies. The uncertainties stem from non-physiologic exposure of apical tubule cell surfaces to proteins in vitro, the extremely high concentrations of various proteins tested in vitro, and non-uniform end points measured. Also noted are a lack of correlation often seen between in vitro and in vivo findings and a lack of uniformity of results, even for seemingly similar in vitro experiments.5.Zandi-Nejad K. Eddy A.A. Glassock R.J. et al.Why is proteinuria an ominous biomarker of progressive kidney disease?.Kidney Int. 2004; 66: S76-S89Abstract Full Text Full Text PDF Scopus (131) Google Scholar Therefore, despite the abundance of in vitro findings suggesting the pathogenic role of urinary proteins in tubule damage, it appeared warranted to design an in vivo study that tests the effect of proteinuria occurring in glomerular disease. Megalin, a member of low-density lipoprotein receptor family, is expressed intensely on the apical membrane of proximal tubule cells (PTCs) and works as a scavenger of filtered proteins by endocytotic process. Most systemic megalin null-mutant mice die perinatally due to defective forebrain development or lung dysfunction.11.Willnow T.E. Hilpert J. Armstrong S.A. et al.Defective forebrain development in mice lacking gp330/megalin.Proc Natl Acad Sci USA. 1996; 93: 8460-8464Crossref PubMed Scopus (394) Google Scholar A strain of transgenic mice carrying a megalin null allele selectively in renal cells was engineered with a use of Cre-loxP system recently.12.Leheste J.R. Melsen F. Wellner M. et al.Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.FASEB J. 2003; 17: 247-269Crossref PubMed Scopus (128) Google Scholar This carries two loxP sites in introns 71 and 74 of the megalin gene and the Cre transgene. The Cre gene is regulated by 3.0-kb promoter fragment of the human ApoE gene, which drives remarkably intensive expression selectively in the kidney, but not in the liver or other organs.13.Simonet W.S. Bucay N. Lauer S.J. et al.In the absence of a downstream element, the Apolipoprotein E gene is expressed at high levels in kidneys of transgenic mice.J Biol Chem. 1990; 265: 10809-10812Abstract Full Text PDF PubMed Google Scholar,14.Simonet W.S. Bucay N. Pitas E. et al.Multiple tissue-specific elements control the Apolipoprotein E/C-I gene locus in transgenic mice.J Biol Chem. 1991; 266: 8651-8654Abstract Full Text PDF PubMed Google Scholar Of note, megalin gene deletion in the above model occurs in a mosaic pattern. As a consequence, the proximal tubule consists of cells with and without intact megalin gene, thereby allowing a direct comparison of megalin-knockout vs megalin-intact PTCs that belong to the same nephron, and hence are exposed to the same quality and quantity of filtered proteins. Using this as a unique opportunity to identify the role of filtered proteins for the tubule injury in glomerular diseases in vivo, Theilig et al.15.Theilig F. Kriz W. Jerichow T. et al.Abrogation of protein through megalin-deficient proximal tubules does not safeguard against tubulointerstitial injury.J Am Soc Nephrol. 2007; 18: 1824-1834Crossref PubMed Scopus (71) Google Scholar induced anti-glomerular basement membrane nephritis, a model of rapidly progressive glomerulonephritis, in this transgenic strain. They found that enhancement in protein endocytosis and upregulation of profibrotic mediators, including intracellular adhesion molecule 1, vascular cellular adhesion molecule 1, and transforming growth factor-β, occurred in a megalin-dependent manner. However, tubule degeneration and interstitial fibrosis were associated with glomerular crescentic lesions, but not with megalin expression, thus it appeared that endocytosis does not play a pathogenic role for the tubulointerstitial injury. Of interest, in this regard, the proteinuria of the anti-glomerular basement membrane nephritis studied was not in nephrotic range and selective, that is primarily of albumin.16.Feith G.W. Assmann K.J. Bogman M.J. et al.Lack of albuminuria in the early heterologous phase of anti-GBM nephritis in beige