AJP - Renal Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 295: F1563-F1573, 2008. First published September 3, 2008; doi:10.1152/ajprenal.90302.2008
0363-6127/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/F1563    most recent
90302.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Verzola, D.
Right arrow Articles by Garibotto, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verzola, D.
Right arrow Articles by Garibotto, G.

Accelerated senescence in the kidneys of patients with type 2 diabetic nephropathy

Daniela Verzola,1 Maria Teresa Gandolfo,1 Gianfranco Gaetani,2 Annamaria Ferraris,2 Rosa Mangerini,2 Franco Ferrario,3 Barbara Villaggio,1 Fabio Gianiorio,1 Fanny Tosetti,1 Ursula Weiss,1 Paolo Traverso,4 Mariano Mji,5 Giacomo Deferrari,1 and Giacomo Garibotto1

1Department of Internal Medicine and Cardionephrology, Azienda Universitaria Ospedale San Martino, University of Genoa; 2Dipartimento di Oncologia, Biologia e Genetica, Università di Genova and Istituto Nazionale per la Ricerca sul Cancro, Genoa; 3Renal Immunopathology Centre, San Carlo Hospital, Milan, Italy; 4Department of Urology, University of Genoa; and 5Nephrology Division, Imperia Hospital, Imperia, Italy

Submitted 11 May 2008 ; accepted in final form 2 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the hypothesis that senescence represents a proximate mechanism by which the kidney is damaged in type 2 diabetic nephropathy (DN). As a first step, we studied whether the senescence-associated β-galactosidase (SA-β-Gal) and the cell cycle inhibitor p16INK4A are induced in renal biopsies from patients with type 2 DN. SA-β-Gal staining was approximately threefold higher (P < 0.05) than in controls in the tubular compartment of diabetic kidneys and correlated directly with body mass index and blood glucose. P16INK4A expression was significantly increased in tubules (P < 0.005) and in podocytes (P = 0.04). Nuclear p16INK4A in glomeruli was associated with proteinuria (P < 0.002), while tubular p16INK4A was directly associated with body mass index, LDL cholesterol, and HbA1c (P < 0.001–0.05). In a parallel set of experiments, proximal tubule cells passaged under high glucose presented a limited life span and an approximately twofold increase in SA-β-Gal and p16INK4A protein. Mean telomere lengths decreased ~20% as an effect of replicative senescence. In addition, mean telomere decreased further by ~30% in cells cultivated under high glucose. Our results show that the kidney with type 2 diabetic nephropathy displays an accelerated senescent phenotype in defined renal cell types, mainly tubule cells and, to a lesser extent, podocytes. A similar senescent pattern was observed when proximal tubule cell cultures where incubated under high-glucose media. These changes are associated with shortening tubular telomere length in vitro. These findings indicate that diabetes may boost common pathways involving kidney cell senescence, thus reinforcing the role of the metabolic syndrome on biological aging of tissues.

tubular cells; telomeres; p16INK4A; senescence-associated β-galactosidase

AGING has been proposed to represent the failure or success of tumor-suppressor mechanisms that depend on the activities of the cyclin-dependent kinase inhibitor p16INK4A and of telomere shortening (2). It has been theorized that the high frequency of end-stage renal disease in the elderly results from an interaction between somatic cell senescence and age-associated diseases, such as hypertension and type 2 diabetes mellitus, which could hinder the limited ability of aged kidney to repair and maintain epithelial functions (24, 25). Cell senescence is characterized by an irreversible growth arrest and functional and morphological changes (2), including enhanced expression of senescence markers, such as senescence-associated β-galactosidase (SA-β-Gal), and different sets of genes, including negative regulators of the cell cycle (2, 17, 18). In vitro studies support the hypothesis that diabetes may accelerate cell and organ senescence in humans. Hyperglycemia induces premature replicative senescence in human skin fibroblasts, an effect that is tightly coupled to larger cell volume in skin fibroblasts from patients with diabetic nephropathy (3, 21). In addition, a role of hyperglycemia in kidney cell senescence has been observed in cultured mesangial cells (1, 34, 35, 37). However, how these observations from in vitro studies apply to human diabetic nephropathy is still unexplored.

