FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080590043> ?p ?o ?g. }

• W2080590043 endingPage "49147" @default.
• W2080590043 startingPage "49142" @default.
• W2080590043 abstract "Mesangial expansion is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Studies indicate that mutations in apolipoprotein E (apoE) might independently contribute to kidney dysfunction. Although the role of apoE as an atheroprotective molecule is well established, its role in kidney is unclear. In this study, we sought to explore whether apoE has a protective function in kidney. Northern blotting and reverse transcriptase-polymerase chain reaction showed apoE expression in kidney, and mesangial cell is a major source of apoE in kidney. In the kidneys of 14–16-month-old apoE-null mice, hematoxylin-eosin (HE) staining revealed increased mesangial cell proliferation and matrix formation compared with wild type mice or apoB-overexpressing mice, which have elevated plasma cholesterol and triglycerides. These data suggest that lack of apoE, rather than hyperlipidemia, contributes to increased mesangial expansion. We isolated mesangial cells from mouse kidney and determined the effect of apoE on cell growth. ApoE (E3, 10 μg/ml) completely inhibited serum, platelet-derived growth factor (10 ng/ml), as well as low density lipoprotein-induced mesangial cell proliferation. Among the three isoforms, E3 was found to be most effective in inhibiting mesangial cell proliferation. ApoE did not show any cytotoxic effect, and moreover, inhibited mesangial cell apoptosis induced by oxidized low density lipoprotein. These data suggest that apoE regulates growth as well as survival of mesangial cells. We previously showed that apoE induces matrix heparan sulfate proteoglycan (HSPG) in vascular cells, which has an antiproliferative effect. Similarly, apoE induced the mesangial matrix HSPG. Perlecan is the major HSPG of mesangial matrix and subendothelial space, and consistent with this, blockade of perlecan reversed the antiproliferative effect of apoE. Immunohistochemistry revealed reduced staining of perlecan in kidney from apoE-null mice. Because the loss of anionic HSPG in the basement membrane and mesangial matrix is associated with disruption of filtration barrier, these data suggest a novel role for kidney apoE in preserving the filtration barrier. In summary, apoE has a protective function in kidney as an autocrine regulator of mesangial expansion and kidney function. Mesangial expansion is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Studies indicate that mutations in apolipoprotein E (apoE) might independently contribute to kidney dysfunction. Although the role of apoE as an atheroprotective molecule is well established, its role in kidney is unclear. In this study, we sought to explore whether apoE has a protective function in kidney. Northern blotting and reverse transcriptase-polymerase chain reaction showed apoE expression in kidney, and mesangial cell is a major source of apoE in kidney. In the kidneys of 14–16-month-old apoE-null mice, hematoxylin-eosin (HE) staining revealed increased mesangial cell proliferation and matrix formation compared with wild type mice or apoB-overexpressing mice, which have elevated plasma cholesterol and triglycerides. These data suggest that lack of apoE, rather than hyperlipidemia, contributes to increased mesangial expansion. We isolated mesangial cells from mouse kidney and determined the effect of apoE on cell growth. ApoE (E3, 10 μg/ml) completely inhibited serum, platelet-derived growth factor (10 ng/ml), as well as low density lipoprotein-induced mesangial cell proliferation. Among the three isoforms, E3 was found to be most effective in inhibiting mesangial cell proliferation. ApoE did not show any cytotoxic effect, and moreover, inhibited mesangial cell apoptosis induced by oxidized low density lipoprotein. These data suggest that apoE regulates growth as well as survival of mesangial cells. We previously showed that apoE induces matrix heparan sulfate proteoglycan (HSPG) in vascular cells, which has an antiproliferative effect. Similarly, apoE induced the mesangial matrix HSPG. Perlecan is the major HSPG of mesangial matrix and subendothelial space, and consistent with this, blockade of perlecan reversed the antiproliferative effect of apoE. Immunohistochemistry revealed reduced staining of perlecan in kidney from apoE-null mice. Because the loss of anionic HSPG in the basement membrane and mesangial matrix is associated with disruption of filtration barrier, these data suggest a novel role for kidney apoE in preserving the filtration barrier. In summary, apoE has a protective function in kidney as an autocrine regulator of mesangial expansion and kidney function. heparan sulfate proteoglycan human apoB transgenic mice wild type low density lipoprotein fetal calf serum phosphate-buffered saline oxidized LDL terminal deoxynucleotidyl transferase end-labeling reverse transcriptase-polymerase chain reaction platelet-derived growth factor lipoprotein lipase Many forms of renal disease that progress to renal failure are characterized by mesangial cell proliferation and more prominently accumulation of mesangial matrix (1Couser W.G. Johnson R.J. Am. J. Kidney Dis. 1994; 23: 193-198Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 2Stockand J.D. Sansom S.C. Am. J. Kidney Dis. 1997; 29: 971-981Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 3Grond J. Weening J.J. Contrib. Nephrol. 