mice.Kidney Int. 1993; 43: 824-827Abstract Full Text PDF PubMed Scopus (14) Google Scholar We therefore studied the effect of massive, nonselective proteinuria on PTCs, using the above transgenic model, with the goals to verify whether megalin is involved in the reabsorption of filtered proteins of large molecular size, and if so, whether megalin participates in tubule injury. To induce massive nonselective proteinuria, the kidney-specific megalin-knockout (hereafter designated as megalin-KO) mice were mated with another strain of transgenic mouse (NEP25), which expresses human CD25 selectively in podocytes. After injection of human CD25-targeted recombinant immunotoxin, anti-Tac (Fv)-PE38 (LMB2),17.Kreitman R.J. Bailon P. Chaudhary V.K. et al.Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of pseudomonas exotoxin procedure complete regression in mice of an interleukin-2 receptor-expressing human carcinoma.Blood. 1994; 83: 426-434Crossref PubMed Google ScholarNEP25 mice develop nephrotic syndrome, focal segmental glomerular sclerosis and secondary tubulointerstitial injury. These injuries are dependent on the dosage of LMB2 and the duration after LMB2 injection, but not on gender.18.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (195) Google Scholar We examined the extent of megalin expression in the kidney of megalin-KO/NEP25 mice by two methods. Western blot analysis on whole kidneys revealed that the amount of megalin in megalin-KO/NEP25 mice was decreased, on average, to 40% compared with that of control mice (megalinloxP/loxP ApoE-Cre(−); NEP25, hereafter designated as megalin-intact/NEP25; Figure 1). Analysis of immunostained sections showed that megalin was uniformly and intensively expressed on the apical membrane of the PTCs and the glomerular parietal epithelial cells in megalin-intact/NEP25 mice. By contrast, megalin staining was defective in mosaic pattern in PTCs and glomerular parietal epithelial cells in megalin-KO/NEP25 mice. Assessment of megalin staining in five randomly selected fields revealed that the proportion of megalin-expressing cells to the whole PTCs was variable among megalin-KO/NEP25 mice, ranging from 34.7 to 50.1%, and on average 41.4±6.4%, whereas that in megalin-intact/NEP25 mice was 100%. Without LMB2, urinary total protein/creatinine ratio of megalin-KO/NEP25 mice was, on average, 33.0±13.1 in male and 5.4±3.3 in female mice, which were comparable to those of megalin-intact/NEP25 mice, 26.3±7.2 (male), 4.5±4.0 (female; NS). However, SDS–polyacrylamide gel electrophoresis analysis of the urine confirmed that megalin-KO/NEP25 mice excreted higher amount of low-molecular-weight proteins, although the value varied among animals (Figure 2). Both megalin-KO/NEP25 and megalin-intact/NEP25 mice showed normal renal histology without LMB2. We performed a pilot study to determine the appropriate dosage of LMB2 and the timing of analysis using NEP25 mice with wild-type megalin gene. We found that 10 days after the injection of 1.25 ng/g BW of LMB2, when mild but significant tubule injury developed, megalin remained readily detectable in 100% of the PTCs. The identity of the latter was verified by their localization in the cortex, presence of brush border, and negative staining for both Tamm-Horsfall protein, a marker of the thick ascending limb, and thiazaide-sensitive NaCl cotransporter, a marker of the distal tubule, on the adjacent sections. With higher doses of LMB2, or at later time points, more severe tubule injury developed. However, megalin was downregulated in severely injured PTCs. To be able to determine megalin genotype of PTCs in megalin-KO/NEP25 mice by megalin staining, we employed the above experimental conditions in the subsequent studies. After injection of 1.25 ng/g BW of LMB2, megalin-KO/NEP25 and megalin-intact/NEP25 mice similarly showed massive proteinuria. Urinary protein/creatinine ratio was, on average, 125.0±43.0 in male and 91.1±38.1 in female megalin-KO/NEP25 mice, which were comparable to those in megalin-intact/NEP25 mice (113.8±64.2 