In this study, we tested the hypothesis that the acceleration of senescence represents a relevant mechanism by which kidney cells are injured in diabetic nephropathy. With this in mind, we used different markers for the study of senescence in kidney biopsies of patients with type 2 diabetic nephropathy at different clinical stages. As a next step, we examined the in vitro effects of hyperglycemia on senescence markers, replicative ability, and telomere length in PTECs stimulated to divide in vitro. Our results show that the kidneys with type 2 diabetic nephropathy and proteinuria display an accelerated senescent phenotype, thus suggesting a role for somatic cell senescence as a mechanism of diabetic nephropathy. In addition, a similar senescent pattern displayed by tubule cells in renal biopsies was observed when incubating proximal tubule cells under high glucose media. These changes were associated with shortening tubular telomere length in vitro. Our findings show that senescence is accelerated in the kidney of patients with type 2 diabetic nephropathy, thus reinforcing the role of the metabolic syndrome on biological aging of tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Media and additives for culture of PTEC cells, 5-bromo-2-β-deoxy-uridine (BrdU) as well as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, acryl amide, and other reagents for SDS-PAGE were purchased from Sigma Chemical (St. Louis, MO). Coomassie protein assay reagent was obtained from Pierce (Rockford, IL). Tissue culture plates and dishes were from Corning. Anti-human p21 monoclonal, anti-human p53 polyclonal, and anti-human p16INK4A monoclonal (Clone F12) antibody (Ab) were obtained by Santa Cruz Biotechnology (Santa Cruz, CA), and anti-human retinoblastoma (Rb) protein monoclonal Ab was obtained by Pharmingen BD (Franklin Lakes, NJ). The biotin-streptavidin-amplified detection system was obtained from Vector Laboratories (Burlingame, CA), and Eukitt was from Kindler (Freiburg, Germany). Horseradish peroxidase-conjugated secondary Ab, nitrocellulose, and positively charged nylon membrane were from GE Healthcare (Buckinghamshire, England). Immobilon Western chemiluminescent horseradish peroxidase substrate was obtained from Millipore (Billerica, MA). The telomere length assay kit was purchased from Roche (Mannheim, Germany).

Patients. Seventeen patients with type 2 diabetic nephropathy entered into this study from the Department of Internal Medicine, Nephrology Division, Genoa University (Table 1). The study was part of a larger study in patients with type 2 diabetic nephropathy, and it was approved by the Ethical Committee of the Department of Internal Medicine, Genoa University. The procedures were in accordance with the Declaration of Helsinki. The age of patients ranged from 50 to 70 yr (mean 61 yr). Diabetic subjects were further classified into two groups based on urine protein excretion and renal function. The first group of subjects had early clinical diabetic nephropathy (defined by proteinuria 500–2,000 mg/day and serum creatinine ≤1.4 mg/dl). The second group of subjects had an advanced clinical nephropathy (proteinuria >2,000 mg/day and/or serum creatinine >1.4 mg/dl). Systolic blood pressure levels were higher, and the duration of diabetes longer in patients with more advanced disease. There were no significant differences in age, body weight, HbA1c, blood glucose, and serum cholesterol between the two groups. Serum triglycerides tended to be greater in subjects with advanced diabetic nephropathy. In every case, the diagnosis of diabetic nephropathy was based on light microscopy and immunofluorescence. Normal portions of kidney tissue from nephrectomies for renal carcinoma (n = 9, 7 male/2 female, mean age 60 yr, range 58–67 yr) were examined as a control group.


View this table:
[in this window]
[in a new window]

  		Table 1. Clinical characteristics of patients with type 2 diabetic nephropathy and controls

 
All kidney biopsy specimens were analyzed by the same pathologist (Dr. F. Ferrario). The morphological changes in kidney biopsies were considered as previously described (23, 32) for the following categories: glomerular sclerosis (nodular or diffuse), glomerular ischemic lesions, mesangial proliferation, interstitial fibrosis, interstitial inflammation, tubular atrophy, atherosclerosis, and arterial hyalinosis. Individual scores ranged from 0 to 3. For interstitial fibrosis, score 0 = no interstitial fibrosis; score 1 = interstitial fibrosis in up to 25%; score 2 = interstitial fibrosis in 26–50%; and score 3 = interstitial fibrosis in >50% of the cross section. For tubular atrophy, score 0 = no tubular atrophy; score 1 = tubular atrophy in up to 25%; score 2 = tubular atrophy in 26–50%; and score 3 = tubular atrophy in >50% of tubules.

Expression of p16INK4a and SA-β-Gal. SA-β-Gal-positive cells were detected in frozen tissue as described by Dimri et al. (9) as cells showing a bright cytoplasmatic blue precipitate. For the analysis of p16INK4A, paraffin sections (5 µm) of 2% paraformaldehyde-fixed renal tissue were deparaffined, hydrated, and treated with 3% methanol hydrogen peroxide solution. Slides were exposed to primary Ab for 1 h (1:100 dilution in PBS), followed by incubation in biotinylated anti rabbit antibody (1:100 diluition in PBS) for 30 min. Immunostaining was completed by incubation for 15 min with extravidin-peroxidase (1:20 in PBS). The biotin-extravidin peroxidase method was performed as described previously (31). p16INK4A/SA-β-Gal coexpression was evaluated in frozen tissue. At least five (range 5–10) glomeruli from each biopsy were systematically serially sectioned. These sections were used to estimate cell number by light microscopy. Endothelial cells were identified as being within the capillary lumen and integral to the filtration surface or mesangiocapillary surface, mesangial cells were defined as those completely within the mesangium, and podocytes were defined as being in the urinary space within the glomerular tuft. Proximal and distal tubular cells were identified primarily by morphological criteria. The percentage of p16INK4A-positive nuclei (nuclear p16INK4A) was assessed for glomeruli and tubules and expressed as percent positive cells. On the average, ~800 glomerular and ~1,200 tubular nuclei were counted in controls and in diabetic subjects, respectively. The nuclear + cytoplasmic expression of p16INK4A in tubules was examined by image analysis and expressed as percent positive areas (30). SA-β-Gal tubular cytoplasmic staining was assessed by counting the percentage of tubular cross sections that showed positive cytoplasmic staining. All specimens for senescence markers detection were analyzed by the same pathologist (Dr. D. Verzola).

