Accelerated expression of senescence associated cell cycle inhibitor p16INK4A in kidneys with glomerular disease
2006; Elsevier BV; Volume: 71; Issue: 3 Linguagem: Inglês
10.1038/sj.ki.5002039
ISSN1523-1755
AutoresB. Sis, Adis Tasanarong, Farhad Khoshjou, Farahnaz Dadras, Kim Solez, Philip F. Halloran,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoThe cell cycle inhibitor p16INK4A (also known as cyclin-dependent kinase inhibitor 2A) is expressed in vivo in many tissues with age. The exposure of certain chronic stresses can trigger p16INK4A expression and a senescence-like phenotype. We studied whether p16INK4A expression is induced in glomerular disease (GD). We performed p16INK4A immunostaining on 35 biopsies with GD, 12 tubulointerstitial nephritis (TIN), and 19 normal live donor kidneys at transplantation. Based on values for 42 normal kidneys, we calculated expected nuclear p16INK4A expression for age and compared the observed values in diseased kidneys to those expected for age. In GD, p16INK4A expression was strikingly increased in glomerular and interstitial cell nuclei compared to normals and TIN, and could not be attributed to age (P<0.05). By multivariate analyses, GD was independently associated with increased nuclear p16INK4a expression in glomeruli (P<0.001) and interstitium (P=0.01). The p16INK4A expression in glomerular and interstitial cell nuclei, and tubular cytoplasm was higher in kidneys with proteinuria and with atrophy/fibrosis (P<0.05). Older age was associated with increased nuclear p16INK4a expression in tubules (P=0.01), and interstitial inflammation was associated with increased nuclear p16INK4a expression in interstitial cells (P=0.001). The p16INK4a staining in tubular cytoplasm was increased in both GD and TIN compared to normals (P 0.05). Thus, kidneys with GD display increased expression of senescence marker p16INK4A in glomerular and interstitial cell nuclei compared to kidneys with normal aging or TIN. The findings suggest a role for somatic cell senescence mechanisms in progression of GD. The cell cycle inhibitor p16INK4A (also known as cyclin-dependent kinase inhibitor 2A) is expressed in vivo in many tissues with age. The exposure of certain chronic stresses can trigger p16INK4A expression and a senescence-like phenotype. We studied whether p16INK4A expression is induced in glomerular disease (GD). We performed p16INK4A immunostaining on 35 biopsies with GD, 12 tubulointerstitial nephritis (TIN), and 19 normal live donor kidneys at transplantation. Based on values for 42 normal kidneys, we calculated expected nuclear p16INK4A expression for age and compared the observed values in diseased kidneys to those expected for age. In GD, p16INK4A expression was strikingly increased in glomerular and interstitial cell nuclei compared to normals and TIN, and could not be attributed to age (P<0.05). By multivariate analyses, GD was independently associated with increased nuclear p16INK4a expression in glomeruli (P<0.001) and interstitium (P=0.01). The p16INK4A expression in glomerular and interstitial cell nuclei, and tubular cytoplasm was higher in kidneys with proteinuria and with atrophy/fibrosis (P<0.05). Older age was associated with increased nuclear p16INK4a expression in tubules (P=0.01), and interstitial inflammation was associated with increased nuclear p16INK4a expression in interstitial cells (P=0.001). The p16INK4a staining in tubular cytoplasm was increased in both GD and TIN compared to normals (P 0.05). Thus, kidneys with GD display increased expression of senescence marker p16INK4A in glomerular and interstitial cell nuclei compared to kidneys with normal aging or TIN. The findings suggest a role for somatic cell senescence mechanisms in progression of GD. The finite limitations in the survival and maintenance of somatic cells could play a role in progressive renal disease and in the development of renal insufficiency in renal transplants.1.Melk A. Schmidt B.M. Vongwiwatana A. et al.Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.Am J Transplant. 2005; 5: 1375-1382Crossref PubMed Scopus (100) Google Scholar Cellular senescence refer to an in vitro phenotype of cultured somatic cells that show permanent and irreversible replication arrest. The features of this state include telomere shortening (in humans) and a sustained increase in the expression of cyclin-dependent kinase inhibitor p16INK4A, also known as cyclin-dependent kinase inhibitor 2A or CDKN2A.2.Sherr C.J. DePinho R.A. Cellular senescence: mitotic clock or culture shock?.Cell. 2000; 102: 407-410Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar,3.Alcorta D.A. Xiong Y. Phelps D. et al.Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.Proc Natl Acad Sci USA. 1996; 93: 13742-13747Crossref PubMed Scopus (746) Google Scholar Recently, we showed that aged human kidneys in vivo show some features of cellular senescence manifested by cultured somatic cells and p16INK4A emerged with the best correlation with kidney age. In addition, p16INK4A was significantly associated with glomerulosclerosis, interstitial fibrosis (IF), and tubular atrophy (TA), some of the principal histological features of renal senescence.4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar In addition to replicative stress and normal aging, a number of environmental stresses, such as DNA damage and oxidative stress, X-rays, ultraviolet irradiation, H2O2, and ectopic expression of certain oncogenes and tumor suppressor genes can trigger a senescence-like phenotype in mammalian cells, known as extrinsic senescence.5.Melk A. Senescence of renal cells: molecular basis and clinical implications.Nephrol Dial Transplant. 2003; 18: 2474-2478Crossref PubMed Scopus (74) Google Scholar, 6.Serrano M. Blasco M.A. Putting the stress on senescence.Curr Opin Cell Biol. 2001; 13: 748-753Crossref PubMed Scopus (339) Google Scholar, 7.Campisi J. Cellular senescence as a tumor-suppressor mechanism.Trends Cell Biol. 2001; 11: S27-S31Abstract Full Text PDF PubMed Scopus (479) Google Scholar, 8.Itahana K. Campisi J. Dimri G.P. Mechanisms of cellular senescence in human and mouse cells.Biogerontology. 2004; 5: 1-10Crossref PubMed Scopus (246) Google Scholar, 9.Famulski K.S. Halloran P.F. Molecular events in kidney ageing.Curr Opin Nephrol Hypertens. 2005; 14: 243-248Crossref PubMed Scopus (41) Google Scholar Many clinical nephrology problems arise in the elderly, including high frequency of end-stage renal disease, could involve an interaction between somatic cell senescence and disease stresses that could accelerate the limitations on the capacity of aged nephron to cope with various injury, repair, and maintain epithelial functions. This may contribute to the acceleration of renal senescence phenotype by age-related diseases, such as hypertension and heart failure.5.Melk A. Senescence of renal cells: molecular basis and clinical implications.Nephrol Dial Transplant. 2003; 18: 2474-2478Crossref PubMed Scopus (74) Google Scholar,10.Melk A. Halloran P.F. Cell senescence and its implications for nephrology.J Am Soc Nephrol. 2001; 12: 385-393PubMed Google Scholar It is also possible that the exposure of certain types of chronic stresses themselves may induce senescence-like changes, even independent of age. The role of cell-cycle regulatory proteins and senescence mechanisms in chronic renal stresses, such as primary glomerular disease (GD), proteinuria, and hypertension remains unknown. We recently showed that chronic deteriorating kidney transplants show accelerated p16INK4A expression, correlating with IF/TA and impaired function.1.Melk A. Schmidt B.M. Vongwiwatana A. et al.Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.Am J Transplant. 2005; 5: 1375-1382Crossref PubMed Scopus (100) Google Scholar Increased p16INK4A expression and/or other cellular senescence elements could cause the development of atrophy and fibrosis, thus might be relevant to the progression of chronic renal diseases. It is probable that GD creates an environmental stress on glomerular cells that exceeds what they would normally experience during aging. Abnormal stresses could include cytokines, growth factors, and reactive oxygen species derived from intrinsic glomerular cells and inflammatory cells. Glomerular injury frequently results in proteinuria and filtered proteins are potential mediators of tubular epithelial cell injury, contributing to chronic tubulointerstitial changes and progression of GD.11.Healy E. Brady H.R. Role of tubule epithelial cells in the pathogenesis of tubulointerstitial fibrosis induced by glomerular disease.Curr Opin Nephrol Hypertens. 1998; 7: 525-530Crossref PubMed Scopus (69) Google Scholar, 12.Eddy A.A. Proteinuria and interstitial injury.Nephrol Dial Transplant. 2004; 19: 277-281Crossref PubMed Scopus (135) Google Scholar, 13.Olson J.L. Progression of renal disease.in: Jennette J.C. Olson J.L. Schwartz M.M. Silva F.G. Heptinstall's Pathology of the Kidney. Lippincott-Raven, Philadelphia, PA1998: 137-167Google Scholar All these disease stresses could theoretically accelerate some senescence changes, including p16INK4A expression in stressed cells, which in turn may limit their function and capacity to replicate and recover. In the present study, we examined whether GD with proteinuria show enhanced expression of p16INK4A compared to tubulointerstitial nephritis (TIN) and normal implantation biopsies. The demographic and clinical data from controls (implantation biopsies) and diseased kidneys are given in Tables 1 and 2. In keeping with our previous reports,4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar normal kidneys showed p16INK4a staining in 2.5±2.8% of glomerular nuclei, 11.9±7.7% of tubular nuclei, and 6.4±7.2% of interstitial cell nuclei. In addition to the nuclear staining, four of 19 normal kidneys (21%) showed tubular cytoplasmic p16INK4a expression (mean score: 0.2±0.4). The mean score for nuclear p16INK4a staining in arteries was 0.3±0.6 (Table 3). Representative stainings are shown in Figure 1a, g, and k.Table 1Demographic and clinical data on groups from which kidney biopsies were derivedNormal kidneysaNormal kidney tissue samples were obtained from live