Dendrin Ablation Prolongs Life Span by Delaying Kidney Failure
2015; Elsevier BV; Volume: 185; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2015.04.011
ISSN1525-2191
AutoresAstrid Weins, Jenny Wong, John M. Basgen, Ritu Gupta, Ilse S. Daehn, Lisette Casagrande, David Lessman, Monica Schwartzman, Kristin Meliambro, Jaakko Patrakka, Andréy S. Shaw, Karl Tryggvason, John Cijiang He, Susanne B. Nicholas, Peter Mündel, Kirk N. Campbell,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoPodocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin. Podocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin. Podocytes are highly specialized cells that constitute a central component of the kidney filtration barrier. Disruption of podocyte function damages the kidney filter, resulting in proteinuria.1Greka A. Mundel P. Cell biology and pathology of podocytes.Annu Rev Physiol. 2012; 74: 299-323Crossref PubMed Scopus (376) Google Scholar Primary and secondary podocytopathies combined account for about 90% of end-stage kidney disease (ESKD) at a cost of $20 billion per year in the United States alone.2Wiggins R.C. The spectrum of podocytopathies: a unifying view of glomerular diseases.Kidney Int. 2007; 71: 1205-1214Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar The health care burden and economic impact is further enhanced by the fact that proteinuria is a strong and independent risk factor for cardiovascular disease.3Agrawal V. Marinescu V. Agarwal M. McCullough P.A. Cardiovascular implications of proteinuria: an indicator of chronic kidney disease.Nat Rev Cardiol. 2009; 6: 301-311Crossref PubMed Scopus (91) Google Scholar Podocytes are terminally differentiated cells that cannot regenerate in response to injury or loss. As a result, podocyte loss causes glomerulosclerosis, in which a reduction in podocyte number below a critical 40% threshold results in high-level sustained proteinuria and decreased renal function.4Kim Y.H. Goyal M. Kurnit D. Wharram B. Wiggins J. Holzman L. Kershaw D. Wiggins R. Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.Kidney Int. 2001; 60: 957-968Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 5Wharram B.L. Goyal M. Wiggins J.E. Sanden S.K. Hussain S. Filipiak W.E. Saunders T.L. Dysko R.C. Kohno K. Holzman L.B. Wiggins R.C. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (582) Google Scholar Clear clinical correlations between podocytopenia and proteinuria have also been established. Podocyte loss contributes to the progression of diabetic nephropathy in patients with type II diabetes mellitus.6Pagtalunan M.E. Miller P.L. Jumping-Eagle S. Nelson R.G. Myers B.D. Rennke H.G. Coplon N.S. Sun L. Meyer T.W. Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (899) Google Scholar Podocyte cell number is also decreased in patients of all ages with type I diabetes, where the reduction in podocyte number correlates with an increased albumin excretion rate.7Steffes M.W. Schmidt D. McCrery R. Basgen J.M. Glomerular cell number in normal subjects and in type 1 diabetic patients.Kidney Int. 2001; 59: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8White K.E. Bilous R.W. Marshall S.M. El Nahas M. Remuzzi G. Piras G. De Cosmo S. Viberti G. Podocyte number in normotensive type 1 diabetic patients with albuminuria.Diabetes. 