1990; 81: 229-239Crossref PubMed Google Scholar). Factors that control mesangial cell function include cytokines and growth mediators, matrix components such as heparan sulfate proteoglycans (HSPG),1 and interactions with other cells such as the endothelial and epithelial cells (2Stockand J.D. Sansom S.C. Am. J. Kidney Dis. 1997; 29: 971-981Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Besides mesangial expansion, a prominent feature of glomerulosclerosis and nephropathy is decreased matrix HSPG (4Jensen T. Diabetes. 1997; 46: S98-S100Crossref PubMed Google Scholar, 5van den Born J. van Kraats A.A. Bakker M.A. Assmann K.J. Dijkman H.B. van der Laak J.A. Berden J.H. Diabetologia. 1995; 38: 1169-1175Crossref PubMed Scopus (39) Google Scholar, 6Tamsma J.T. van den Born J. Bruijn J.A. Assmann K.J. Weening J.J. Berden J.H. Wieslander J. Schrama E. Hermans J. Veerkamp J.H. Diabetologia. 1994; 37: 313-320Crossref PubMed Scopus (177) Google Scholar, 7Makino H. Ikeda S. Haramoto T. Ota Z. Nephron. 1992; 61: 415-421Crossref PubMed Scopus (51) Google Scholar). Because HSPG is a key regulator of mesangial growth (8Castellot Jr., J.J. Hoover R.L. Harper P.A. Karnovsky M.J. Am. J. Pathol. 1985; 120: 427-435PubMed Google Scholar), it is conceivable that the decreased HSPG in part contributes to increased mesangial proliferation. Understanding the regulation of mesangial proliferation is important for the design of therapeutic strategies to alleviate or arrest proliferative glomerular disease. ApoE is a major protein component of plasma lipoproteins and plays a key role in lipoprotein clearance (9Mahley R.W. Science. 1988; 29: 622-630Crossref Scopus (3386) Google Scholar, 10Mahley R.W Huang Y. Curr. Opin. Lipidol. 1999; 10: 207-217Crossref PubMed Scopus (324) Google Scholar). A lack of apoE results in hyperlipidemia and in the development of atherosclerosis (11Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1840) Google Scholar, 12Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). Several studies now show that apoE can be atheroprotective even in a hyperlipidemia setting (13Shimano H. Ohsuga J. Shimada M. Namba Y. Gotoda T. Harada K. Katsuki M. Yazaki Y. Yamada N. J. Clin. Invest. 1995; 95: 469-476Crossref PubMed Scopus (128) Google Scholar, 14Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar, 15Fazio S. Babaev V.R. Murray A.B. Hasty A.H. Carter K.J. Gleaves L.A. Atkinson J.B. Linton M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar). This atheroprotective effect of apoE could be because of any of the recently identified novel functions, prominent among these is their ability to inhibit smooth muscle cell proliferation and increase vascular HSPG (16Paka L. Kako Y. Obunike J.C. Pillarisetti S. J. Biol. Chem. 1999; 274: 4816-4823Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 17Ishigami M. Swertfeger D.K. Granholm N.A. Hui D.Y. J. Biol. Chem. 1998; 273: 20156-20161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Atherosclerosis and glomerulosclerosis have several common features including loss of HSPG, endothelial dysfunction, and unregulated cell proliferation. Thus, kidney apoE could be protective against the development of glomerulosclerosis. Although apoE expression was shown in kidney (19Wallis S.C. Rogne S. Gill L. Markham A. Edge M. Woods D. Williamson R. Humphries S. EMBO J. 1983; 2: 2369-2373Crossref PubMed Scopus (34) Google Scholar,20Blue M.L. Williams D.L. Zucker S. Khan S.A. Blum C.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 283-287Crossref PubMed Scopus (138) Google Scholar), its role in the kidney is not known. In the current study, we explored the contribution of apoE to kidney function. Our studies show that apoE regulates mesangial expansion and HSPG levels, and a lack of apoE contributes to increased mesangial proliferation and matrix accumulation. Kidneys used in these studies were obtained from 14–16-month-old mice (Jackson Laboratories) of the following strains: C57BL/6 mice (n = 4), apoE-null mice on C57BL/6 background (n = 6), and human apoB transgenic (HuBTg) mice (n = 6). HuBTg mice had a mixed genetic background of predominantly C57BL/6J (>75%) and FVB/N strains (21Kako Y. Huang L.S. Yang J. Katopodis T. Ramakrishnan R. Goldberg I.J. J. Lipid Res. 1999; 40: 2185-2194Abstract Full Text Full Text PDF PubMed Google Scholar) and were fed on a western-type diet. The cholesterol and triglyceride levels