in male and 78.6±79.1 in female, NS). SDS–polyacrylamide gel electrophoresis analysis confirmed that proteinuria was nonselective, that is not only low but also intermediate and high-molecular-weight proteins were excreted and the quantity was large (Figure 2). At the time of assessment, both megalin-KO/NEP25 and megalin-intact/NEP25 mice similarly showed mild edema and ascites with body weight gain, on average, 16.8±22.3% in megalin-KO/NEP25 mice and 15.6±17.1% in megalin-intact/NEP25 mice (NS), respectively. Light microscopic analyses revealed that both megalin-KO/NEP25 and megalin-intact/NEP25 mice mostly developed mild glomerular injury, and less than 5.3% of glomeruli were modestly or severely damaged (Figure 3). Semiquantification of glomerular injury revealed that the indices for glomerular mesangial and epithelial injury were, on average, 1.04±0.76 and 1.05±0.97, respectively, in megalin-KO/NEP25 mice, and 0.96±0.80 and 0.93±0.82, respectively, in megalin-intact/NEP25 mice. There was no significant difference between the two groups, indicating that similar mild glomerular injury was induced in both groups. Both types of mice similarly showed mild tubulointerstitial changes with occasional tubule dilatation and rare fibrosis. As approximately 40% of PTCs still express megalin in megalin-KO/NEP25 mice, we compared protein reabsorption by megalin-intact (+) vs megalin-deficient (−) PTCs within the same megalin-KO/NEP25 mouse kidney. Without LMB2, small albumin droplets were seen in a small fraction of PTCs whereas immunoglobulin (Ig) κ-light chain (IgL), IgA (α-heavy chain), and IgG (whole molecule) were rarely stained in PTCs. After LMB2 injection, small to large albumin droplets were seen in PTCs in variable degrees in megalin-KO/NEP25 mice (Figure 4b). Immunostaining for IgL (Figure 4d), IgA (Figure 4f), and IgG (Figure 4h) showed small to large reabsorption droplets in a fashion similar to albumin in megalin-KO/NEP25 mice. Figure 4a and b are from adjacent sections of a megalin-KO/NEP25 mouse kidney stained for megalin and albumin. In Figure 4a, megalin (+, black arrows) and (-, yellow arrows) PTCs are next to each other. The albumin staining in the adjacent section (Figure 4b) demonstrates that megalin (+) PTCs contain albumin droplets whereas megalin (−) PTCs do not. Morphometrical analysis revealed that, on average, 25.2±14.6% of megalin (+) PTCs accumulated albumin within the cell whereas only 5.0±4.6% of megalin (−) cells showed albumin accumulation (P=0.004, Figure 5a). Analysis for IgL (Figure 4c and d), IgA (Figure 4e and f), and IgG (Figure 4g and h) demonstrated that, likewise, these proteins were preferentially accumulated in megalin (+) PTCs. Thus, the ratio of protein accumulation in megalin (+) vs (−) cells was, on average, 14.5±8.7 vs 1.3±1.4% (P=0.003) for IgL (Figure 5b), 18.8±7.3 vs 2.1±3.2% (P=0.003) for IgA (Figure 5c), and 7.3±4.6 vs 0.1±0.3% (P=0.005) for IgG (Figure 5d), respectively. Serial section analysis revealed that protein reabsorption droplets in megalin (+) cells of megalin-KO/NEP25 mice (Figure 4i, black arrows) contain IgL (Figure 4j), IgA (Figure 4k), and IgG (Figure 4l). We next compared tubule insult/injury markers in megalin (+) and (−) PTCs within the same megalin-KO/NEP25 mouse kidney. Thus, we assessed expressions of heme-oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and aquaporin-1 (AQP-1) and the evidence of apoptosis. As shown in Figure 6b, HO-1 staining shows numerous small round-shaped materials within the PTCs, which resemble protein reabsorption droplets on periodic acid Schiff-stained tissues. All of the HO-1-expressing cells shown in this figure are megalin positive (Figure 6a). Quantitative analysis in all megalin-KO/NEP25 mice revealed that, on average, 2.32±2.26% of megalin (+) PTCs expressed HO-1 whereas only 0.24±0.20% of megalin (−) PTCs expressed HO-1 (P=0.003, Figure 7a).Figure 7Percentage of early injury marker-expressing cells in megalin (+) vs (−) proximal tubule cells of megalin-KO/NEP25 mice given LMB2. In (a, b, c), each