2002; 51: 3083-3089Crossref PubMed Scopus (269) Google Scholar In IgA nephropathy, podocyte loss correlates closely with the degree of proteinuria, glomerulosclerosis, and renal dysfunction.9Lemley K.V. Lafayette R.A. Safai M. Derby G. Blouch K. Squarer A. Myers B.D. Podocytopenia and disease severity in IgA nephropathy.Kidney Int. 2002; 61: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar Podocytopenia is also associated with disease progression in membranous nephropathy, focal segmental glomerulosclerosis (FSGS), amyloidosis, and the aging kidney.10Shankland S.J. The podocyte's response to injury: role in proteinuria and glomerulosclerosis.Kidney Int. 2006; 69: 2131-2147Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar Despite remarkable advances in clarifying the details of the molecular architecture of podocytes and their interdigitating foot processes, mechanisms underlying podocyte injury and loss remain poorly understood. This results in a significant unmet clinical need. We previously identified dendrin as a dual-compartment signaling molecule anchored at the slit diaphragm under normal conditions.11Asanuma K. Campbell K.N. Kim K. Faul C. Mundel P. Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes.Proc Natl Acad Sci U S A. 2007; 104: 10134-10139Crossref PubMed Scopus (77) Google Scholar Dendrin has no known homologous partners and an unclear role in normal physiology. Under the influence of cell death–inducing high-dose transforming growth factor-β (TGF-β), dendrin relocates to the podocyte nucleus and promotes apoptosis, which is ameliorated after gene silencing of dendrin.11Asanuma K. Campbell K.N. Kim K. Faul C. Mundel P. Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes.Proc Natl Acad Sci U S A. 2007; 104: 10134-10139Crossref PubMed Scopus (77) Google Scholar Of note, dendrin-mediated podocyte apoptosis is inhibited by the prosurvival Yes associated protein (YAP).12Campbell K.N. Wong J.S. Gupta R. Asanuma K. Sudol M. He J.C. Mundel P. Yes-associated protein (YAP) promotes cell survival by inhibiting proapoptotic dendrin signaling.J Biol Chem. 2013; 288: 17057-17062Crossref PubMed Scopus (47) Google Scholar In rodents, nuclear dendrin has been observed in inflammatory glomerulonephritis, in CD2-associated protein (Cd2ap)-deficient (Cd2ap−/−) mice and in adriamycin (ADR) nephropathy.11Asanuma K. Campbell K.N. Kim K. Faul C. Mundel P. Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes.Proc Natl Acad Sci U S A. 2007; 104: 10134-10139Crossref PubMed Scopus (77) Google Scholar, 13Yaddanapudi S. Altintas M.M. Kistler A.D. Fernandez I. Moller C.C. Wei C. Peev V. Flesche J.B. Forst A.L. Li J. Patrakka J. Xiao Z. Grahammer F. Schiffer M. Lohmuller T. Reinheckel T. Gu C. Huber T.B. Ju W. Bitzer M. Rastaldi M.P. Ruiz P. Tryggvason K. Shaw A.S. Faul C. Sever S. Reiser J. CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival.J Clin Invest. 2011; 121: 3965-3980Crossref PubMed Scopus (114) Google Scholar, 14Asanuma K. Akiba-Takagi M. Kodama F. Asao R. Nagai Y. Lydia A. Fukuda H. Tanaka E. Shibata T. Takahara H. Hidaka T. Asanuma E. Kominami E. Ueno T. Tomino Y. Dendrin location in podocytes is associated with disease progression in animal and human glomerulopathy.Am J Nephrol. 2011; 33: 537-549Crossref PubMed Scopus (34) Google Scholar Nuclear dendrin has also been found in biopsies from patients with FSGS, membranous nephropathy, and class V lupus nephritis.14Asanuma K. Akiba-Takagi M. Kodama F. Asao R. Nagai Y. Lydia A. Fukuda H. Tanaka E. Shibata T. Takahara H. Hidaka T. Asanuma E. Kominami E. Ueno T. Tomino Y. Dendrin location in podocytes is associated with disease progression in animal and human glomerulopathy.Am J Nephrol. 2011; 33: 537-549Crossref PubMed Scopus (34) Google Scholar Conversely, dendrin-null (Ddn−/−) mice display no overt pathology; they have a normal life span and do not develop proteinuria or renal failure.15Xiao Z. Rodriguez P.Q. He L. Betsholtz C. Tryggvason K. Patrakka J. Wtip- and gadd45a-interacting protein dendrin is not crucial for the development or maintenance of the glomerular filtration barrier.PLoS One. 2013; 8: e83133Crossref PubMed Scopus (5) Google Scholar Here, we tested the hypothesis that the ablation of dendrin would improve renal survival in progressive glomerulosclerosis. We deleted the dendrin