of these mice are as follows: cholesterol (mg/dl) −70 ± 23 (WT), 315 ± 45 (apoE-null), and 260 ± 27 (HuBTg) and triglycerides (mg/dl) −64 ± 29 (WT), 91 ± 30 (apoE-null), and 140 ± 38 (HuBTg). Kidney tissues were cut into small pieces, fixed in 10% formalin, embedded in paraffin, and 4-μm sections were stained with HE and examined under microscope. At least 20 glomeruli for each tissue were evaluated for cell count. The extracellular matrix expansion was analyzed and quantitated as described previously (22Raij L. Azar S. Keane W. Kidney Int. 1984; 26: 137-143Abstract Full Text PDF PubMed Scopus (655) Google Scholar). Mesangial cells from C57BL/6 mice were isolated and cultured as described previously (23Wolf G. Haberstroh U. Neilson E.G. Am. J. Pathol. 1992; 140: 95-107PubMed Google Scholar). The cells were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with insulin-transferrin-sodium selenite media (Sigma) and 10% fetal calf serum (FCS). Cells used in experiments were from passages 5–10. Glomerular epithelial cells were maintained in 10% FCS-RPMI 1640 medium. Tubular epithelial cell lines were obtained from American Type Culture Collection (Madin-Darby canine kidney) and grown in Dulbecco's modified Eagle's medium containing 10% FCS. LDL (d < 1.063) was isolated from fresh human plasma in the presence of EDTA by ultracentrifugation and dialyzed against phosphate-buffered saline (PBS) containing 0.5 mm EDTA. Oxidized LDL (OxLDL) was prepared by dialyzing the LDL with 10 μm copper sulfate for 24 h at room temperature as described previously (24Heinecke J.W. Rosen H. Chait A. J. Clin. Invest. 1984; 74: 1890-1894Crossref PubMed Scopus (432) Google Scholar). Apolipoprotein E isoforms, E2, E3, and E4 were obtained from Calbiochem. Unless indicated otherwise, apoE3 was used in all experiments. Cell proliferation was assessed by [3H]thymidine incorporation as described previously (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mesangial cells were seeded at a density of 2 × 104/well in a 48-well plate, and experiments were performed the following day (40–50% confluency). Cells were treated with serum-free medium for 24 h followed by serum medium with or without various agents for 24 h. The cells were then labeled with [3H]thymidine for 6 h, and the radioactivity incorporated into DNA was determined by a scintillation counter. Apoptotic cells were determined by counting the terminal deoxynucleotidyl transferase end-labeling (TUNEL)-positive nuclei (Dead End Colorimetric Apoptosis Detection System, Promega). Equal numbers of cells were plated on chamber slides (Nalgene-Nunc) and cultured for 24 h followed by 24 h of serum starvation. The cells were treated with 1) normal media as control, 2) media with 50 μg/ml LDL, 3) media with 50 μg/ml OxLDL, 4) media with 50 μg/ml LDL and 10 μg/ml apoE, or 5) media with 50 μg/ml OxLDL and 10 μg/ml apoE. Two days later, the cells were washed with PBS and fixed in 4% paraformaldehyde. TUNEL-positive cells were identified according to protocol by the manufacturer. Total RNA was extracted from kidneys of C57BL/6 and apoE-null mice and used for Northern analysis. Plasmid (pJS381) containing apoE clone was kindly provided by Dr. Jonathan Smith (Rockefeller University, NY). A 1053-bp apoE fragment was generated by restriction digestion of pJS381 by XbaI. The probe was labeled with [α-32P]dCTP by random primer labeling according to the manufacturer (Roche Molecular Biochemicals). Hybridization was carried out for 6 h at 68 °C in Perfect-Hyb Plus hybridization solution (Sigma). The same membrane was rehybridized with glyceraldehyde-3-phosphate dehydrogenase as internal control. Total RNA (2 μg) was subjected to RT-PCR using the following primers: forward 5′-CCAATCACAGGCAGGAAGAT-3′ and reverse 5′-CTCCTGCACCTGCTCAGAC-3′. The predicted polymerase chain reaction product using these primers is a 261-bp fragment. Confluent mesangial cells were incubated in culture medium containing either [3H]glucosamine or 35SO4 with or without 10 μg/ml apoE for 24 h. Heparan sulfate proteoglycans associated with cells and those secreted into medium were determined as described previously following purification by DEAE-cellulose chromatography and digestion with heparinase (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). ApoE effects on proteoglycans in different pools, pericellular (trypsin-releasable representing cell surface and extracellular proteoglycans), cellular (Triton X-100/NH4 releasable), and matrix (guanidine hydrochloride extractable following Triton X-100 treatment), were determined as described previously (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Pericellular proteoglycans minus matrix proteoglycans was represented as cell surface proteoglycans. Rat monoclonal antiperlecan antibody was purchased from Neo Markers Inc., and conjugated with fluorescein by QuickTag FITC conjugation kit (Roche Molecular Biochemicals) according to the recommended procedures. Kidneys from WT and apoE-null mice were embedded in OCT and snap frozen in liquid nitrogen. 