pair of dots connected with a line represents total values for megalin (+) vs (−) cells from each mosaic mouse. (a) Percentage of heme-oxygenase-1 (HO-1)-expressing cells. (b) Percentage of monocyte chemoattractant protein-1 (MCP-1)-expressing cells. (c) Percentage of apoptotic cells. HO-1 expression, MCP-1 expression, and apoptosis were observed preferentially in megalin (+) proximal tubule cells (PTCs). (d) Percentage of aquaporin-1 (AQP-1) diminishing cells. Each pair of dots connected with a line represents average values for megalin (+) vs (−) cells from each mosaic mouse. Diminished AQP-1 was observed similarly in megalin (+) and (−) PTCs.View Large Image Figure ViewerDownload (PPT) Likewise, most MCP-1-expressing cells and apoptotic cells were positive for megalin staining (Figure 6d and e). Thus, on average, 0.37±0.57% of megalin (+) PTCs expressed MCP-1 whereas only 0.08±0.12% of megalin (−) PTCs expressed MCP-1 (P=0.043, Figure 7b). Moreover, on average, 0.43±0.52% of megalin (+) PTCs underwent apoptosis whereas only 0.13±0.19% of megalin (−) PTCs underwent apoptosis (P=0.026, Figure 7c). AQP-1 is normally expressed on both apical and basolateral membranes of PTCs. In a variety of glomerular diseases including immunofluorescence-negative mesangial proliferative glomerulonephritis, IgA nephropathy, and Lupus nephritis, expression of AQP-1 is diminished along the basolateral membrane.19.Bedford J.J. Leader J.P. Walker R.J. Aquaporin expression in normal human kidney and in renal disease.J Am Soc Nephrol. 2003; 14: 2581-2587Crossref PubMed Scopus (79) Google Scholar As shown in Figure 6g, PTCs in megalin-KO/NEP25 mice injected with LMB2 showed diminution of basolateral AQP-1 expression. However, AQP-1 diminution was not limited to megalin (+) PTCs. Quantitative analysis revealed that, on average, 34.7±25.4% of megalin (+) PTCs and 38.0±14.8% of megalin (−) PTCs showed diminution of basolateral AQP-1 (NS, Figure 7d). The present study explored the function of megalin in PTCs exposed to massive nonselective proteinuria following glomerular podocyte injury. Analysis in megalin-KO/NEP25 mice, which lack tubule megalin in mosaic fashion, revealed that uptake of albumin, IgL, IgA, and IgG was dependent on megalin. In addition, megalin (+) PTCs preferentially expressed three injury markers, namely HO-1, MCP-1, and apoptosis more frequently, compared to megalin (−) PTCs within the same kidney. Previous studies showed that albumin and IgL are ligands of megalin;20.Christensen E.I. Birn H. Megalin and cubilin: multifunctional endocytic receptors.Nature Reviews. 2002; 3: 258-268Crossref Scopus (603) Google Scholar however, it was not known whether macromolecules including Ig heavy chains are ligands of megalin. The anti-IgA antibody used in the present study was specific for α-heavy chain. The finding that most IgA staining was in megalin (+) PTCs indicates that uptake of α-heavy chain is dependent on megalin. As our study did not directly test that α-heavy chain has significant affinity to megalin, it is conceivable that heavy chain may be incorporated into PTCs through interaction between light chain and megalin. Of importance, in our study, megalin (+) PTCs showed more HO-1, that is, an oxidative stress marker, more MCP-1, that is, an inflammatory marker, and more apoptosis than megalin (−) PTCs within the same kidney under massive nonselective protein overload. Expression of these markers has been shown to be closely associated with tubule injury in a variety of settings including protein overload.21.Murali N.S. Ackerman A.W. Croatt A.J. et al.Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease.Am J Physiol Renal Physiol. 2007; 292: F837-F844Crossref PubMed Scopus (38) Google Scholar,22.Shimizu M. Ohta K. Yang Y. et al.Glomerular proteinuria induces heme oxygenase-1 gene expression within renal epithelial cells.Pediatr Res. 2005; 58: 666-671Crossref PubMed Scopus (18) Google Scholar,23.Kuusniemi A.M. Lapatto R. Holmberg C. et al.Kidneys with heavy proteinuria show fibrosis, inflammation, and oxidative stress, but no tubular phenotypic change.Kidney Int. 2005; 68: 121-132Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,24.Erkan E. Garcia C.D. Patterson L.T. et al.Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: roles of proteinuria and Fas-dependent pathways.J Am Soc Nephrol. 2005; 16: 398-407Crossref PubMed Scopus (54) Google Scholar Our study indicated that there is a causal relationship between macromolecule reabsorption through megalin and the injury markers. As HO-1 activation has been shown to cause nuclear factor κ-B activation,8.Wang Y. Rangan G.K. Tay Y.C. et al.Induction of monocyte chemoattractant protein-1 by albumin is mediated by Nuclear Factor κB in proximal tubule cells.J Am Soc Nephrol. 1999; 10: 1204-1213PubMed Google Scholar,21.Murali N.S. Ackerman A.W. Croatt A.J. et al.Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease.Am J Physiol Renal Physiol. 2007; 292: F837-F844Crossref PubMed Scopus (38) Google Scholar,24.Erkan E. Garcia C.D. Patterson L.T. et al.Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: roles of proteinuria and Fas-dependent pathways.J Am Soc Nephrol. 2005; 16: 398-407Crossref PubMed Scopus (54) Google Scholar possible mechanism underlying tubule injury is that massive macromolecule uptake somehow induces oxidative stress, which then causes nuclear factor κ-B activation and inflammatory responses, leading to tubule injury. However, further studies are necessary to clarify precise molecular mechanisms. Our findings are in accordance with previous reports showing close association between high-molecular-weight proteinuria and tubule injury. Thus, in the remnant kidney model and passive Heymann nephritis model, interstitial infiltrates are observed in the vicinity of tubule segment that contains casts or PTCs reabsorbing IgG.25.Abbate M. Zoja C. Corna D. et al.In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.J Am Soc Nephrol. 1998; 9: 1213-1224PubMed Google Scholar An in vitro study utilizing cultured PTCs showed that high-concentration IgG induced expression of inflammatory and fibrogenic mediators, including MCP-1 and RANTES.26.Zoja C. Donadelli R. Colleoni S. et al.Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation.Kidney Int. 1998; 53: 1608-1615Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar Moreover, Morais et al.27.Morais C. Westhuyzen J. Metharom P. et al.High molecular weight plasma proteins induce apoptosis and Fas/FasL expression in human proximal tubular cells.Nephrol Dial Transplant. 2005; 20: 50-58Crossref PubMed Scopus (28) Google Scholar documented a relationship between high-molecular-weight proteins and apoptosis in a study with human PTCs. Exposure to 100–440 and 30–440 kDa plasma protein fractions produced significant increases in apoptosis whereas PTCs exposed to a 30–100 kDa fraction were not significantly different from control cells. Some clinical studies have also described that nonselective proteinuria, but not selective proteinuria, is closely associated with tubule injury and poor prognosis.6.Bazzi C. Petrini C. Rizza V. et al.A modern approach to selectivity of proteinuria and tubulointerstitial damage in nephrotic syndrome.Kidney Int. 2000; 58: 1732-1741Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,7.Bakoush O. Grubb A. Rippe B. et al.Urine excretion of protein HC in proteinuric glomerular diseases correlates to urine IgG but not to albuminuria.Kidney Int. 2001; 60: 1904-1909Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar In the recent study by Theilig et al.15.Theilig F. Kriz W. Jerichow T. et al.Abrogation of protein through megalin-deficient proximal tubules does not safeguard against tubulointerstitial injury.J Am Soc Nephrol. 2007; 18: 1824-1834Crossref PubMed Scopus (71) Google Scholar discussed earlier, evidence was lacking to indicate that endocytosis plays a pathogenic role in the development of the tubulointerstitial damage in anti-glomerular basement membrane nephritis, a model for rapidly progressive glomerular nephritis. In that, proteinuria was mild and presumed to be primarily albuminuria, and tubule injury was closely associated with an encroachment of the crescents into glomerulotubular transition and tubule obstruction.15.Theilig F. Kriz W. Jerichow T. et al.Abrogation of protein through megalin-deficient proximal tubules does not safeguard against tubulointerstitial injury.J Am Soc Nephrol. 