Cell culture. The effect of hyperglycemia on senescence and telomere length was studied in primary cultures of proximal tubular cells obtained from normal portions of kidney tissue obtained from nephrectomies for renal carcinoma (n = 7, 6 male/1 female, mean age 62 yr, range 60–67 yr). Subjects were nonsmokers, nondiabetics, and free of cardiovascular disease or hypertension, variables that could influence telomere length (5). Human PTECs were isolated as previously described (31). Briefly, renal cortical tissue was obtained from kidneys that were removed for circumscribed tumors. Cortical specimens were cut into small cubes and passed through a series of mesh sieves of diminishing pore size. PTECs were collected on the 53-µm sieve and digested with collagenase (750 U/ml) at 37°C for 15 min. Tubular cells were isolated by centrifugation and grown in modified Eagle's medium supplemented as previously described (33).

Cell treatments. PTECs were passaged three times for 28 days in normal growth medium. During this period, the cultures were regularly fed twice weekly. PTECs were then passaged under different conditions as follows: 1) baseline medium containing 5.5 mmol/l glucose [normoglycemia (NG)]; 2) addition of supplemental glucose to a concentration of 30 mmol/l [hyperglycemia (HG)]; and 3) addition of mannitol to a concentration of 24.5 mmol/l [osmotic control, mannitol (M)]. To determine if HG is involved in replicative arrest and senescence, cells were grown in NG, HG, or M for 5, 10, or 15 days. After this time period, telomere length was assessed.

Cell proliferation. Incorporation of BrdU was measured as an index of DNA synthesis. PTECs were grown for 5, 10, or 15 days in NG, HG, or M medium. BrdU (10 µmol/l) was directly added to the culture medium for 1 h. The cells were detached and incubated on poly-L-lysine coated glass slides for 40 min at 4°C. Then, the spots were fixed in 70% ethanol. The slides were immersed in 0.07 N NaOH for 2 min, and the base was neutralized in PBS, pH 8.5. 20 µl of antibody to BrdU was mixed with 50 µl of 0.5% Tween 20/PBS and incubated for 30 min. Cells were exposed to biotinylated conjugated secondary antibody (1:100 in PBS) for 30 min and, then, to FITC-streptavidin (1:400 in PBS) for 30 min. Nuclei were counterstained with propidium iodide. Slides were observed and counted under a fluorescence microscope, and positive cells were expressed as a percentage of total cells counted. For the calculation of population doubling time (11), the cell number was measured manually with a hemocytometer.

Western blot analysis. The cells were scraped and suspended in cold lysis buffer [20 mmol/l HEPES; 150 mmol/l NaCl; 10% (vol/vol) glycerol; 0.5% (vol/vol) NP-40; 1 mmol/l EDTA; 2.5 mmol/l DTT; 10 µg/l aprotinin, leupeptin, and pepstatin A; and 1 mmol/l PMSF and Na3VO4]. Protein concentration was determined by using the Coomassie protein reagent, and the same amounts were resolved on 7–15% SDS-polyacrylamide gels and electrotransferred to a nitrocellulose membrane. Blots were blocked for 1 h at room temperature in PBS 5% milk. The membrane was incubated overnight at 4°C with anti-human p16INK4A, anti-human Rb protein, anti-human p53, and anti-human p21 antibodies. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were developed with ECL detection system.

Telomere length. Mean telomere terminal restriction fragment (TRF) length analysis was performed with the TAGGG telomere length assay kit. Ten micrograms of genomic DNA was digested overnight with a high concentration (50 U/ml) of HinfI and RsaI and separated on 0.8% agarose gel. After electrophoresis, DNA was denatured with 0.5M NaOH/1.5 M NaCl, neutralized with 0.5 M Tris·HCl/3 M NaCl and transferred onto a positively charged nylon membrane. The membrane was hybridized to a digoxigenin-labeled telomere specific probe for 3 h at 65°C and then washed twice in 2x SSC/0.1% SDS for 15 min at room temperature and twice in 0.5x SSC/0.025% SDS for 20 min at 39°C. The membrane was finally incubated with a digoxigenin-specific antibody, covalently coupled to alkaline phosphatase. The telomeric probe was visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase CDP-star (phenylphosphate substituted 1,2 dioxetane). To measure the mean TRF length of DNA samples, the membrane was exposed to Hyper film ECL (Amersham). The mean telomere length, defined as the strongest density peak, of the samples examined was determined compared with DNA of standard lengths, using KODAK 1D image analysis software (Carestream Health, Rochester, NY; Ref. 12). The coefficient of variation range for the mean TRF length by this method is 4%.