gene in Cd2ap−/− mice that develop podocyte injury with proteinuria at 2 to 3 weeks of age and die of renal failure at 8 to 9 weeks of age.16Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar The phenotype of Cd2ap−/− mice was completely reversed with podocyte-specific expression of CD2AP,17Grunkemeyer J.A. Kwoh C. Huber T.B. Shaw A.S. CD2-associated protein (CD2AP) expression in podocytes rescues lethality of CD2AP deficiency.J Biol Chem. 2005; 280: 29677-29681Crossref PubMed Scopus (55) Google Scholar confirming podocyte injury as the root cause of kidney failure and death. Clinical evidence for the role of CD2AP as an essential component of the glomerular filtration barrier has been provided by the identification of patients with FSGS-causing Cd2ap−/− gene mutations.18Benoit G. Machuca E. Nevo F. Gribouval O. Lepage D. Antignac C. Analysis of recessive CD2AP and ACTN4 mutations in steroid-resistant nephrotic syndrome.Pediatr Nephrol. 2010; 25: 445-451Crossref PubMed Scopus (27) Google Scholar, 19Gigante M. Pontrelli P. Montemurno E. Roca L. Aucella F. Penza R. Caridi G. Ranieri E. Ghiggeri G.M. Gesualdo L. CD2AP mutations are associated with sporadic nephrotic syndrome and focal segmental glomerulosclerosis (FSGS).Nephrol Dial Transplant. 2009; 24: 1858-1864Crossref PubMed Scopus (97) Google Scholar, 20Lowik M.M. Groenen P.J. Pronk I. Lilien M.R. Goldschmeding R. Dijkman H.B. Levtchenko E.N. Monnens L.A. van den Heuvel L.P. Focal segmental glomerulosclerosis in a patient homozygous for a CD2AP mutation.Kidney Int. 2007; 72: 1198-1203Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar Here, we show that dendrin ablation delays the onset of proteinuria, glomerulosclerosis, renal failure, and death in Cd2ap−/− mice. These results implicate dendrin as a potential podocyte-specific therapeutic target in slowing the progression from proteinuria to ESKD and death. All studies involving mice were performed in accordance with Icahn School of Medicine institutional guidelines under an institutional animal care and use committee–approved protocol. DNA for genotyping was extracted using a sodium hydroxide extraction protocol from The Jackson Laboratory (Bar Harbor, ME). Briefly, 2-mm tail was incubated with 25 mmol/L NaOH/0.2 mmol/L EDTA at 98°C for 1 hour in a thermocycler with subsequent temperature reduction to 15°C. After adding 40 mmol/L Tris-HCl (pH 5.5), the mixture was centrifuged at 16,000 × g for 3 minutes and an aliquot removed for PCR. The following primers were used for genotyping Cd2ap in the mice: forward 5′-GTTGACTATATTGTGGAATATGACTATGA-3′ and reverse 5′-CAGCAGGTTGCCAATCACAAATGTACCTATA-3′ for wild type; forward 5′-CAAGTCTGTCATAGGTCACACTCAAGAAG-3′ and reverse 5′-AGGTGCCACTCCCACTGTCCTTTCCTAATAA-3′ for knockout. The expected sizes for wild type is around 300 bp and for knockout is around 400 bp. The following primers were used for genotyping dendrin in the mice: forward 5′-GGAGGATCTCAGCGTCCATA-3′ and reverse 5′-AGGTTCAAGGCCTCTCCATT-3′ for wild type; forward 5′-AATTCCATCAGACCTCGACCT-3′ and reverse 5′-AGGTTCAAGGCCTCTCCATT-3′ for knockout. The expected size for wild type is 574 bp and for knockout is 405 bp. Urine albumin was quantified by enzyme-linked immunosorbent assay according to the manufacturer's protocol (Bethyl Laboratory, Montgomery, TX). Urine creatinine levels were measured in the same samples using the creatinine assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's protocol. Urine albumin excretion rate was expressed as the ratio of albumin to creatinine. Serum creatinine measurements were performed by liquid chromatography-tandem mass spectrometry at the UAB-UCSD O'Brien Core Center for Acute Kidney Injury Research (Birmingham, AL). Mice were perfused with filtered phosphate-buffered saline containing 3% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) at