2007; 18: 1824-1834Crossref PubMed Scopus (71) Google Scholar In contrast, NEP25 is characterized by massive nonselective proteinuria, in which focal segmental glomerular sclerosis and tubule damage develop without obstruction of glomerulotubular junction. It appears, then, that, when compared to low-molecular-weight proteins, high-molecular-weight proteins have substantially greater injurious impact on PTCs, and in nephrosis when compared to nephritis, proteinuria has a greater pathogenic significance in the development of tubule damage. When LMB2 is administered into wild-type mice, it is accumulated in the kidney and excreted into the urine, suggesting that LMB2 is filtered through glomeruli and reabsorbed by tubule cells.28.Kreitman R.J. Pastan I. Accumulation of a recombinant immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell are sufficient for complete responses.Cancer Res. 1998; 58: 968-975PubMed Google Scholar One may concern that reabsorption of LMB2 may be mediated through megalin, so that megalin (+) PTCs are preferentially injured through reabsorption of LMB2. However, this is unlikely as LMB2 was previously shown to have no toxic effect on tubule cells in wild-type mice.18.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (195) Google Scholar We verified this aspect in the present study, also. Thus, we injected 1.25 ng/g BW of LMB2 into megalin-KO and megalin-intact mice without human CD25 transgene. Again, none of these mice showed proteinuria, abnormal histology, expression of HO-1, MCP-1, or apoptosis in the tubule. AQP-1 is expressed in both apical and basolateral membranes of PTCs and works as a water channel. Diminution of its expression was shown previously in cisplatin nephritis,29.Kishore B.K. Krane C.M. Iulio D.D. et al.Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria.Kidney Int. 2000; 58: 701-711Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar unilateral ureteral obstruction,30.Li C. Wang W. Knepper M.A. et al.Downregulation of renal aquaporins in response to unilateral ureteral obstruction.Am J Physiol Renal Physiol. 2003; 284: F1066-F1079Crossref PubMed Scopus (9) Google Scholar and several human renal diseases.19.Bedford J.J. Leader J.P. Walker R.J. Aquaporin expression in normal human kidney and in renal disease.J Am Soc Nephrol. 2003; 14: 2581-2587Crossref PubMed Scopus (79) Google Scholar Although we did observe AQP-1 diminution in basolateral membrane dominantly, there was no significant correlation with megalin expression or protein reabsorption by PTCs. The finding is consistent with the existence of tubule injury processes independent of protein reabsorption, such as one triggered by tubule obstruction.31.Kriz W. Hosser H. Hähnel B. et al.From segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies.Nephrol Dial Transplant. 1998; 13: 2781-2798Crossref PubMed Scopus (127) Google Scholar Of note, our study was limited to assessment of early stages of tubule injury, that is, at stages when the genotype marker, megalin, still remains intact and appreciable, and the damage of PTCs of a given genotype has yet to bring about secondary injury to the PTCs of the other genotype. In fact, in our study, when studied in late stages, severe tubule damage was always accompanied by lack/loss of megalin expression. In that, one may erroneously conclude that lack of megalin brought about injury instead of the other way around. In any case, further studies are required to determine whether the effect of megalin on the PTCs demonstrated in the present study by several early injury markers continues to be significant throughout the development of histologically full-blown tubulointerstitial changes, and whether reabsorption