Statistical analysis Data are presented as means ± SE or, when skewed, median (range). The Statview statistical package (Cary, NC) was used for the analysis. Means were compared for statistically significant differences by t-test, Mann-Whitney, or ANOVA when two or more than two groups, respectively, were involved. Relationships between parameters were analyzed using simple regression analysis or Spearman test, as required. Multiple regression analysis was performed to assess which clinical metabolic parameters [body mass index (BMI), LDL cholesterol, HbA1c, and blood glucose] or structural changes (glomerular ischemic changes, tubular atrophy, and interstitial fibrosis), which were significantly related in simple regression analysis, were independent predictors of senescence. A two-tailed P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
p16 INK4A protein expression in type 2 diabetic nephropathy. Normal kidneys showed p16INK4A staining in 1.3 ± 0.63% of glomerular cells (Fig. 1A, arrowheads), 1.5 ± 0.27% of tubular cells (Fig. 1B), and 0.3 ± 0.6% of vessels (Table 2). Only scanty interstitial cells were p16INK4A positive (0.1 ± 0.2%). In addition to the nuclear staining, 70% of normal kidneys showed tubular cytoplasmic p16INK4A expression (mean score: 0.1 ± 0.04). These findings are in accordance with what has been previously reported (28, 18, 19).


Figure 1
View larger version (100K):
[in this window]
[in a new window]

  		Fig. 1. p16INK4A expression in normal kidneys and type 2 diabetic nephropathy biopsies. A: normal kidney (male, 60 yr) with rare p16INK4A staining in mesangium (arrowhead). B: normal kidney tubules (male, 60 yr) showing focal p16INK4A staining. C: type 2 diabetic nephropathy biopsy from a 62-yr-old man, showing increased nuclear p16INK4A expression in podocytes (arrowheads). D: type 2 diabetic nephropathy biopsy from a 55-yr-old man, showing increased p16INK4A staining in podocytes and mesangial-endothelial cells. E–F: type 2 diabetic nephropathy biopsy from a 63-yr-old man, showing p16INK4A staining in tubular cytoplasm and nuclei. G–H: type 2 diabetic nephropathy biopsy from a 65-yr-old woman, showing cytoplasmatic and nuclear p16INK4A expression in endothelial cells of arterial vessels. (original magnification = x1,000). PC, podocyte; MC, mesangial cell; EC, endothelial cell.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Comparison of p16INK4A expression (%p16INK4A positive nuclei) in normal kidneys and type 2 diabetic nephropathy

 
The percentage of p16INK4A positive glomeruli (19.8 ± 0.5 vs. 13.7 ± 0.5% in patients and controls, respectively; P > 0.05), as well as the percentage of glomerular p16INK4A positive nuclei (1.5 ± 0.37 vs. 1.3 ± 0.63% in patients and controls, respectively; P > 0.05), was similar in patients with diabetic nephropathy and controls (Table 2). When nuclear p16INK4A expression in glomeruli was analyzed by cell type (Fig. 1; Table 2), podocyte nuclear p16INK4A staining was increased in diabetic nephropathy compared with normal kidneys (P = 0.04). In contrast, the p16INK4A signal in mesangial-endothelial cell nuclei and in the arterial vessels was similar to controls.

Diabetic patients showed a strikingly increased p16INK4A expression in tubular nuclei (~3-fold increase; Fig. 1, E and F; Table 2) and tubular nuclei and cytoplasm (mean score: 0.30 ± 0.08 vs 0.1 ± 0.04 in controls; P < 0.05 ).

SA-β-Gal expression is enhanced in the kidney of patients with type 2 diabetic nephropathy. SA-β-Gal staining was approximately threefold higher (32.6 ± 17 vs. 7.35 ± 5.8% positive tubuli in patients vs. controls; P < 0.05) in the tubular compartment of diabetic kidneys with early disease than in controls (Fig. 2) and did not increase further in advanced diabetic nephropathy. The SA-β-Gal signal was predominantly confined to tubular cells, and it was also only weakly detected in glomerular parietal cells, podocytes, and vascular endothelial cells (Fig. 2). Tubular SA-β-Gal staining was directly related to the nuclear + cytoplasmic expression of p16INK4A (r= 0.63; P < 0.03), but not nuclear p16INK4A, in the tubular compartment.