6 μm of frozen sections were fixed in cold acetone, rinsed in PBS, and incubated in PBS containing 1% bovine serum albumin for 20 min. The sections were then incubated with FITC-conjugated perlecan antibody (1:100 dilution) followed by detection by fluorescence microscope. For negative control, slides were preincubated with 5× non-FITC-labeled perlecan antibody. Results are expressed as the mean ± S.D. Experiments were done in triplicates and repeated at least once. Statistical analyses were performed by Student's t test to determine the significance of change. A significance difference was considered for p values that are equal to or less than 0.05. ApoE is known to be antiproliferative in smooth muscle cells (17Ishigami M. Swertfeger D.K. Granholm N.A. Hui D.Y. J. Biol. Chem. 1998; 273: 20156-20161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Because the mesangial cell is phenotypically similar to the smooth muscle cell, we first investigated whether a lack of apoE results in altered mesangial morphology in kidney. ApoE-null mice have both high plasma cholesterol and triglycerides, and in order to distinguish the effects of apoE from those of hyperlipidemia, we also examined HuBTg mice. These mice are transgenic for human apoB and are a distinct model for hyperlipidemia. Kidney sections from wild type mice (WT, C57BL/6), apoE-null mice, and HuBTg mice were examined by HE staining (Fig.1). Significantly increased mesangial proliferation was seen in kidneys from apoE-null mice compared with kidneys from WT mice (p < 0.01) or HuBTg mice (p < 0.05). The most striking change in apoE-null mice was the matrix overproduction in comparison with WT and apoB mice (p < 0.01). Interestingly, the gross kidney of HuBTg mouse is much bigger than that of WT and apoE-null mice, and enlarged glomerulus was observed with mild cell proliferation, which was significantly different from that of WT mouse. However, there was no prominent matrix expansion in HuBTg mice (Fig. 1, TableI). These data suggest that a lack of apoE contributes to mesangial cell proliferation and matrix expansion in the kidney.Table IMorphological changes in glomeruli from wild type apoE-null and apoB transgenic miceCellSclerosisWT (n = 4)35.1 ± 0.10.5 ± 0.2ApoE-null (n = 6)59.8 ± 14.21-ap < 0.01versus WT.1-bp < 0.05 versusApoB-Tg.2.0 ± 0.51-cp < 0.01 versusWT.1-dp < 0.01 versusApoB-Tg.ApoB-Tg (n= 4)42.9 ± 2.51-ep < 0.05 versusWT.0.5 ± 0.3Glomerular cell numbers and sclerosis score of kidneys from wild type (WT), apoE-null and apoB transgenic (ApoB-Tg) mice. Kidney sections were stained with hematoxylin-eosin and examined under microscope. At least 20 glomeruli for each tissue were evaluated for cell count and extracellular matrix expression as described under “Materials and Methods.”1-a p < 0.01versus WT.1-b p < 0.05 versusApoB-Tg.1-c p < 0.01 versusWT.1-d p < 0.01 versusApoB-Tg.1-e p < 0.05 versusWT. Open table in a new tab Glomerular cell numbers and sclerosis score of kidneys from wild type (WT), apoE-null and apoB transgenic (ApoB-Tg) mice. Kidney sections were stained with hematoxylin-eosin and examined under microscope. At least 20 glomeruli for each tissue were evaluated for cell count and extracellular matrix expression as described under “Materials and Methods.” To correlate apoE expression to mesangial expansion, we determined apoE expression in kidney and kidney cell types. Northern blot analysis of total kidney RNA showed apoE expression in kidneys of WT but not of apoE-null mice (Fig. 2A). RT-PCR of total RNA isolated from different kidney cell types revealed a strong expression of apoE in mesangial cells (Fig. 2B). A weak band was also seen in glomerular epithelial cells. Both tubular epithelial cells and endothelial cells were negative for apoE expression. The results suggest that mesangial cell is a major contributor of kidney apoE. To determine the effects of apoE on cell proliferation, we isolated mesangial cells from wild type mouse kidney and tested whether apoE inhibits mesangial cell proliferation. ApoE (10 μg/ml) completely inhibited serum-stimulated cell growth (Fig. 3A). At a similar dose, apoE did not affect glomerular epithelial cell proliferation (Fig. 3B). We previously reported that apoE does not inhibit endothelial proliferation (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, the antiproliferative effect of apoE is specific for mesangial cells. Although the trigger for mesangial proliferation is not clear, studies have identified several possible candidates including PDGF, interleukin-6, and LDL (25Schocklmann H.O. Lang S. Sterzel R.B. Kidney Int. 1999; 56: 1199-1207Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Eitner F. Westerhuis R. Burg M. Weinhold B. Grone H.J. Ostendorf T. Ruther U. Koch K.M. Rees A.J. Floege J. Kidney Int. 1997; 51: 69-78Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 