of filtered macromolecules alone can bring about the histological changes. In summary, at least at the stage we analyzed, when glomeruli are injured and a large quantity of proteins is filtered, a variety of proteins are reabsorbed by PTCs by megalin, the latter leading to expression of early injury markers in PTCs. It should be noted that the model we studied is unique, namely it is characterized by massive nonselective proteinuria, and the assessment was made shortly after the onset of proteinuria. It is, therefore, entirely conceivable that other mechanisms also operate in tubule injuries, which take place more chronically with less severe proteinuria. The present study does not determine the quantitative importance of those mechanisms. The Animal Experimentation Committee at Tokai University Medical School approved the protocol, in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Both kidney-specific megalin-knockout mice (megalinloxP/loxPApoE-Cre(+)) and NEP25 mice were maintained on C57BL/6 strain. These were mated more than twice to obtain mice homozygous for megalin-loxP and heterozygous for ApoE-Cre and NEP25 (megalinloxP/loxP ApoE-Cre(+); NEP25). In total, 11 (4 males and 7 females) megalin-KO/NEP25 mice and 10 (5 males and 5 females) megalin-intact/NEP25 mice, 6–22 weeks of age, were weighed and housed in metabolic cages for 24 h for urine collection. Then, under anesthesia with diethyl ether, mice were intravenously injected with 1.25 ng/g BW LMB2 diluted in 0.1 ml of 0.1% bovine serum albumin/phosphate-buffered saline. Nine days after the injection, mice were weighed, and 24-h urine was collected. At 10 days after the injection, mice were killed, and kidney samples were harvested. For western blot analysis, kidney samples were snap frozen in liquid nitrogen and stored at -80 °C. For histological analysis, kidney samples were fixed in buffered 4% paraformaldehyde and embedded in paraffin. Concentration of total protein and creatinine were determined in an outside laboratory using an automatic analyzer (SRL, Tokyo, Japan). SDS–polyacrylamide gel electrophoresis was performed on urine samples (2 μl) using 2–15% gradient gel. Kidney samples from megalin-KO/NEP25 and megalin-intact/NEP25 mice were homogenized in a lysis buffer, which is one tablet of Complete Protease Inhibitor Cocktail (Roche Diagnostic, Basel, Switzerland) dissolved in 25 ml phosphate-buffered saline, and the protein concentration was determined using RC DC Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). Samples, each containing 50 μg protein, were separated in NuPAGE 3–8% Tris-Acetate gel (Invitrogen, Carlsbad, CA, USA) under a denatured condition. After electrophoresis, proteins were transferred onto PVDF membrane (Immobilon-P, Millipore, Tokyo, Japan) using a wet transfer system at 15 V, overnight. Then, the membrane was blocked in 3% bovine serum albumin/0.1% NaN3/phosphate-buffered saline for 1 h at 37 °C, incubated with anti-rat megalin antibody32.Saito A. Kazama J.J. Iino N. et al.Bioengineered implantation of megalin-expressing cells: a potential intracorporeal therapeutic model for uremic toxin protein clearance in renal failure.J Am Soc Nephrol. 2003; 14: 2025-2032Crossref PubMed Scopus (31) Google Scholar diluted at 1:5000 at 37 °C overnight. The membrane was then, washed in 0.05% Tween 20/phosphate-buffered saline for 60 min 3 times, incubated in HRP-anti rabbit IgG (1:5000, GE Healthcare, Chalfont St Giles, UK) for 1 h, washed in 0.05% Tween 20/phosphate-buffered saline again. Signals were detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare). To quantify glomerular damage, injuries of epithelial cells and mesangial cells were graded separately for each glomerulus on periodic acid Schiff-stained 2-μm sections, using a score of 0–4, with 0 representing no lesion and 4 representing severest lesion as we described previously.18.