a flow rate of 9 mL per minute for 3 minutes. After perfusion, kidneys were harvested and further fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4; Electron Microscopy Services, Hatfield, PA). Four-micrometer paraffin-processed, formalin-fixed kidney sections were stained with periodic acid-Schiff or, after antigen retrieval [pressure cooker or microwave in citrate buffer (pH 6)], with antibodies against synaptopodin (G1 mouse monoclonal antibody, dilution 1:10; supplied by P.M.), PAX2 (paired box protein Pax-2; rabbit polyclonal antibody, dilution 1:75; Invitrogen/Life Technologies, Carlsbad, CA), WT1 (rabbit polyclonal antibody, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX), cleaved caspase 3 (dilution 1:20; Abcam, Cambridge, UK), cathepsin L (dilution 1:100; Sigma-Aldrich), and dendrin (dilution 1:50; EMD Millipore, Billerica, MA), followed by horseradish peroxidase–conjugated secondary antibodies (Dako, Carpinteria, CA), and counterstained with hematoxylin. One-micrometer sections of resin-embedded tissue were stained with toluidine blue. Images were taken with an Olympus BX53 microscope with a DP72 camera (Olympus, Tokyo, Japan) and processed using Adobe Photoshop software (Adobe Systems, San Jose, CA). Histology of three to six animals in each group was assessed. Periodic acid-Schiff– and toluidine blue–stained sections were analyzed in a blinded fashion (A.W.). Ultra-thin 80-nm sections of resin-embedded kidney tissue were mounted on copper grids, treated with uranyl acetate and lead citrate, and examined by a pathologist (A.W.) in a blinded fashion using a JEOL 1010 transmission electron microscope (JEOL, Tokyo, Japan). Electron micrographs were assessed in a blinded fashion (A.W.). One-millimeter cubes of kidney cortex were fixed in glutaraldehyde and embedded in EPON resin. One-micrometer-thick sections were cut using an ultramicrotome fitted with a Histo Jumbo Diamond Knife (Diatome US, Hatfield, PA). Serial sections were cut, and every tenth section and the adjacent section were saved to a microscope slide and stained with toluidine blue.21Bai X.Y. Basgen J.M. Podocyte number in the maturing rat kidney.Am J Nephrol. 2011; 33: 91-96Crossref PubMed Scopus (19) Google Scholar A total of 20 pairs of sections were saved per kidney. Images were obtained using the 100× objective lens (numerical aperture = 1.40) of an Olympus BX51 microscope fitted with a DP71 digital camera and DP Controller software DP-BSW-Ver3.2 (Olympus America, Center Valley, PA). Images were observed using Photoshop (Adobe Systems) on a 24-inch monitor at a window magnification of 100%. Ten glomeruli per kidney were analyzed for all morphometric parameters. Podocytes were counted using the fractionator/disector principle.22Gundersen H.J. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson.J Microsc. 1986; 143: 3-45Crossref PubMed Scopus (1637) Google Scholar, 23Nyengaard J.R. Stereologic methods and their application in kidney research.J Am Soc Nephrol. 1999; 10: 1100-1123Crossref PubMed Google Scholar Briefly, images of adjacent sections from throughout a glomerulus were observed, and the number of profiles from podocyte nuclei present in the second section of each pair, but not present in the first section, was counted. The number of podocyte nuclei was used as a surrogate for the number of podocytes. It was assumed there was only one nucleus per podocyte. The number of podocytes per glomerulus was calculated using the equation NumberPodocyte=10×ΣQ−, where 10 is the reciprocal of the fraction of the glomerulus sampled and ΣQ− is the sum of nuclei counted in the second section, but not present in the first section, of each section pair. An average of 137 Q− (range, 48 to 212) was counted per animal. Glomerular volume was measured using the Cavalieri principle.24Gundersen H.J. Jensen E.B. The efficiency of systematic sampling in stereology and its prediction.J Microsc. 