27Sharma P. Reddy K. Franki N. Sanwal V. Sankaran R. Ahuja T.S. Gibbons N. Mattana J. Singhal P.C. Kidney Int. 1996; 50: 1604-1611Abstract Full Text PDF PubMed Scopus (34) Google Scholar). We tested whether apoE can suppress the growth-promoting effects of these agents. Incubation of mesangial cells with PDGF (10 ng/ml) resulted in a 2–2.5-fold increase in [3H]thymidine incorporation into DNA (Fig.4A). Similarly, LDL (20 μg/ml) increased [3H]thymidine incorporation into DNA by ∼2-fold (Fig. 4B). The addition of apoE completely reversed this effect. These data show that the antiproliferative effects of apoE extend to a variety of mesangial growth inducers. We next tested whether the antiproliferative effects of apoE are isoform-specific. At similar concentrations, apoE3 was found to be most effective (∼50%) in inhibiting mesangial cell proliferation (Fig.4C). ApoE4 also showed significant inhibition (∼30%,p < 0.05). ApoE2 showed moderate effect (∼19%) on mesangial proliferation. Several antiproliferative agents can also induce cell apoptosis. To rule out the possibility that the antiproliferative effect of apoE is because of apoptosis, we determined the effect of apoE on OxLDL-induced apoptosis. Confluent mesangial cells were incubated with OxLDL (50 μg/ml) and LDL (50 μg/ml) in the presence or absence of apoE for 48 h. Under these conditions only OxLDL but not LDL induced mesangial cell apoptosis as determined by TUNEL staining. OxLDL-induced cell apoptosis was completely blocked by apoE, and virtually no apoptotic cells were observed (Fig. 5). These data show that apoE plays a key role in both the regulation of proliferation as well as the survival of mesangial cells. The mechanism by which apoE inhibits mesangial cell proliferation is not clear. Heparan sulfate and HSPG are potent inhibitors of mesangial proliferation, and we previously showed that apoE induced HSPG in smooth muscle cells (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We next tested whether apoE inhibits mesangial cell proliferation by increasing HSPG. Subconfluent monolayers of mesangial cells were incubated with apoE, and cellular HSPG were expressed as a ratio to cell number. ApoE3 increased HSPG to cell ratio by >2-fold (Fig.6A). E2 and E4 were less effective, and this may in part explain their effects on mesangial cell proliferation (Fig. 4C). The kidney, like other tissues, contain different HSPGs including syndecan and glypican (cell surface) and perlecan (extracellular matrix). ApoE treatment resulted in the induction of HSPG in all pools (Fig. 6B). Perlecan is the major HSPG in the glomerular basement membrane as well as mesangial matrix and may contribute to the regulation of mesangial cell proliferation (28Iozzo R.V. Cohen I.R Grassel S. Murdoch A.D. Biochem. J. 1994; 302: 625-639Crossref PubMed Scopus (341) Google Scholar, 29Groffen A.J. Veerkamp J.H. Monnens L.A. van den Heuvel L.P. Nephrol. Dial. Transplant. 1999; 14: 2119-2129Crossref PubMed Scopus (72) Google Scholar). We next tested whether the induction of perlecan mediates the antiproliferative effect of apoE (Fig.6C). Perlecan antibody but not mouse IgG reversed the antiproliferative effect of apoE, suggesting that perlecan contributes to the antiproliferative effect of apoE. We next determined whether the absence of apoE results in altered perlecan expression in kidney. Immunohistochemical analysis of perlecan in WT and apoE-null mice revealed significantly decreased staining in apoE-null mice (Fig. 7). These data suggest that the morphological changes seen in apoE-null kidney may in part be due to decreased perlecan. Our data for the first time identify a protective role for apoE in preventing a proliferative phenotype in kidney. Mice that are deficient in apoE have both increased proliferation as well as matrix overproduction, a hallmark of kidney pathogenesis. Several human studies identified an association between apoE polymorphism and nephropathy (30Chowdhury T.A. Dyer P.H. Kumar S. Gibson S.P. Rowe B.R. Davies S.J. Marshall S.M. Morris P.J. Gill G.V. Feeney S. Maxwell P. Savage D. Boulton A.J. Todd J.A. Dunger D. Barnett A.H. Bain S.C. Diabetes. 1998; 47: 278-280Crossref PubMed Scopus (76) Google Scholar, 31Werle E. Fiehn W. Hasslacher C. Diabetes Care. 1998; 21: 994-998Crossref PubMed Scopus (56) Google Scholar, 32Eto M. Horita K. Morikawa A. Nakata H. Okada M. Saito M. Nomura M. Abiko A. Iwashima Y. Ikoda A. Makino I. Clin. Genet. 1995; 48: 288-292Crossref PubMed Scopus (75) Google Scholar, 33Onuma T. Laffel L.M. Angelico M.C. Krolewski A.S. J. Am. Soc. Nephrol. 1996; 7: 1075-1078PubMed Google Scholar). A recent large case-controlled study with 223 subjects showed a 3.1-fold increase in the risk of diabetic nephropathy in subjects carrying E2 allele of apoE (34Araki S. Moczulski D.K. Hanna L. Scott L.J. Warram J.H. Krolewski A.S. Diabetes. 