Figure 2
View larger version (134K):
[in this window]
[in a new window]

  		Fig. 2. Staining for senescence-associated β-galactosidase (SA-β-Gal) in human renal tissue. Staining appears as bright-blue granular staining into the cytoplasm of tubular epithelial cells, mainly proximal tubules. SA-β-Gal staining was only weakly expressed in normal kidney tissue (A). Representative staining from patients with type 2 diabetic nephropathy showed intense signals in tubules (B–E) and, partially, in endothelia of peritubular capillaries (E). Glomeruli showed weak signals. Expression of SA-β-Gal was similarly enhanced both in patients with early and in those with advanced clinical impairment (F; original magnification = x1,000). SA-β-Gal tubular staining was assessed by counting the percentage of tubular cross sections that showed positive cytoplasmic staining. *P < 0.05 vs. controls. GL, glomeruli; V, vessels. Data are means ± SE.

 
When we examined the SA-β-Gal and p16 coexpression in single sections, some tubule cells showed a complete coexpression of these two markers, while others showed an isolated expression of SA-β-Gal or p16 (Fig. 3). This suggests that aging phenotypes may be differently expressed at the same time in the same tissue or that, alternatively, also p16-independent mechanisms may account for tubular senescent changes. In addition, it is also possible that hyperglycemia-stimulated p16INK4A expression is transient but causes permanent changes.


Figure 3
View larger version (142K):
[in this window]
[in a new window]

  		Fig. 3. Kidney SA-β-Gal and p16INK4A stainings in kidney tubules of patients with type 2 diabetic nephropathy. A: rare isolated nuclear p16INK4A (arrow) and SA-β-Gal (*) stainings, as well as p16INK4A/SA-β-Gal coexpression (arrowheads). B: rare isolated SA-β-Gal expression (arrow). C: rare SA-β-Gal and p16INK4A expressions. D: diffuse SA-β-Gal expression (arrowhead) contrasting with a modest p16INK4A/SA-β-Gal coexpression (asterisk; original magnification = x1,000).

 
Determinants of p16INK4A and SA-β-Gal expressions. By univariate linear regression analysis, nuclear p16INK4A expression was strongly associated with proteinuria in glomeruli (r = 0.72; P < 0.002) but not in tubuli (r = 0.13; P > 0.05; Table 3; Fig. 4). In addition, glomerular p16INK4A was not related to estimated glomerular filtration rate (GFR) at the time of biopsy, blood pressure, BMI, or other metabolic parameters. The enhanced tubular cell nuclear + cytoplasmic p16INK4A expression was directly associated with BMI (r = 0.82; P < 0.001; Table 3; Fig. 5), plasma LDL cholesterol (r = 0.49; P < 0.05), and HbA1c (r = 0.49; P < 0.05; Fig. 5). Similarly SA-β-Gal staining in tubules was directly associated with BMI and blood glucose at the time of the biopsy (Fig. 6). All these parameters played an independent role in senescent changes in the kidney, as assessed by multiple regression analysis (Table 4).


View this table:
[in this window]
[in a new window]

  		Table 3. Univariate analysis of the correlation between some clinical parameters and p16INK4A expression in glomeruli and tubules in patients with type 2 diabetic nephropathy

 

Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 4. Nuclear p16INK4A expression in the glomeruli (A) but not in tubule cells (B) was directly associated with proteinuria in patients with type 2 diabetic nephropathy.

 

Figure 5
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 5. Nuclear + cytoplasmic (N +C) tubular p16INK4A expression was directly associated with body mass index (BMI; left) and HbA1c (right) in patients with type 2 diabetic nephropathy.

 

Figure 6
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 6. Tubular SA-β-Gal expression was directly associated with BMI (A) and fasting blood glucose (B) in patients with type 2 diabetic nephropathy.

 

View this table:
[in this window]
[in a new window]

  		Table 4. Results of the multiple regression analyses modeling some clinical parameters, p16INKA, and SA-β-Gal expression in the kidney tubules of patients with type 2 diabetic nephropathy

 
Histological associations. Both p16INK4A and SA-β-Gal expressions were already upregulated in patients with proteinuria and did not further change in advanced diabetic disease. A few significant relations were observed between individual structural kidney changes and senescence markers (Table 5). SA-β-Gal staining in tubules was directly associated with interstitial fibrosis (r = 0.49; P < 0.05), while tubular p16INK4A expression (%positive nuclei) was directly associated with tubule atrophy (r = 0.64; P < 0.02) and glomerular ischemic lesions (r = 0.76; P < 0.01). Multiple regression analysis was used to discover which proportion of variations in tubular p16INK4A or SA-β-Gal staining in tubules (as dependent variables) was accounted for by glomerular ischemic lesions, interstitial fibrosis, and tubular atrophy (independent variables), which were significantly related in simple regression analysis. The aforementioned structural changes did not play an independent role in variations in tubule senescence markers (model r2 = 0.050; P = NS; individual data not shown).