1987; 147: 229-263Crossref PubMed Scopus (3078) Google Scholar Briefly, using the Layers function of Photoshop, a counting grid was randomly placed over the image of the first section of each section pair from a glomerulus, and the number of grid point falling on the glomerulus was counted.21Bai X.Y. Basgen J.M. Podocyte number in the maturing rat kidney.Am J Nephrol. 2011; 33: 91-96Crossref PubMed Scopus (19) Google Scholar Glomerular volume was calculated using the equation: VolumeGlomerulus=10×ΣP×(d/mag)2μm3, where 10 is the distance in micrometers between the measured sections, ΣP is the sum of grid points falling on all of the measured images from the glomerulus, d is the distance in micrometers between points on the grid, and mag is the image magnification. An average of 726 (range, 337 to 2721) grid points on glomeruli were counted per animal. Podocyte numerical density was calculated by dividing podocyte number by glomerular volume and expressed as number per cubic micrometer. First, glomerular component volume densities [Vv(Component X/glomerulus)] were measured using electron microscopy.25Basgen J.M. Sobin C. Early chronic low-level lead exposure produces glomerular hypertrophy in young C57BL/6J mice.Toxicol Lett. 2014; 225: 48-56Crossref PubMed Scopus (24) Google Scholar Briefly, silver-gold sections were cut with an Ultra Diamond Knife (Diatome US) placed on single-slot grids and stained with uranyl acetate and lead citrate. Images were obtained using a JEM-1200EX electron microscope (JEOL). Images were used at a final magnification of ×3200 and were taken for the 3-, 4-, and 5-week-old animals. Measurements were performed on five animals per group and 10 glomeruli per animal. A glomerulus was defined as the area within a minimal string polygon drawn around a glomerulus including all of the podocytes. Only glomeruli with a complete profile were used. Five components within the glomerulus were defined: i) mesangium including mesangial matrix, mesangial cell, and mesangial glomerular basement membrane (GBM); ii) podocytes (excluding cysts); iii) capillary lumen including endothelial cells and other cells within the lumen; iv) cysts within the podocytes; and v) remainder tissues, including Bowman's space, GBM, and nonresolvable tissue. Using the Layer function of Photoshop, a grid of points was randomly placed over an image of the complete glomerular profile, and the number of points falling on each of the five glomerular components was counted. The volume density for component X was calculated using the equation: Vv(ComponentX/glomerulus)=PComponentX/ΣPFivecomponents, where PComponent X is the number of points falling on a component X (ie, mesangium) and ΣPFive components is the number of points falling on all five components. The volume of each component was calculated by multiplying each appropriate volume density by the glomerular volume. For example, for mesangial volume: VolumeMesangium=Vv(Mesangium/glomerulus)×VolumeGlomerulusμm3. An average of 882 points (range, 388 to 1665) were counted per animal. An average podocyte volume for the mouse was calculated by dividing the total podocyte volume within a glomerulus by the average number of podocytes per glomerulus and was reported as cubic micrometers. Conditionally immortalized murine podocytes26Mundel P. Reiser J. Zuniga Mejia Borja A. Pavenstadt H. Davidson G.R. Kriz W. Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) and 100 U/mL penicillin–streptomycin (Life Technologies) on type I collagen (Corning, Corning, NY) coated dishes or flasks. Undifferentiated podocytes were maintained at 33°C in the presence of 10 U/mL mouse γ-interferon (Cell Sciences, Canton, MA) to drive T-antigen expression. To induce differentiation, podocytes were thermo-shifted to a 37°C incubator and switched to medium lacking γ-interferon. Mouse mesangial cells (MMCs) were cultured in DMEM containing 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin–streptomycin. pLKO.1 lentiviral shRNA plasmids were purchased from Addgene (Cambridge, MA) for scramble control and from Sigma-Aldrich for CD2AP and dendrin. We used sequence 5′-CCGGCAAACCTAAACCCAGATATTTCTCGAGAAATATCTGGGTTTAGGTTTGTTTTTG-3′ to target CD2AP. We used sequence 5′-CCGGGATTGAAGTGAAGACTATTTCCTCGAGGAAATAGTCTTCACTTCAATCTTTTTG-3′ to target dendrin. The pLKO.1 plasmids, along with the helper plasmids psPAX2 and pMD2.G, were transfected into HEK293T cells at 70% confluence using FuGENE 6 Transfection Reagent (Promega, Madison, WI). Medium was replaced 16 to 18 hours after transfection. Virus was collected at 48 hours and 72 hours post-transfection. Cell debris was removed by passing the supernatant through a 0.45-μ filter. After podocytes were differentiated for 7 days, virus was added to the differentiated podocytes in the presence of 4 μg/mL Polybrene (Sigma-Aldrich) overnight. The medium was changed the next day. After 4 days of infection, cells were serum starved in DMEM medium containing 0.1% heat-inactivated fetal bovine serum. Twenty-four hours after serum starvation, the supernatant was collected and spun down to remove cell debris. The collected supernatants (conditioned podocyte medium) were then used to treat MMCs that were themselves previously made quiescent by serum starvation. MMCs (1000) in 100 μL of medium were plated into each well of the opaque-bottom 96-well plate (Corning). Cells were allowed to adhere overnight. The next day, the MMCs were serum starved overnight in DMEM containing 0.1% heat-inactivated fetal bovine serum. The following day, 100 μL of supernatant from serum-starved podocytes was used to treat the MMCs. After 24 hours of incubation, cells were allowed to equilibrate to room temperature for 30 minutes. Cell viability was then measured by adding 100 μL of CellTiter-Glo Luminescent Reagent (Promega) to each well, according to the manufacturer's protocol. After 10 minutes of incubation at room temperature, the luminescence of each sample was measured in a plate-reading luminometer. MMCs (40,000) were plated in each well of a 6-well plate. Cells were then serum starved overnight in DMEM medium containing 0.1% heat-inactivated fetal bovine serum. The following day, serum-starved podocyte medium was added to the MMCs. Conditioned media from experimental cultures were collected 24 hours after treatment, spun down at 2300 × g for 5 minutes to remove cell debris, and analyzed by Western immunoblot for fibronectin. Conditioned media were also diluted 1:10 and quantified in duplicates using a Fibronectin Mouse ELISA Kit (Abcam) according to the manufacturer's protocol. MMCs (40,000) were plated in each well of a 6-well plate. Cells were then serum starved overnight in DMEM medium containing 0.1% heat-inactivated fetal bovine serum. The following day, serum-starved podocyte medium was added to the MMCs. MMCs were trypsinized, harvested, and counted after 48 hours of treatment. An equal number of cells were lysed with 100 μL of radioimmunoprecipitation assay buffer by incubating on ice for 30 minutes. After 30 minutes, lysates were spun down at 16,000 × g for 30 minutes. Supernatants were transferred to a new tube and quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's protocol. Data are given as means ± SD. Statistical analyses were performed using analysis of variance to compare data among groups and t-tests to determine differences between two groups. P < 0.05 was considered statistically significant. We reported previously the relocation of dendrin to the podocyte nucleus in murine inflammatory glomerulonephritis.11Asanuma K. Campbell K.N. Kim K. Faul C. Mundel P. Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes.Proc Natl Acad Sci U S A. 2007; 104: 10134-10139Crossref PubMed Scopus (77) Google Scholar Now, we found nuclear redistribution of dendrin in podocytes during disease progression in Cd2ap−/− mice (Supplemental Figure S1), suggesting that nuclear dendrin promotes podocyte injury and renal failure in the absence of CD2AP. To directly test this hypothesis, we generated Cd2ap-null mice with and without dendrin deletion: Cd2ap−/−;Ddn−/− and Cd2ap−/−;Ddn+/+. Wild-type littermates (Cd2ap+/+;Ddn+/+) served as controls (Supplemental Figure S2A). The deletion of dendrin in Cd2ap−/− mice was confirmed by genotyping PCR (Supplemental Figure S2B). Because Cd2ap−/− mice die from renal failure,16Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar we determined the effect of dendrin ablation on life span and found that Cd2ap−/−;Ddn−/− mice survived significantly longer compared to Cd2ap−/−;Ddn+/+ littermates (mean life span: Cd2ap−/−;Ddn+/+: 8.83 ± 0.28 weeks; Cd2ap−/−;Ddn−/−: 13.52 ± 0.38; P < 0.001, t-test) (Figure 1A). No wild-type littermate controls died during the study period (Figure 1A). To test whether the improved survival of Cd2ap−/−;Ddn−/− mice resulted from prolonged preservation of renal function, we assessed serum creatinine levels. At 3 weeks of age, when proteinuria develops in Cd2ap−/− mice,16Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar serum creatinine values were comparable between Cd2ap−/−;Ddn+/+ (0.78 ± 0.198 μg/mL), Cd2ap−/−;Ddn−/− (0.72 ± 0.086 μg/mL), and wild-type mice (NS, analysis of variance) (Figure 1B). By contrast, at 6 weeks of age, Cd2ap−/−;Ddn+/+ mice had significantly higher mean serum creatinine levels (4.3 ± 0.552 μg/mL) compared to Cd2ap−/−;Ddn−/− littermates (0.64 ± 0.103 μg/mL; P < 0.001, t-test) (Figure 2B). The simplest interpretation of these results is that dendrin ablation increases the life span of Cd2ap−/− mice by delaying the onset of chronic kidney disease and renal failure.Figure 2Genetic ablation of dendrin delays severity of glomerular and tubulointerstitial injury in Cd2ap−/− mice. A: At 3 weeks of age, Cd2ap−/−;Ddn+/+ mice develop mesangial expansion, which is milder in Cd2ap−/−;Ddn−/− mice. By 4 weeks of age, Cd2ap−/−;Ddn+/+ mice develop segmental glomerulosclerosis, and global glomerulosclerosis and focal collapsing lesions at 5 weeks of age. By contrast, hardly any glomerular lesions are found in Cd2ap−/−;Ddn−/− mice at 5 weeks. By 6 weeks of age, Cd2ap−/−;Ddn+/+ mice show extensive chronic changes affecting most glomeruli, whereas Cd2ap−/−;Ddn−/− animals have significantly fewer glomerular lesions. B: Quantitative analysis of histological changes. Cd2ap−/−;Ddn−/− mice (gray bars) show fewer segmental and global glomerulosclerosis than Cd2ap−/−;Ddn+/+ mice (black bars) by 4 and 5 weeks of age; the differences reach statistical significance by 6 weeks. Fewer collapsing lesions are also seen in 5- and 6-week-old Cd2ap−/−;Ddn−/− mice compared to age-matched Cd2ap−/−;Ddn+/+ littermates. No interstitial fibrosis and tubular atrophy (IFTA) is seen in animals at 4 weeks, whereas IFTA is significantly more widespread in Cd2ap−/−;Ddn+/+ mice at 5 and 6 weeks. Data are expressed as means ± SD. ∗P < 0.05, ∗∗P < 0.01. Scale bar = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In humans, proteinuria is a risk factor for cardiovascular disease, kidney failure, and death.3Agrawal V. Marinescu V. Agarwal M. McCullough P.A. Cardiovascular implications of proteinuria: an indicator of chronic kidney disease.Nat Rev Cardiol. 2009; 6: 301-311Crossref PubMed Scopus (91) Google Scholar, 27Tonelli M. Jose P. Curhan G. Sacks F. Braunwald E. Pfeffer M. Pr
Referência(s)