donor biopsies at the time of transplantation. Recipient age: 43.5±17.5 years, recipient gender (m/f): 13/6. (n=19)TIN (n=12)Glomerular disease (n=35)Kidney (donor) age (years)36.4±9.652.8±20.5*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).47.9±19.5*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).Kidney (donor) gender (m/f)8/112/1023/12Creatinine (μmol/l)74.8±16.8399.9±352.3*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).122.6±91.8*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).Proteinuria (g/l/24 h)0.12±0.030.68±0.86*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).5.4±3.7*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).IF/TA0.15±0.371.33±0.88*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).1.60±0.7*Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).IF, interstitial fibrosis; TA, tubular atrophy; TIN, tubulointerstitial nephritis.IF/TA was assessed from 0 to 3.Values are given as mean±s.d.* Significant difference between normal kidneys and TIN, glomerular disease (Mann–Whitney, P<0.05).a Normal kidney tissue samples were obtained from live donor biopsies at the time of transplantation. Recipient age: 43.5±17.5 years, recipient gender (m/f): 13/6. Open table in a new tab Table 2Demographic and clinical data of the patients with proteinuriaPatient noBiopsy noAge (years)SexProteinuria (g/l/24 h)Creatinine at biopsy (μmol/l)DiagnosisLast creatinine (μmol/l)Follow-up after the biopsy (months)1139M10.0113MGP121122187F3.5108MGP12973156M4.384MGP117154141M3.569MGP95635157F2.075MGP66396180M2.286MGP76457135M3.688MGP95308173F9.586MGP10549168F3.576MGP622010144M17.483MGP339462479.0200MGP—011127F2.027MGP351812139F5.083MGP139602443.4139MGP155613159M3.575MGP1061314139F0.1781MGP7842715135M10.669MGP1002816120M8.594MGP911417164M8.094MGP021918148M4.086MGP971919118M10.081MGP4003822111.0400MGP147920139M3.580MCD869021170F2.560MCD654322176M6.0101MCD1125823170F7.5130MCD138224138M4.4100MCD98425148M3.580FSGS1204726115M3.590FSGS350172171.0350FSGS12322327147M1.697FSGS835228126F4.068FSGS913229154F1.070FSGS987530166M9.6300FSGS6401731171M3.5171FSGS14573FSGS, focal segmental glomerular sclerosis; MCD, minimal change disease; MGP, membranous glomerulopathy. Open table in a new tab Table 3Comparison of p16INK4a expression in normal kidneys, TIN, and GD.p16INK4a expression (%, mean±s.d.)Normal kidneys (n=19)TIN (n=12)Glomerular disease (n=35)Glomeruli-N2.5±2.84.6±317.1±12.2**P≤0.001 Mann–Whitney U test.Tubules-N11.9±7.77.0±6.317.3±20Tubules-Cap16INK4a positivity scores (mean±s.d.).0.2±0.41.5±1**P≤0.001 Mann–Whitney U test.1.3±0.9**P≤0.001 Mann–Whitney U test.Interstitium-N6.4±7.213.8±9.4**P≤0.001 Mann–Whitney U test.30.1±24**P≤0.001 Mann–Whitney U test.Arteries-Nap16INK4a positivity scores (mean±s.d.).0.3±0.60.5±0.40.5±0.5C, cytoplasmic staining; GD, glomerular disease; TIN, tubulointerstitial nephritis; N, nuclear.Normal kidneys were compared to TIN or GD.** P≤0.001 Mann–Whitney U test.a p16INK4a positivity scores (mean±s.d.). Open table in a new tab IF, interstitial fibrosis; TA, tubular atrophy; TIN, tubulointerstitial nephritis. IF/TA was assessed from 0 to 3. Values are given as mean±s.d. FSGS, focal segmental glomerular sclerosis; MCD, minimal change disease; MGP, membranous glomerulopathy. C, cytoplasmic staining; GD, glomerular disease; TIN, tubulointerstitial nephritis; N, nuclear. Normal kidneys were compared to TIN or GD. GD cases showed a strikingly increased p16INK4a expression in glomerular nuclei (GD: 17.1±12.2% vs normal kidneys: 2.5±2.8%; P<0.001) (Figure 1b–e), interstitial cell nuclei (GD: 30.1±24% vs normal kidneys: 6.4±7.2%; P<0.001) (Figure 1h and i), and tubular cytoplasm (GD: 80% of cases, mean score: 1.3±0.9 vs normal kidneys: 21% of cases, mean score: 0.2±0.4; P 0.05) (Table 3, Figure 1h, i and l). The demographic data are given in Table 1. TIN biopsies showed higher p16INK4a staining in interstitial cell nuclei (13.8±9.4%, P=0.001) and tubular cytoplasm (mean score: 1.5±1, P 0.05) (Table 3). The trivial increase in glomerular p16INK4a in TIN compared to normals (4.6±3 vs 2.5±2.8%; P=0.047) (Figure 1f) probably reflects aging (mean age: 52.8±20.5 vs 36.4±9.6) (Table 3). Comparison of nuclear p16INK4a expression in GD and TIN (Table 3) revealed that GD showed higher p16INK4a staining in glomerular (P=0.001) and interstitial cell nuclei (P=0.02). We used regression formulas with age derived from 42 normal kidney specimens, as previously published,4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar to calculate the expected p16INK4a expression for each biopsy in the current study. For normal kidneys (implantation biopsies), measured and expected p16INK4a expression was not significantly different (P>0.05; Figure 2; Figure S1), that is, p16INK4a expression at implantation was as expected for the age of the donor. Download .ppt (7.95 MB) Help with ppt files Supplementary Figure 1 Kidneys with GD showed increased p16INK4a expression in glomerular and interstitial cell nuclei beyond that expected for age (for glomeruli, measured: 17.1±12.2% vs expected: 4.3±0.7%; for interstitial