View this table:
[in this window]
[in a new window]

  		Table 5. Associations between structural changes and senescence-associated markers in patients with type 2 diabetic nephropathy (n = 17)

 
Cultured proximal tubular cells show accelerated senescence when exposed to high glucose media. After the observation of enhanced expression of senescence markers in the tubular compartment of renal biopsies and of their association with BMI, blood glucose, or HbA1c, we tested the hypothesis that high ambient glucose boosts the pathways involved in cellular senescence in tubule cells. PTECs were cultured for 5, 10, and 15 days in NG (5.5 mM, HG; 30 mM), or M (24.5 mM; osmotic control) containing media. Figure 7 shows that both NG and HG decreased the nuclear incorporation of BrdU (an expression of proliferative capacity) with time. However, DNA syntheses were significantly decreased by HG after 15 days. The PTEC population doubling time was 34 ± 11 and 70 ± 7 h at 5 days under NG and HG, respectively (P = NS). The population doubling time increased by threefold as an effect of replicative senescence in cells cultivated under NG for 15 days (P < 0.05). However, PTECs proliferated at even lower rates under HG (population doubling time 10 days: NG = 74 ± 13, HG = 115 ± 13 h, P = NS; 15 days NG = 125 ± 13, HG = 206 ± 15 h, P < 0.05). These data indicate that the passaged PTECs had limited life span in vitro, which is further decreased by high glucose media.


Figure 7
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 7. Under conditions in which proximal tubular cells (PTECs) are stimulated to divide in vitro 5-bromo-2-β-deoxy-uridine (BrdU) is incorporated into newly synthesized DNA strands. Shown is PTECs proliferation rate by BrdU test after 5–15 days replication under glucose 5 mmol (NG; {blacklozenge}), glucose 30 mmol/l (HG; {blacksquare}), or mannitol 24.5 mM/l (M; {blacktriangleup}). Proliferation was assessed by counting the number of BrdU incorporating cells. Both NG and HG decreased nuclear incorporation of BrdU an expression of proliferative capacity) with time. However, DNA syntheses were significantly decreased by HG, compared with those grown in NG. Results are expressed as means ± SE and are representative of 7 separate experiments *P < 0.001 vs respective basal values; °P < 0.001 vs NG (d, days).

 
Next, we used the measurement of SA-β-Gal activity to determine whether the replicative arrest was associated with senescence. Replicative senescence was associated with a marked increase in the cytochemically detectable SA-β-Gal activity (Fig. 8, A and B). In addition, after 15 days SA-β-Gal activity was significantly increased by HG, compared with NG (HG 23.1 ± 4 and NG 12.3 ± 3.6; P = 0.007; Fig. 8C).


Figure 8
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 8. Tubular expression of SA-β-Gal after exposure to HG medium (A–C). Cells were grown in NG medium for three passages. Beginning with the third passage, PTEC were treated under different conditions as follows: baseline medium containing 5.5 mmol/l glucose (NG) or 30 mmol/l glucose (HG) or mannitol (M) to a concentration of 24.5 mmol. To investigate the role of the cell cycle check points regulators in controlling HG-induced senescence, we evaluated the activation of the two pathways potentially implicated in senescence induction: the p53/p21 pathway and the retinoblastoma (a downstream target of p16INK4A) pathway in PTECS cultured in NG or HG media up to 15 days (D and E). Extracts prepared from early passage human PTECs contained barely detectable levels of p16INK4A and Rb proteins (Fig. 7, D–E). In sharp contrast, both p16INK4A and Rb proteins were highly expressed in PTECs grown under high glucose medium (Fig. 7, D–E). Contrarywise, p53 and p21 were not expressed, neither at the baseline nor after HG (data not shown). Tubulin was loading control for both pRB and p16. Results are means ± SE obtained from triplicate experiments.

 
HG upregulates p16INK4A and Rb proteins in PTECs. To investigate the role of the cell cycle check point regulators in controlling HG-induced senescence, we evaluated the activation of the two pathways potentially implicated in senescence induction: the p53/p21 pathway and the retinoblastoma (a downstream target of p16INK4A) pathway. Therefore, we studied the p53, p21, p16INK4A, and Rb expressions (Western blot) in PTECs cultured in NG or HG media up to 15 days. Extracts prepared from early passage human PTECs contained barely detectable levels of p16INK4A and Rb proteins (Fig. 8D). In sharp contrast, both p16INK4A and Rb proteins were highly expressed in PTECs grown under HG medium (Fig. 8, D–E). Of note, p53 and p21 were not expressed, neither at the baseline nor after HG (data not shown). According to these observations, HG media activate both the p16 telomere-dependent pathway and the p16/pRB-senescent pathway in renal epithelial cells in vitro.