2000; 49: 2190-2195Crossref PubMed Scopus (81) Google Scholar). Although the molecular mechanisms underlying this increased risk are unclear, it is often thought to be because of dyslipidemia, in particular, to increased triglycerides. However, our data on mesangial matrix accumulation in HuBTg mice suggest lipid-independent effects on mesangial expansion in apoE-null mice. HuBTg mice, despite having hypercholesterolemia and hypertriglyceredemia, clearly did not show changes in mesangial morphology that are seen in apoE-null mice. ApoE2, as we previously showed, has no significant antiproliferative effect on smooth muscle cells (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, it is conceivable that the increased risk of nephropathy in E2 subjects is in part attributed to the inability of apoE2 to regulate mesangial expansion. Apart from liver, studies showed that kidney is a major source of apoE (19Wallis S.C. Rogne S. Gill L. Markham A. Edge M. Woods D. Williamson R. Humphries S. EMBO J. 1983; 2: 2369-2373Crossref PubMed Scopus (34) Google Scholar, 20Blue M.L. Williams D.L. Zucker S. Khan S.A. Blum C.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 283-287Crossref PubMed Scopus (138) Google Scholar). Our Northern data confirmed this finding and showed an intense band of apoE in WT mice but not in apoE-null mice. Although kidney expression of apoE has been clearly demonstrated, the source of apoE in kidney is not known. Early studies showed relatively greater amounts of apoE synthesis in the cortex compared with medulla (20Blue M.L. Williams D.L. Zucker S. Khan S.A. Blum C.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 283-287Crossref PubMed Scopus (138) Google Scholar). RT-PCR analysis of different kidney cell types in the current study showed that mesangial cells are a major source of apoE expression. Mesangial cell proliferation and matrix overproduction are the predominant pathological features of many forms of glomerulonephritis, such as IgA nephropathy, lupus nephropathy, and diabetic nephropathy and frequently precedes the increase of extracellular matrix in the mesangium and the development of glomerulosclerosis (2Stockand J.D. Sansom S.C. Am. J. Kidney Dis. 1997; 29: 971-981Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 3Grond J. Weening J.J. Contrib. Nephrol. 1990; 81: 229-239Crossref PubMed Google Scholar). When exposed to injurious stimuli such as hyperglycemia, glycated proteins, or oxidants, the mesangial cell responds by cellular proliferation and matrix synthesis. A key example of this is the pro-sclerotic cytokine-transforming growth factor-β, which is induced by many diabetic stimuli (35Rossert J. Terraz-Durasnel C. Brideau G. Diabetes Metab. 2000; 26: S16-S24PubMed Google Scholar, 36Sharma K. McGowan T.A. Cytokine Growth Factor Rev. 2000; 11: 115-123Crossref PubMed Scopus (75) Google Scholar). The up-regulation of transforming growth factor-β may be a necessary event in the removal and repair of damaged matrix. However, a loss of proper regulation of this repair process may ultimately progress to the development of glomerulosclerosis. Expression of apoE in mesangial cells may serve as an autocrine regulator of such uncontrolled mesangial expansion. Our data clearly establish apoE as an antiproliferative molecule for mesangial cells. It inhibited mesangial proliferation induced by different stimuli including growth factors and lipids. This finding is consistent with the observed apoE effects on smooth muscle cells in which the proliferative effects of serum, PDGF, OxLDL, and lysolecithin were inhibited by apoE (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The antiproliferative effects of apoE, however, were not attributed to the induction of apoptosis. Instead, apoE was found to be antiapoptotic and prevented oxidant-induced apoptosis. Proliferation as well as apoptosis of mesangial cells has been shown in glomerular diseases (37Makino H. Sugiyama H. Kashihara N. Kidney Int. 2000; 58: S67-S75Abstract Full Text Full Text PDF Scopus (68) Google Scholar, 38Saleh H. Schlatter E. Lang D. Pauels H.G. Heidenreich S. Kidney Int. 2000; 58: 1876-1884Abstract Full Text Full Text PDF PubMed Google Scholar). Both processes may regulate the cellular content of the mesangium by closely influencing each other. Thus, apoE may offer a dual protection against proliferation and apoptosis. Increasing matrix HSPG (perlecan) is a probable mechanism by which apoE can be antiproliferative to mesangial cells. Consistent with previous studies with smooth muscle cells (18Paka L. Goldberg I.J. Obunike J.C. Choi S.Y. Saxena U. Goldberg I.D. Pillarisetti S. J. Biol. Chem. 1999; 274: 36403-36408Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), perlecan antibody significantly blocked the antiproliferative effect of apoE on mesangial cells. Obunike et al. (39Obunike J.C. Pillarisetti S. Paka L. Kako Y. Butteri M.J. Ho Y.Y. Wagner W.D. Yamada N. Mazzone T. Deckelbaum R.J Goldberg I.J. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 111-118Crossref PubMed Scopus (37) Google Scholar) previously showed that lipoprotein lipase (LpL), like apoE, can induce proteoglycans in other cell types. However, it is not known whether this increase is in HSPG or in perlecan. Recent studies (40Mamputu J.C. Levesque L. Renier G. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2212-2219Crossref PubMed Scopus (35) Google Scholar, 41Stevenson F.T. Shearer G.C. Atkinson D.N. Kidney Int. 2001; 59: 2062-2068Abstract Full Text Full Text PDF PubMed Google Scholar) from two different laboratories showed that LpL, in contrast to apoE, stimulates proliferation of smooth muscle and mesangial cells. The antiproliferative effects of apoE on smooth muscle cells appear to require LpL activity. It is conceivable that residual serum lipids (lipoprotein-derived) or cellular lipids are hydrolyzed by LpL leading to the generation of fatty acids or lysolipids, both of which can activate protein kinase C, a mitogenic signal. Not surprisingly, these authors found that protein kinase C inhibitors completely blocked LpL-induced proliferation. The ability of ApoE to increase mesangial (current study) and endothelial HSPG (16Paka L. Kako Y. Obunike J.C. Pillarisetti S. J. Biol. Chem. 1999; 274: 4816-4823Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) may have other implications. Apart from being antiproliferative to mesangial cells, HSPG plays an important role in barrier function (4Jensen T. Diabetes. 1997; 46: S98-S100Crossref PubMed Google Scholar, 5van den Born J. van Kraats A.A. Bakker M.A. Assmann K.J. Dijkman H.B. van der Laak J.A. Berden J.H. Diabetologia. 1995; 38: 1169-1175Crossref PubMed Scopus (39) Google Scholar, 6Tamsma J.T. van den Born J. Bruijn J.A. Assmann K.J. Weening J.J. Berden J.H. Wieslander J. Schrama E. Hermans J. Veerkamp J.H. Diabetologia. 1994; 37: 313-320Crossref PubMed Scopus (177) Google Scholar). Increasing evidence supports the hypothesis that a loss of heparan sulfate may play a pathophysiological role in the development of diabetic vascular complications. Our data show decreased staining for perlecan in apoE-null mice. Perlecan, the major HSPG of the basement membrane and mesangial matrix, plays an important role in the assembly and structure of the basement membrane and regulation of basement membrane permeability (42Pillarisetti S. Trends Cardiovasc. Med. 2000; 10: 60-65Crossref PubMed Scopus (63) Google Scholar). In diabetic patients with mesangial cell expansion and clinical nephropathy, a negative correlation was observed among the number of anionic sites representing HSPG in the glomerular basement membrane, and urinary albumin secretion was also observed (6Tamsma J.T. van den Born J. Bruijn J.A. Assmann K.J. Weening J.J. Berden J.H. Wieslander J. Schrama E. Hermans J. Veerkamp J.H. Diabetologia. 1994; 37: 313-320Crossref PubMed Scopus (177) Google Scholar). Heparin, which increases HSPG, has been shown to reduce albuminuria in patients with incipient diabetic nephropathy (43van Der Woude F.J. Haemostasis. 1999; 29: S61-S67PubMed Google Scholar). Thus, apoE by virtue of its ability to induce perlecan may help preserve barrier function and inhibit the hyperpermeability associated with kidney dysfunction. To determine whether morphological changes seen in apoE-null mice correlate with permeability changes, we determined urine albumin in wild type and apoE-null mice. Serum creatinine levels were similar in wild type and apoE-null mice (27.27 ± 6.53 μmol/literversus 28.32 ± 5.01 μmol/liter). Although it did not reach statistical significance, urinary albumin was elevated in apoE-null mice (1.7 ± 0.24 mg/mmol creatinine in apoE-null mice compared with 1.38 ± 0.1 mg/mmol creatinine in wild type mice,p = 0.09, n = 4). Further studies requiring large number of animals are needed to conclusively show kidney dysfunction in apoE-null mice. Several non-traditional and novel functions of apoE are only beginning to be realized (42Pillarisetti S. Trends Cardiovasc. Med. 2000; 10: 60-65Crossref PubMed Scopus (63) Google Scholar). Data presented here add to the growing list of protective effects that apoE possesses. In addition to its antiproliferative and HSPG-inducing effects, apoE can also be an antioxidant (44Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (801) Google Scholar). Because oxidant stress is a major player in nephropathy, it will be of interest to see whether overexpression of apoE offers protection against the development of nephropathy and kidney dysfunction. We thank Drs. Rick Timmer and Uday Saxena for comments on the manuscript." @default.
• W2080590043 created "2016-06-24" @default.
• W2080590043 creator A5004398523 @default.
• W2080590043 creator A5025548638 @default.
• W2080590043 creator A5034860785 @default.
• W2080590043 creator A5055516364 @default.
• W2080590043 creator A5072228368 @default.
• W2080590043 creator A5091583711 @default.
• W2080590043 date "2001-12-01" @default.