cells, measured: 30.1±24% vs expected: 7.3±3.5%) (P<0.001; Figure 2; Figure S1). In TIN biopsies, the measured p16INK4a expression was similar to the amount predicted for age in glomerular nuclei (4.6±3 vs 4.5±0.8%; P=0.90), but was higher in interstitial cell nuclei (13.8±9.4 vs 8.2±3.7%; P=0.04) and lower in tubular nuclei (7±6.3 vs 22.2±8.8%; P<0.001) (Figure 2; Figure S1) than the amount predicted by age. By univariate linear regression analysis, p16INK4a expression in glomerular and interstitial cell nuclei was associated with GD, proteinuria, and IF/TA (P<0.05; Table 4). In addition, age correlated with nuclear p16INK4a expression in glomerular cells and interstitial inflammation correlated with nuclear p16INK4a expression in interstitial cells (P 0.05). Age was the only determinant of nuclear p16INK4a expression in tubules (P=0.01, Table 4) and arteries (P=0.03, R2=0.070) (P>0.05).Table 4Univariate and multivariate linear regression analyses for nuclear p16INK4a expression in glomeruli, tubules, interstitium, and arteriesGlomeruliInterstitiumTubulesArteriesp16INK4a expressionR2P-valueR2P-valueR2P-valueR2P-valueUnivariate linear regression analysis GD vs no GDaNo GD, includes normals and TIN.0.372<0.0010.258<0.0010.0650.220.0200.27 TIN vs no TINbNo TIN, includes normals and GD.0.3720.550.2580.280.0650.400.0200.55 GD vs TIN0.2140.0010.1030.020.0630.080.0020.77 Age0.0620.040.0440.090.0660.030.0700.03 Serum creatinine at biopsy0.0450.080.0350.130.0360.120.0090.44 Proteinuria0.1590.0010.1320.0030.0110.400.0050.59 IF/TA0.1270.0030.0980.010.0000.890.0260.20 Inflammation0.0280.170.1250.0040.0000.990.2900.17 FIT0.0090.440.0010.790.0120.390.0000.97 Global glomerulosclerosis0.0040.620.0050.570.0170.300.0040.61Multivariate linear regression analysis, P-value GD vs no GDaNo GD, includes normals and TIN.<0.0010.010.490.96 TIN vs no TINbNo TIN, includes normals and GD.0.170.090.430.51 GD vs TIN0.0020.0010.050— Age0.120.350.010.07 Serum creatinine at biopsy0.120.060.058— Proteinuria0.850.38—— IF/TA0.500.12—0.98 Inflammation0.250.001—0.46FIT, fibrous intimal thickening in arteries; GD, glomerular disease; IF, interstitial fibrosis; TA, tubular atrophy; TIN, tubulointerstitial nephritis. Bold P-values are less than 0.05.a No GD, includes normals and TIN.b No TIN, includes normals and GD. Open table in a new tab FIT, fibrous intimal thickening in arteries; GD, glomerular disease; IF, interstitial fibrosis; TA, tubular atrophy; TIN, tubulointerstitial nephritis. Bold P-values are less than 0.05. Using multivariate linear regression analysis, GD was found the best independent contributor of increased p16INK4a expression in glomeruli (P 0.05). When only patients with GD were analyzed, proteinuria did not correlate with p16INK4a expression in glomerular, tubular, interstitial cell nuclei, and tubular cytoplasm (P>0.05). However, IF/TA correlated with the amount of p16INK4a expression in tubular nuclei and interstitial cell nuclei (P 0.05).Table 5Comparison of nuclear p16INK4a positivity scores in normal kidneys, GD, and TIN according to the glomerular cell typesp16INK4a positive cell typeScoreNormal kidneyGDTINMesangial–endothelial cells0–117 (90%)21 (60%)10 (83.3%)2–32 (10%)14 (40%)*P=0.04, χ2 test.2 (16.7%)Parietal cells0–119 (100%)32 (91%)11 (91.7%)2–3—3 (9%)1 (8.3%)Podocytes—11 (58%)16 (46%)8 (66.7%)+8 (42%)19 (54%)4 (33.3%)GD, glomerular disease; TIN, tubulointerstitial nephritis.* P=0.04, χ2 test. Open table in a new tab GD, glomerular disease; TIN, tubulointerstitial nephritis. The overall expression of p16INK4a in glomeruli, interstitium, tubules, and arteries was not significantly different among membranous glomerulopathy (MGP), focal segmental glomerular sclerosis (FSGS), and minimal change disease (MCD) (P>0.05) (Table 6). Nuclear staining for p16INK4a was detected in all three glomerular cell types in MGP (Figure 1b and c), FSGS (Figure 1d), and MCD (Figure 1e). The amount of p16INK4a positive mesangial–endothelial cell, podocyte, and parietal cell nuclei was similar in MGP, FSGS, and MCD (P>0.05; Table 7). Thus, both immune complex mediated (MGP) and non-immune complex mediated GD (FSGS and MCD) showed a similar pattern and amount of glomerular p16INK4a expression.Table 6Distribution of p16INK4a among GD groupsp16INK4a expression (%, mean±s.d.)MGP (n=22)FSGS (n=8)MCD (n=5)Glomeruli-N17.9±12.418.2±13.411.8±9.9Tubules-N22±23.510.7±7.27.3±7.6Tubules-Cap16INK4a positivity scores (mean±s.d.).1.4±11.6±0.70.6±0.8Interstitium-N32.5±25.425.1±22.627.4±23.3Vessels-Nap16INK4a positivity scores (mean±s.d.).0.6±0.60.5±0.30.2±0.2C, cytoplasmic staining; FSGS, focal segmental glomerular sclerosis; GD, glomerular disease; MCD, minimal change disease; MGP, membranous glomerulopathy; N, nuclear.Kruskal–Wallis test, P>0.05.a p16INK4a positivity scores (mean±s.d.). Open table in a new tab Table 7Localization and quantitative assessment of nuclear p16INK4a expression in glomerular cell types in GD groupsp16INK4a expression ap16INK4a expression is given as the percentage of p16INK4a-positive mesangial–endothelial or podocyte or parietal cell nuclei according