High glucose accelerates telomere shortening in cultured PTECs. The reduction of telomere length is one of the events leading to activation of p53 and downstream p21 signaling, while p16INK4A has been shown to induce cellular senescence also independently of telomere attrition (2). We thus investigated the effect of hyperglycemia on telomere length in PTECs, as described in the MATERIALS AND METHODS. Before expansion, TRF lengths were ~13 kb, a value similar to those previously observed in the human kidney (18, 19). Four lines, which were maximally expanded in culture with NG or HG media, were harvested at growth arrest for genomic DNA analysis. Figure 9 shows the change in telomere length of cell cultures as a function of glucose concentration. Senescent, HG grown PTECs had lower TRF length than those grown in NG. Mean TRF lengths decreased from 11.25 to 8.92 kb as an effect of replicative senescence; in addition mean TRF decreased further to 4.39 kb in expanded cells cultivated under HG (Fig. 9). These results revealed significant loss of telomere DNA in the experimental conditions simulating diabetes mellities.


Figure 9
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 9. Southern blots (A) comparing terminal restriction fragment (TRF) lengths of unexpanded (lane 1) and maximally expanded (lanes 2–3) human PTECs exposed to normal (5 mmol; NG) or high (HG; 30 mmol) glucose media. Telomere length in human PTEC (B) exposed to high glucose media. Genomic DNAs from PTECs deriving from seven different kidneys were extracted for the determination of TRF length. Of these, four lines were maximally expanded. Mean TRF length was measured by densitometric scanning the image. Telomere lengths were shorter in PTECs cells at late passage compared with unexpanded PTEC lines. However, incubation with HG was associated with a further decreases in TRF length. Results are means ± SE obtained in triplicate experiments. Size (kb) is indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we tested the hypothesis that diabetic nephropathy is associated with an acceleration of senescent phenotype in kidney cells. With this in mind, we used different assays for the study of senescence markers in kidney biopsies of patients with type 2 diabetic nephropathy at different clinical stages and examined the association of several clinical parameters as potential predictors of kidney cell senescence. Previous studies (18) have shown that the nuclear p16INK4A expression increases progressively with age in several kidney cell types and that this increase is more pronounced in tubular cells. In our study, an acceleration of senescent phenotype was observed mainly in tubule cells and, to a lesser extent, podocytes. As a complement to the observed upregulation of senescence-associated SA-β-Gal staining and the cell cycle inhibitor p16INK4A, a prominent senescent phenotype was shown by human proximal tubular cells stimulated to divide in vitro under high glucose media, suggesting that the hyperglycemic milieu per se induces tubular senescence. Finally, the hyperglycemia-associated senescent phenotype was associated with a consistent decrease in tubule cell telomeric length and replication. Taken together, these findings strongly suggest that diabetic nephropathy is associated with premature kidney aging and that hyperglycemia is, at least in part, responsible for the observed changes.

The glomerular expression of p16INK4A, a major cell cycle regulatory protein associated with replicative senescence, increases with age (18) and in stress conditions, such as chronic kidney transplant rejection (20). In our study, kidneys with diabetic nephropathy displayed an increased expression of senescence markers p16INK4A and SA-β-Gal compared with age-matched controls, indicating that an age effect could not be responsible for our findings. In addition, the estimated GFR at the time of biopsy did not correlate with p16INK4A or SA-β-Gal staining (P > 0.05), suggesting that renal failure per se is not responsible for the activation of senescence. Glomerular injury frequently results in proteinuria, and filtered proteins are potential mediators of tubular epithelial cell injury, contributing to chronic tubulointerstitial changes (6). In patients with glomerulonephritis, p16INK4A expression in glomerular and tubular cells has been observed to be higher in kidneys with proteinuria (28). In patients with diabetic nephropathy studied here, the p16INK4A response appeared to vary in different cell types. A small number (1–3%) of glomerular cells were p16INK4A positive. In addition, glomerular p16INK4A was similarly expressed in proteinuric patients and controls. However, it was expressed mainly in podocytes, which are considered to be terminally differentiated cells. It is of note that podocyte loss in diabetic nephropathy is believed to result in permanent alterations in the glomerular filtration barrier (6, 8, 22). In our study, we found an association between glomerular p16INK4A and proteinuria, suggesting that a senescent phenotype in glomerular cells is associated with altered permeability.

SA-β-Gal is a well-known marker of cellular senescence (9). The presence of SA-β-Gal is independent of DNA synthesis and reflects the change in cell function that accompanies senescence (9). In accordance with the p16INK4A data, SA-β-Gal expression was markedly upregulated in tubular cells, whereas the signal was weak and mainly confined to podocytes in the glomeruli. Similar findings have been observed in animal models of kidney senescence (15). Of note, accelerated senescence was already observed in proteinuric patients with normal or subnormal GFR and only modest chronic changes at the time of their biopsy, suggesting that cell senescence is an early finding in human clinical diabetic nephropathy.