• W2080590043 modified "2023-10-09" @default.
• W2080590043 title "A Protective Role for Kidney Apolipoprotein E" @default.
• W2080590043 cites W1855999912 @default.
• W2080590043 cites W1967362524 @default.
• W2080590043 cites W1980355423 @default.
• W2080590043 cites W1985859760 @default.
• W2080590043 cites W1990815703 @default.
• W2080590043 cites W1992980650 @default.
• W2080590043 cites W1993404941 @default.
• W2080590043 cites W1993563498 @default.
• W2080590043 cites W1996324680 @default.
• W2080590043 cites W1999968358 @default.
• W2080590043 cites W2000951003 @default.
• W2080590043 cites W2012900183 @default.
• W2080590043 cites W2017315967 @default.
• W2080590043 cites W2021304013 @default.
• W2080590043 cites W2034159586 @default.
• W2080590043 cites W2034968148 @default.
• W2080590043 cites W2035252462 @default.
• W2080590043 cites W2048890665 @default.
• W2080590043 cites W2053097440 @default.
• W2080590043 cites W2063576773 @default.
• W2080590043 cites W2066837348 @default.
• W2080590043 cites W2083074809 @default.
• W2080590043 cites W2085382182 @default.
• W2080590043 cites W2093771931 @default.
• W2080590043 cites W2096755911 @default.
• W2080590043 cites W2099346887 @default.
• W2080590043 cites W2107087264 @default.
• W2080590043 cites W2121775710 @default.
• W2080590043 cites W2127014900 @default.
• W2080590043 cites W2132243176 @default.
• W2080590043 cites W2134010411 @default.
• W2080590043 cites W2136431568 @default.
• W2080590043 cites W2142403172 @default.
• W2080590043 cites W2146233088 @default.
• W2080590043 cites W2157621288 @default.
• W2080590043 cites W2165548747 @default.
• W2080590043 cites W2166453795 @default.
• W2080590043 cites W2278269068 @default.
• W2080590043 cites W2315335674 @default.
• W2080590043 cites W251730109 @default.
• W2080590043 doi "https://doi.org/10.1074/jbc.m104879200" @default.
• W2080590043 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11579084" @default.
• W2080590043 hasPublicationYear "2001" @default.
• W2080590043 type Work @default.
• W2080590043 sameAs 2080590043 @default.
• W2080590043 citedByCount "76" @default.
• W2080590043 countsByYear W20805900432012 @default.
• W2080590043 countsByYear W20805900432013 @default.
• W2080590043 countsByYear W20805900432014 @default.
• W2080590043 countsByYear W20805900432015 @default.
• W2080590043 countsByYear W20805900432016 @default.
• W2080590043 countsByYear W20805900432017 @default.
• W2080590043 countsByYear W20805900432018 @default.
• W2080590043 countsByYear W20805900432019 @default.
• W2080590043 countsByYear W20805900432020 @default.
• W2080590043 countsByYear W20805900432023 @default.
• W2080590043 crossrefType "journal-article" @default.
• W2080590043 hasAuthorship W2080590043A5004398523 @default.
• W2080590043 hasAuthorship W2080590043A5025548638 @default.
• W2080590043 hasAuthorship W2080590043A5034860785 @default.
• W2080590043 hasAuthorship W2080590043A5055516364 @default.
• W2080590043 hasAuthorship W2080590043A5072228368 @default.
• W2080590043 hasAuthorship W2080590043A5091583711 @default.
• W2080590043 hasBestOaLocation W20805900431 @default.
• W2080590043 hasConcept C126322002 @default.
• W2080590043 hasConcept C185592680 @default.
• W2080590043 hasConcept C2778163477 @default.
• W2080590043 hasConcept C2780091579 @default.
• W2080590043 hasConcept C55493867 @default.
• W2080590043 hasConcept C62746215 @default.
• W2080590043 hasConcept C71924100 @default.
• W2080590043 hasConcept C86803240 @default.
• W2080590043 hasConceptScore W2080590043C126322002 @default.
• W2080590043 hasConceptScore W2080590043C185592680 @default.
• W2080590043 hasConceptScore W2080590043C2778163477 @default.
• W2080590043 hasConceptScore W2080590043C2780091579 @default.
• W2080590043 hasConceptScore W2080590043C55493867 @default.
• W2080590043 hasConceptScore W2080590043C62746215 @default.
• W2080590043 hasConceptScore W2080590043C71924100 @default.
• W2080590043 hasConceptScore W2080590043C86803240 @default.
• W2080590043 hasIssue "52" @default.
• W2080590043 hasLocation W20805900431 @default.
• W2080590043 hasOpenAccess W2080590043 @default.
• W2080590043 hasPrimaryLocation W20805900431 @default.
• W2080590043 hasRelatedWork W1531601525 @default.
• W2080590043 hasRelatedWork W2319480705 @default.
• W2080590043 hasRelatedWork W2384464875 @default.

• next