to the total number of nuclei in 10 randomly counted glomeruli. Kruskal–Wallis test, P>0.05. (%, mean±s.d.)P16INK4a−positive cell typeMGP (n=22)FSGS (n=8)MCD (n=5)Mesangial–endothelial cells12.2±11.814.2±12.36.7±7.5Parietal cells3.4±3.73.6±2.42.1±1.6Podocytes3.5±2.92.7±2.72.5±2.1FSGS, focal segmental glomerular sclerosis; GD, glomerular disease; MCD, minimal change disease; MGP, membranous glomerulopathy.a p16INK4a expression is given as the percentage of p16INK4a-positive mesangial–endothelial or podocyte or parietal cell nuclei according to the total number of nuclei in 10 randomly counted glomeruli. Kruskal–Wallis test, P>0.05. Open table in a new tab C, cytoplasmic staining; FSGS, focal segmental glomerular sclerosis; GD, glomerular disease; MCD, minimal change disease; MGP, membranous glomerulopathy; N, nuclear. Kruskal–Wallis test, P>0.05. FSGS, focal segmental glomerular sclerosis; GD, glomerular disease; MCD, minimal change disease; MGP, membranous glomerulopathy. Of the 35 GD, nine showed a mild increase in mesangial matrix (eight MGP, one MCD) and seven showed a mild mesangial hypercellularity (five MGP, one FSGS, one MCD). None of the biopsies with GD showed glomerular inflammation. The presence of mesangial matrix expansion and mild hypercellularity did not correlate with p16INK4a expression in glomeruli, tubules, interstitium, or arteries (P>0.05). For cases with MGP, stage and underlying mechanism (idiopathic vs secondary) did not relate to p16INK4a expression in any compartment (P>0.05). In three of eight FSGS, the representative sections stained for p16INK4a showed occasional segmental sclerotic lesions. First, the p16INK4a staining in these segmentally sclerotic glomeruli was mainly observed in non-sclerotic tuft area rather than the sclerotic lesion (may be because of the hypo/acellular nature of the lesion). Second, the amount of p16INK4a staining appeared to be similar in glomeruli with or without segmental sclerosis, but the number of sclerotic glomeruli was not enough to do a statistical comparison. Our results indicate that GD may act as an environmental stress inducing senescence in glomerular and interstitial cells, which in turn may contribute the progression by arresting replication needed to sustain the glomerulus. Kidneys with GD displayed a greatly increased expression of senescence marker p16INK4a in glomerular and interstitial cell nuclei and tubular cytoplasm compared to normal kidneys or those with TIN. In multivariate analysis, GD itself accounted for the increased p16INK4a expression in glomeruli and interstitial cells. These relationships are likely to be complex: GD causes proteinuria, which was also associated with p16INK4a expression. We do not know why the level of proteinuria did not correlate with p16INK4a expression, but the stresses related to GD itself and proteinuria are probably not dissectible and both may cooperate in inducing senescence. Perhaps the proteinuria would have been expected to induce p16INK4a expression in tubular nuclei, but this did not occur. Nevertheless, proteinuria may link between GD to p16INK4a expression in interstitial cell nuclei and tubular cytoplasm: filtered proteins injure tubular epithelial cells and trigger pro-inflammatory and pro-fibrotic effects.11.Healy E. Brady H.R. Role of tubule epithelial cells in the pathogenesis of tubulointerstitial fibrosis induced by glomerular disease.Curr Opin Nephrol Hypertens. 1998; 7: 525-530Crossref PubMed Scopus (69) Google Scholar,12.Eddy A.A. Proteinuria and interstitial injury.Nephrol Dial Transplant. 2004; 19: 277-281Crossref PubMed Scopus (135) Google Scholar It is noteworthy that the location and amount of p16INK4a expression is similar in both immune complex GD (MGP) and non-immune complex mediated GD (FSGS, MCD). In normal human glomeruli, we observed p16INK4a staining in all resident cell types, including mesangial cells, endothelial cells, podocytes, and parietal cells, and its expression increased with age. Biopsies with MGP, FSGS, and MCD showed higher p16INK4a expression than normals or TIN kidneys, notably in mesangial–endothelial cells. Although the underlying pathogenetic mechanisms are different, the amount of p16INK4a expression in mesangial–endothelial cells, podocytes, and parietal epithelium was similar in both immune complex GD and non-immune GD. Another interesting finding in the present study is the diffuse and global pattern of p16INK4a expression in FSGS. The p16INK4a expression in the glomeruli affected by FSGS was mainly observed in non-sclerotic tuft areas. Although FSGS is a focal disease, these observations suggest that the stresses related to disease processes induce a more diffuse and global p16INK4a expression in glomeruli, which is not limited to the glomeruli affected by focal sclerosis. It is known that podocyte alterations in FSGS are relatively diffuse and global at the electron microscopy level, although the lesions are focal and segmental at the light microscopy level.14.D'Agati V.D. Fogo A.B. Bruijn J.A. Jennette J.C. Pathologic classification of focal segmental glomerulosclerosis: a working proposal.Am J Kidney Dis. 2004; 43: 368-382Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar The regulation of the cell-cycle by proteins such as cyclin-dependent kinase inhibitors determines the cellular response to various immune and non-immune injury, and thus the development of histopathological lesions.15.Shankland S.J. Cell cycle regulatory proteins in glomerular disease.Kidney Int. 1999; 56: 1208-1215Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 16.Shankland S.J. Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis.Am J Physiol Renal Physiol. 2000; 278: F515-F529PubMed Google Scholar, 17.Nagata M. Tomari S. Kanemoto K. et al.Podocytes, parietal cells, and glomerular pathology: the role of cell cycle proteins.Pediatr Nephrol. 2003; 18: 3-8Crossref PubMed Scopus (23) Google Scholar Shankland et al.16.Shankland S.J. Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis.Am J Physiol Renal Physiol. 2000; 278: F515-F529PubMed Google Scholar suggested that the role of each cell-cycle regulator protein is cell-type specific and dependent on the type of injury. The proliferation of rat mesangial cells induced by platelet-derived growth factor was significantly inhibited by p16INK4a and moderately inhibited by p21WAF1.18.Terada Y. Yamada T. Nakashima O. et al.Overexpression of cell cycle inhibitors (p16INK4 and p21Cip1) and cyclin D1 using adenovirus vectors regulates proliferation of rat mesangial cells.J Am Soc Nephrol. 1997; 8: 51-60PubMed Google Scholar In human diseases without podocyte proliferation (MCD, MGP), p21WAF1, p27KIP1, and p57KIP2 expression did not change. In contrast, in diseases with podocyte proliferation (cellular FSGS, HIV-associated nephropathy) there was decreased p27KIP1 and p57KIP2 expression with an increase in p21WAF1 expression by podocytes.19.Shankland S.J. Eitner F. Hudkins K.L. et al.Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.Kidney Int. 2000; 58: 674-683Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar These results underscore the complex roles of cyclin-dependent kinase inhibitors in renal diseases. The pattern and location of p16INK4a expression may be an indicator of the primary site of injury in chronic renal disease. Aged kidneys display increased nuclear p16INK4a expression in tubules, glomeruli, interstitium, and vessels, and some show tubular cytoplasmic staining for p16INK4a, which is not observed in young kidneys.4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar Kidney transplants with IF/TA manifested increased p16INK4a expression in interstitial, glomerular, tubular cell nuclei, and tubular cytoplasm beyond normal aging.1.Melk A. Schmidt B.M. Vongwiwatana A. et al.Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.Am J Transplant. 2005; 5: 1375-1382Crossref PubMed Scopus (100) Google Scholar In this study, native kidneys with GD showed increased p16INK4a expression in glomerular and interstitial cell nuclei, and tubular cytoplasm, which further confirms our previous findings. On the other hand, tubular nuclear p16INK4a staining in GD biopsies was similar to the amount predicted for age. TIN biopsies showed an increased p16INK4a staining in interstitial cell nuclei and tubular cytoplasm but not in glomeruli. Perhaps somatic cell senescence and irreversible cell-cycle arrest prevent one renal compartment from sustaining itself, leading to nephron loss as further insults accrue. The tubular cytoplasmic p16INK4a staining observed in kidneys with GD and TIN was beyond the changes of normal aging, suggesting that the stress of proteinuria or of TIN may induce this change in tubular epithelium. Cytoplasmic p16INK4a staining was previously observed in various cancer types.20.Ghiorzo P. Villaggio B. Sementa A.R. et al.Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients.Hum Pathol. 2004; 35: 25-33Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21.Zhao P. Hu Y.C. Talbot I.C. Expressing patterns of p16 and CDK4 correlated to prognosis in colorectal carcinoma.World J Gastroenterol. 2003; 9: 2202-2206Crossref PubMed Scopus (61) Google Scholar, 22.Milde-Langosch K. Bamberger A.M. Rieck G. et al.Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a more malignant phenotype.Breast Cancer Res Treat. 2001; 67: 61-70Crossref PubMed Scopus (128) Google Scholar, 23.Murphy N. Heffron C.C. King B. et al.p16INK4A positivity in benign, premalignant and malignant cervical glandular lesions: a potential diagnostic problem.Virchows Arch. 2004; 445: 610-615Crossref PubMed Scopus (57) Google Scholar, 24.Shiozawa T. Nikaido T. Shimizu M. et al.Immunohistochemical analysis of the expression of cdk4 and p16INK4 in human endometrioid-type endometrial carcinoma.Cancer. 1997; 80: 2250-2256Crossref PubMed Scopus (59) Google Scholar However, localization in non-neoplastic cells is unclear. Here, we observed tubular cytoplasmic staining not only in cells with nuclear staining or vice versa, which may be compatible with dysregulation of the nuclear transport mechanism or a mislocalization owing to overexpression. As a small protein, p16INK4a may freely diffuse in and out of the nucleus to interact with its partners.20.Ghiorzo P. Villaggio B. Sementa A.R. et al.Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients.Hum Pathol. 2004; 35: 25-33Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Thus, excess production, abnormal nuclear transport, and decreased catabolism may all contribute to increased tubular cytoplasmic p16INK4a staining, possibly indicating cells subjected to unique disease stresses. On the basis of the association of p16INK4a expression in aged kidneys,4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar damaged kidney transplants,1.Melk A. Schmidt B.M. Vongwiwatana A. et al.Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.Am J Transplant. 2005; 5: 1375-1382Crossref PubMed Scopus (100) Google Scholar and GD with proteinuria, we hypothesize that injury exhausts finite replicative capacity in key cell populations, triggering p16INK4a expression and irreversible loss of replicative ability and thus repair capacity. This in turn will contribute to loss of nephrons in the face of continuing stress. Senescent cells may drop out completely or persist, for example, in atrophic tubules or interstitial cells, with persisting cells potentially adding to dysfunction in the tissue.5.Melk A. Senescence of renal cells: molecular basis and clinical implications.Nephrol Dial Transplant. 2003; 18: 2474-2478Crossref PubMed Scopus (74) Google Scholar Either cell loss or cell dysfunction in a nephron may trigger the regulatory systems that prevent nephrons from operating when glomerular–tubular balance is disturbed, leading to nephron shutdown. This would mean that the sequence is ‘injury–p16INK4a–nephron loss’. However, p16INK4a could also follow obsolescence, providing a way of preventing harmful behaviors of cells from nephrons that are no longer functioning. Thus, the sequence ‘injury–nephron shutdown–p16INK4a expression’ is also possible. In summary, this study shows that kidneys with GD and proteinuria display an accelerated expression of p16INK4a in glomerular and tubulointerstitial compartments, correlating with the degree of IF/TA, thus suggesting a role for somatic cell senescence in progress of GD. It will also be interesting to learn how somatic cell senescence mechanisms are balanced against the other options of damaged renal epithelium, such as epithelial–mesenchymal transition or apoptosis. A total of 31 patients (mean age 47.9, range 15–87) biopsied during the period of 1996–2003 for investigation of proteinuria, with or without renal insufficiency were included. The magnitude of proteinuria based on 24-h urine collections at the time of renal biopsy and serum creatinine levels were recorded. Thirty-five biopsy of 31 patients were diagnosed as MGP (N=22), FSGS (N=8), and MCD (N=5). Of the 22 MGP cases, five were stage I, 11 stage II, five stage III, and one was stage IV. The pertinent clinical data from the medical records of patients with MGP (n=19) were reviewed to determine whether MGP is idiopathic or associated with an underlying disease: Twelve (63%) were idiopathic and seven (37%) were presumed to be secondary MGP (one is associated with adenocarcinoma of the lung, two with hepatitis B or C infection, one with systemic lupus erythematosis, one with rheumatoid arthritis, and two with diabetes mellitus type I or II). Twelve native kidney biopsies were selected from patients with impaired renal function, diagnosed with TIN: acute TIN (n=6), acute and chronic TIN (n=3), and chronic TIN (n=3). Nineteen normal human kidney tissue samples were obtained from zero biopsies at the time of transplantation (implantation biopsies). All biopsies are reviewed and the amount of IF, TA, interstitial inflammation, fibrous intimal thickening in arteries, the percentage of global glomerulosclerosis, and the presence of mesangial matrix increase and cellularity or glomerular inflammation (if any) are recorded. We previously described a study of 42 human kidney samples, mostly nephrectomy specimens from patients with kidney tumors, in which we calculated the observed increase in p16INK4a with age.4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar To adjust for age differences among the groups in the current study, we used that equation to calculate the expected nuclear staining of p16INK4a in the present cases. The study was covered by approval (Protocol No. 2627) from the Institutional Review Board (Health Research Ethics Board, University of Alberta, Edmonton, Alberta, Canada). Immunoperoxidase staining for p16INK4a was performed using 2 μM sections of paraffin embedded tissue. Briefly, sections were deparaffinized and rehydrated. The sections were immersed in 3% H2O2 in methanol to inactivate endogenous peroxidase. Slides were blocked with 20% normal goat serum. Tissue sections were then incubated for 1 h at room temperature with the primary antibody (mouse monoclonal antibody, Clone F-12, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and rinsed with phosphate-buffered saline. Following 30 min of incubation with the Envision Monoclonal System (Dako, Mississauga, Ontario, Canada), sections were washed again in phosphate-buffered saline. Visualization was performed using the DAB substrate kit (Dako, Mississauga, Ontario, Canada). The slides were counterstained with hematoxylin and mounted. Tonsil was used as a positive control and sections stained with immunoglobin G isotype were used as a negative control. Analysis was carried out by counting 10 high-power fields. Percentage of positive nuclei was assessed for glomeruli, tubules, and interstitium. In interstitium, fibroblast-like spindle cells with positive staining were counted. Tubular cytoplasmic staining was assessed by counting the percentage of tubular cross-sections that showed positive cytoplasmic staining for p16INK4a and scored on a scale from 0 to 3 as >50% positive (score 3), 25–50% positive (score 2), <25% positive (score 1), and negative (score 0). Each artery was graded on a scale from 0 to 3, with 0 being no staining, 1 up to 30% stained nuclei, 2 between 30 and 60% stained nuclei, and 3 more than 60% stained nuclei. The mean score was then calculated for each section. Because of the difficulties in finding enough tissue for double stainings with specific cell markers, the glomerular cell types were identified morphologically. Because the distinction of mesangial cells from endothelial cells may be difficult, even impossible, by morphology alone, we defined cells lying within the mesangium and along the inner walls of glomerular capillary loops as mesangial–endothelial cells. Cells attached to the outer aspect of the glomerular capillary loops and lying along the inner wall of Bowman's capsule were identified as podocytes and parietal cells, respectively. We are aware of the fact that morphological assessment alone may not be a very precise method for quantification of staining. On the other hand, we think that it is helpful to show the overall pattern and location of expression. This determination can be made with considerable accuracy as it is clear which side of the basement membrane cells are on. The exact determination of which cell are endothelial and which mesangial is much more difficult (both on the inner aspect of the basement membrane) and this is why these two categories were combined. The morphological identification of glomerular cell types has been also previously used to show the differential expression and location of cyclin-dependent kinase inhibitors in human GD.19.Shankland S.J. Eitner F. Hudkins K.L. et al.Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.Kidney Int. 2000; 58: 674-683Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar In glomeruli, p16INK4a positive nuclei were scored semiquantitatively according to cell types (0–3: negative, 50%). For podocytes, p16INK4a staining was only noted as 0: negative or 1: positive. The semiquantitative analysis of p16INK4a staining in glomerular cell types was carried out for normal kidneys, GD, and TIN. Then, the glomerular p16INK4a staining in MGP, FSGS, and MCD was analyzed separately and the percentage of p16INK4a-positive mesangial–endothelial cell, podocyte, and parietal cell nuclei in 10 glomeruli was determined. Data analyses were performed using SPSS 12.0 (Chicago, IL, USA). p16INK4a expression in normal kidneys, GD and TIN were compared using Mann–Whitney U, Kruskal–Wallis or χ2 test. Kruskal–Wallis or χ2 test were used to compare p16INK4a expression among different glomerular diseases. Expected p16INK4a expression was calculated using the regression formulas as published previously:4.Melk A. Schmidt B.M. Takeuchi O. et al.Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.Kidney Int. 2004; 65: 510-520Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar glomerular p16INK4a=0.04 × age+2.40; tubular p16INK4a=0.43 × age-0.47; interstitial p16INK4a=0.18 × age-1.25. Expected and measured p16INK4a expression was also compared using paired t-test. Univariate and multivariate linear regression analyses were used to select the significant determinants of increased nuclear p16INK4a expression. Cytoplasmic tubular p16INK4a staining was not entered to the linear regression analysis because the values were categorical. Univariate correlates of the nuclear p16INK4a expression with a P<0.3 were entered into the multivariate model to determine the best independent determinants of increased nuclear p16INK4a expression. The cutoff P-value <0.3 for the multivariate analysis was chosen arbitrarily. The level of significance was set at P<0.05. All values are given as mean±s.d. unless noted otherwise. This research has been supported by funding and/or resources from Genome Canada, Genome Alberta, the University of Alberta, the University of Alberta Hospital Foundation, Roche Molecular Systems, Hoffmann-La Roche Canada Ltd, Alberta Innovation and Science, the Roche Organ Transplant Research Foundation, the kidney Foundation of Canada, and Astellas Canada. Dr Halloran also holds a Canada Research Chair in Transplant Immunology and the Muttart Chair Immunology. Figure S1. Comparision of measured with expected p16INK4a expression.
Referência(s)