Acute kidney injury is a major clinical problem and advanced age is associated with ineffective renal regeneration and poor functional outcome. Data from kidney injury models suggest that a loss of tubular epithelial proliferation contributes to a decrease in renal repair capacity with aging, but aging can also lead to a higher severity of inflammation and damage which may influence repair. In this study we tested intrinsic age-dependent changes in tubular epithelial proliferation in young and old mice, by injecting low-dose lead acetate as a non-injurious mitogen. In parallel, we explored in vitro techniques of studying cellular senescence in primary tubular epithelial cells (PTEC). Lead acetate induced tubular epithelial proliferation at a significantly higher rate in young as compared to old mice. Old kidneys showed significantly more senescence as demonstrated by increased p16INK4a, senescence associated β-galactosidase, and γH2AX+/Ki-67？ cells. This was paralleled in old kidneys by a higher number of Cyclin D1 positive tubular cells. This finding was corroborated by a positive correlation between Cyclin D1 positivity and age in human renal biopsies. When tubular cells were isolated from mouse kidneys they rapidly lost their age-associated differences under culture conditions. However, senescence was readily induced in PTEC by γ-irradiation representing a future model for study of cellular senescence in the renal epithelium. Together, our data indicate that the tubular epithelium of aged kidney has an intrinsically reduced proliferative capacity probably due to a higher load of senescent cells. Moreover, stress induced models of cellular senescence are preferable for study of the renal epithelium in vitro. Finally, the positive correlation of Cyclin D1 with age and cellular senescence in PTEC needs further evaluation as to a functional role of renal epithelial aging.
References
[1]
Braun H, Schmidt BM, Raiss M, Baisantry A, Mircea-Constantin D, et al. (2012) Cellular Senescence Limits Regenerative Capacity and Allograft Survival. J Am Soc Nephrol. 2012 Sep 23(9): 1467–73. doi: 10.1681/asn.2011100967
[2]
Schmitt R, Marlier A, Cantley LG (2008) Zag expression during aging suppresses proliferation after kidney injury. J Am Soc Nephrol 19: 2375–2383. doi: 10.1681/asn.2008010035
[3]
Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, et al. (2008) Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2: 284–291. doi: 10.1016/j.stem.2008.01.014
[4]
Melk A, Schmidt BM, Braun H, Vongwiwatana A, Urmson J, et al. (2009) Effects of donor age and cell senescence on kidney allograft survival. Am J Transplant 9: 114–123. doi: 10.1111/j.1600-6143.2008.02500.x
[5]
Witzgall R, Brown D, Schwarz C, Bonventre JV (1994) Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 93: 2175–2188. doi: 10.1172/jci117214
[6]
Melk A, Schmidt BM, Takeuchi O, Sawitzki B, Rayner DC, et al. (2004) Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney. Kidney Int 65: 510–520. doi: 10.1111/j.1523-1755.2004.00438.x
[7]
Westhoff JH, Schildhorn C, Jacobi C, Homme M, Hartner A, et al. (2010) Telomere shortening reduces regenerative capacity after acute kidney injury. J Am Soc Nephrol 21: 327–336. doi: 10.1681/asn.2009010072
[8]
Melk A (2003) Senescence of renal cells: molecular basis and clinical implications. Nephrol Dial Transplant 18: 2474–2478. doi: 10.1093/ndt/gfg322
[9]
Naesens M (2011) Replicative senescence in kidney aging, renal disease, and renal transplantation. Discov Med 11: 65–75.
[10]
Schmitt R, Melk A (2012) New Insights on Molecular Mechanisms of Renal Aging. Am J Transplant. 2012 Nov 12(11): 2892–900. doi: 10.1111/j.1600-6143.2012.04214.x
[11]
Melk A, Kittikowit W, Sandhu I, Halloran KM, Grimm P, et al. (2003) Cell senescence in rat kidneys in vivo increases with growth and age despite lack of telomere shortening. Kidney Int 63: 2134–2143. doi: 10.1046/j.1523-1755.2003.00032.x
[12]
Melk A, Ramassar V, Helms LM, Moore R, Rayner D, et al. (2000) Telomere shortening in kidneys with age. J Am Soc Nephrol 11: 444–453.
[13]
Verzola D, Gandolfo MT, Gaetani G, Ferraris A, Mangerini R, et al. (2008) Accelerated senescence in the kidneys of patients with type 2 diabetic nephropathy. Am J Physiol Renal Physiol 295: F1563–1573. doi: 10.1152/ajprenal.90302.2008
[14]
Westhoff JH, Hilgers KF, Steinbach MP, Hartner A, Klanke B, et al. (2008) Hypertension induces somatic cellular senescence in rats and humans by induction of cell cycle inhibitor p16INK4a. Hypertension 52: 123–129. doi: 10.1161/hypertensionaha.107.099432
[15]
Joosten SA, van Ham V, Nolan CE, Borrias MC, Jardine AG, et al. (2003) Telomere shortening and cellular senescence in a model of chronic renal allograft rejection. Am J Pathol 162: 1305–1312. doi: 10.1016/s0002-9440(10)63926-0
[16]
Miura K, Goldstein RS, Morgan DG, Pasino DA, Hewitt WR, et al. (1987) Age-related differences in susceptibility to renal ischemia in rats. Toxicol Appl Pharmacol 87: 284–296. doi: 10.1016/0041-008x(87)90290-0
[17]
Qiao X, Chen X, Wu D, Ding R, Wang J, et al. (2005) Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/reperfusion injury. J Gerontol A Biol Sci Med Sci 60: 830–839. doi: 10.1093/gerona/60.7.830
[18]
Choie DD, Richter GW (1972) Cell proliferation in rat kidneys after prolonged treatment with lead. Am J Pathol 68: 359–370.