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (195) Google Scholar Immunostaining was performed on 2-μm serial paraffin sections. Primary antibodies used were polyclonal rabbit anti-rat megalin antibody (1:1000), polyclonal rabbit anti-human Tamm-Horsfall protein antibody (1:600, Biomedical Technologies, Stoughton, MA, USA), polyclonal rabbit anti-mouse TSC antibody (1:1000, Chemicon, Temecula, CA, USA), polyclonal rabbit anti-mouse albumin antibody (1:2500, Nordic Bioscience, Herlev, Denmark), polyclonal goat anti-mouse κ-light chain (IgL) antibody (1:500, Cappel, Solon, OH, USA), polyclonal goat anti-mouse IgA (α-heavy chain) antibody (1:500, Cappel), polyclonal goat anti-mouse IgG (whole molecule) antibody (1:100, Cappel), polyclonal rabbit anti-rat AQP-1 antibody (1:300, Chemicon), polyclonal rabbit anti-HO-1 antibody (1:1000, Stressgen, Ann Arbor, MI, USA), and polyclonal goat anti-mouse MCP-1 antibody (1:70, R&D systems, Minneapolis, MN, USA). For albumin, IgL, IgG, and HO-1, sections were heated in citrate buffer by autoclave or microwave. For TSC and MCP-1, sections were treated with 0.1% trypsin for 10 min at 37 °C. Immunoreactive signals were visualized by biotin-secondary antibodies, Vectastain ABC Reagent (Vector Laboratories, Burlingame, CA, USA) and diaminobentidine (Nichirei Bioscience, Tokyo, Japan). Sections stained for megalin was counterstained with periodic acid Schiff, and for others with hematoxylin. For detection of apoptosis, terminal transferase dUTP nick-end labeling staining was performed utilizing a detection kit (ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100, Chemicon) and visualized with diaminobentidine. For analysis, terminal transferase dUTP nick-end labeling staining was performed following megalin staining performed on the same sections, visualized with Vector VIP Substrate Kit for Peroxidase (Vector Laboratories). In sections stained for megalin, five nonoverlapping high power fields were randomly chosen in the cortex and photographed. In each field, all PTCs (approximately 100 per a field) were assessed, and the megalin genotype was determined by its staining. On the adjacent sections stained for albumin, IgL, IgA, or IgG, each PTC was assessed for cytoplasmic accumulation of these proteins. The ratio of protein-accumulating vs nonaccumulating cells was determined for megalin (+) and (−) PTCs separately. Similarly, AQP-1 expression was assessed on the sections adjacent to megalin-stained sections. The ratio of AQP-1 diminishing vs nondiminishing cells was determined for megalin (+) and (−) PTCs. For HO-1 examination, all PTCs expressing HO-1 on tissue sections were assessed and megalin genotype was determined for each cell on the adjacent sections stained for megalin. Assessment for MCP-1 was performed in a similar fashion. Apoptosis was assessed on sections double stained for megalin and terminal transferase dUTP nick-end labeling. All PTCs positive for terminal transferase dUTP nick-end labeling staining were assessed, and megalin genotype was determined. For these analyses, the proportion of megalin (+) or (−) PTCs to total PTCs was determined in five randomly selected representative fields. It was utilized to calculate the proportion of HO-1 or MCP-1-expressing cells or apoptotic cells in megalin (+) and (−) PTCs, separately. Results are expressed as means±s.d. Mann–Whitney U-test was used to compare megalin-KO/NEP25 and megalin-intact/NEP25 groups. Wilcoxon signed-rank test was used to compare megalin (+) vs (−) cells within megalin-KO/NEP25 mouse. Values were considered significant at P<0.05. It is indicated as NS if there was no statistical difference. All the authors declared no competing interests. We thank Mr Tsukamoto, Ms Sakurai, and Ms Imai for their expert technical assistance. This study was supported by Grant-in Aid for Scientific Research of the Japan Society for the Promotion of Science, 16109005, and in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. A part of this study was presented in abstract form at the International Pediatric Nephrology Association meeting (Budapest, Hungary, 31 August–4 September 2007) and the American Society of Nephrology meeting (San Francisco, CA, 2–5 November 2007).