[19]
Choie DD, Richter GW (1972) Cell proliferation in rat kidney induced by lead acetate and effects of uninephrectomy on the proliferation. Am J Pathol 66: 265–275.
[20]
Vogetseder A, Palan T, Bacic D, Kaissling B, Le Hir M (2007) Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney. Am J Physiol Cell Physiol 292: C807–813. doi: 10.1152/ajpcell.00301.2006
[21]
Vogetseder A, Picard N, Gaspert A, Walch M, Kaissling B, et al. (2008) Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells. Am J Physiol Cell Physiol 294: C22–28. doi: 10.1152/ajpcell.00227.2007
[22]
Witzgall R (2008) Are renal proximal tubular epithelial cells constantly prepared for an emergency? Focus on “the proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells”. Am J Physiol Cell Physiol 294: C1–3. doi: 10.1152/ajpcell.00470.2007
[23]
Yang L, Besschetnova TY, Brooks CR, Shah JV, Bonventre JV (2010) Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury. Nat Med 16: : 535–543, 531p following 143.
[24]
Diehl JA (2002) Cycling to cancer with cyclin D1. Cancer Biol Ther 1: 226–231.
[25]
Lawless C, Wang C, Jurk D, Merz A, Zglinicki T, et al. (2010) Quantitative assessment of markers for cell senescence. Exp Gerontol 45: 772–778. doi: 10.1016/j.exger.2010.01.018
[26]
Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, et al. (2011) The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature 477: 90–94. doi: 10.1038/nature10357
[27]
Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, et al. (2005) Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433: 760–764. doi: 10.1038/nature03260
[28]
Davalos AR, Kawahara M, Malhotra GK, Schaum N, Huang J, et al. (2013) p53–dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes. J Cell Biol 201: 613–629. doi: 10.1083/jcb.201206006
[29]
Coppe JP, Patil CK, Rodier F, Krtolica A, Beausejour CM, et al. (2010) A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. PLoS One 5: e9188. doi: 10.1371/journal.pone.0009188
[30]
Liu J, Xu K, Chase M, Ji Y, Logan JK, et al. (2012) Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells. J Cell Sci 125: 376–386. doi: 10.1242/jcs.089466
[31]
Freund A, Laberge RM, Demaria M, Campisi J (2012) Lamin B1 loss is a senescence-associated biomarker. Mol Biol Cell 23: 2066–2075. doi: 10.1091/mbc.e11-10-0884
[32]
Miya M, Maeshima A, Mishima K, Sakurai N, Ikeuchi H, et al. (2012) Age-related decline in label-retaining tubular cells: implication for reduced regenerative capacity after injury in the aging kidney. Am J Physiol Renal Physiol 302: F694–702. doi: 10.1152/ajprenal.00249.2011
[33]
Deng Q, Liao R, Wu BL, Sun P (2004) High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts. J Biol Chem 279: 1050–1059. doi: 10.1074/jbc.m308644200
[34]
Sherr CJ, DePinho RA (2000) Cellular senescence: mitotic clock or culture shock? Cell 102: 407–410. doi: 10.1016/s0092-8674(00)00046-5
[35]
Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24: 2463–2479. doi: 10.1101/gad.1971610
Rodier F, Munoz DP, Teachenor R, Chu V, Le O, et al. (2011) DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion. J Cell Sci 124: 68–81. doi: 10.1242/jcs.071340
[38]
Han EK, Ng SC, Arber N, Begemann M, Weinstein IB (1999) Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis. Apoptosis 4: 213–219. doi: 10.1023/a:1009618824145
[39]
Burton DG, Sheerin AN, Ostler EL, Smith K, Giles PJ, et al. (2007) Cyclin D1 overexpression permits the reproducible detection of senescent human vascular smooth muscle cells. Ann N Y Acad Sci 1119: 20–31. doi: 10.1196/annals.1404.026
[40]
Ledda-Columbano GM, Pibiri M, Cossu C, Molotzu F, Locker J, et al. (2004) Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP. Hepatology 40: 981–988. doi: 10.1002/hep.1840400429
[41]
Suzuki A, Sakaguchi T, Inaba K, Suzuki S, Konno H (2012) Impact of cell cycle disruption on impaired hepatic regeneration in aged livers with ischemic insult. J Surg Res 173: 267–277. doi: 10.1016/j.jss.2010.10.012
Sorensen I, Rong S, Susnik N, Gueler F, Shushakova N, et al. (2011) Bbeta(15-42) attenuates the effect of ischemia-reperfusion injury in renal transplantation. J Am Soc Nephrol 22: 1887–1896. doi: 10.1681/asn.2011010031
[44]
Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4: 1798–1806. doi: 10.1038/nprot.2009.191
[45]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262