In vitro studies show that tubular cells undergoing senescence express TGF-β1 with advancing age (10). In keeping with these findings, in the aging kidney, p16INK4A gene expression independently correlates with aging and age-associated histological changes (19). In our study, at univariate analysis, tubular p16INK4A expression was directly related to the degree of glomerular ischemic lesions, suggesting that glomerular ischemia favors the downstream occurrence of senescence in the tubular compartment. In addition, tubular p16INK4A expression was directly associated with tubule atrophy, while tubular SA-β-Gal staining was directly associated with interstitial fibrosis. These findings raise the possibility that p16INK4A and SA-β-Gal play different roles (or, alternatively, are an expression of different mechanisms) in chronic tubulointerstitial changes in type 2 diabetic kidney disease. However, in multiple regression neither glomerular ischemic lesions nor interstitial fibrosis/tubular atrophy played an independent role on variations in tubule senescence markers. Of note, these lesions often progress sinchronously in patients with type 2 diabetic nephropathy (23).

In our study, although both p16INK4A and SA-β-Gal were both overexpressed in the diabetic kidney, their association was not very strong, with only 40% of changes in SA-β-Gal being accounted for the increase in p16INK4A in linear regression analysis. When we examined the SA-β-Gal and p16INK4A coexpression, some tubule cells showed a complete coexpression of these two markers, while others showed an isolated expression of SA-β-Gal or p16INK4A. This suggests that aging phenotypes may be differently expressed in the same tissue or that, alternatively, p16-independent mechanisms may account for tubular senescent changes in some tubule cells.

In our study, senescent changes in the kidney were related to several clinical parameters of metabolic stress, suggesting that a positive energy balance can cause kidney senescence. It is interesting that after an increase in extracellular pyruvate levels, human fibroblasts undergo a fast induction of cellular senescence (26, 36), implying that an increased metabolic supply causes cell senescence. To better understand the effects of changes observed in renal biopsies, we evaluated the effects of high glucose media on cell cycle regulatory proteins and senescent markers in PTECs. We detected SA-β-Gal activity in PTECs cultured in high glucose as one of many hallmark measures of cell replicative senescence or physiological aging. However, PTECs cultured in high glucose were associated with the upregulation of cell cycle inhibitors p16INK4A but not p21 at variance with findings obtained in mesangial cells (2, 34, 35). It has been suggested that each cell cycle regulator protein is cell and injury specific (18, 27). In senescent fibroblasts, G1 arrest is initially accomplished by p21 alone and occurs before p16INK4A accumulation (27, 29). In our study, hyperglycemia appears to activate both the p16 telomere-dependent pathway and the p16/pRB-senescent pathway in PTECs in vitro. Here we can speculate that, paralleling changes observed in fibroblasts (4), p16INK4A is upregulated as part of a terminal program that is involved in the maintenance of the senescent arrest.

The antiproliferative signals provoking the elevation of p16INK4A levels in senescent cells are thought to be generated by telomere shortening (2) but may be also telomere-independent (18) . Telomeres consist of a repetitive base sequence and are necessary for protection of the coding parts of the DNA from degradation and replicative damage. Telomere loss is accelerated by oxidative stress (26). Human adult kidney cells express scarce telomerase activity and contain short telomeres. In humans, telomere DNA is lost with age in kidney and the rate of loss in cortex is greater than in the medulla (18). With this in mind, we analyzed renal telomere length in response to hyperglycemia in renal tubular cells. We observed that high glucose accelerates the telomere loss observed during PTEC replicative senescence. Therefore, according to our findings, both age and hyperglycemic metabolic stress could hinder the ability of the kidney to sustain normal repair functions.

The results of the present study also strengthen the role of the metabolic syndrome in the biological aging of tissues. Of note, in humans, weight gain is associated with accelerated telomere attrition and a low calorie intake prevents various age-dependent changes, such as diabetes and atherosclerosis (22). Because the plasma levels of glucose and insulin are decreased by calorie restriction, changes of these pathways may underlie the improvement of longevity related to a lower calorie intake (24). In addition, insulin resistance largely accounts for the observed relationship between BMI and white blood cell telomere length (13, 14).

Why do telomeres shorten in cells exposed to high glucose? A possible explanation is direct damage to telomeres. This hypothesis is supported by studies showing accelerated telomere attrition caused by oxidative stress (13) or due to direct exposure to hyperglycemic conditions (7). With this regard, we (30, 31) have previously observed that hyperglycemia triggers the generation of free radicals and oxidant stress in human kidney tubular cells, a mechanism that is also linked to apoptosis.

Tissue repair and regeneration are processes that are essential for maintaining tissue integrity. In response to acute damage, the normal kidney has a remarkable capacity for regeneration, as evidenced by complete recovery of function after acute renal failure. Our results suggest that hyperglycemia, either directly or indirectly, exhausts the finite replicative capacity in selected kidney cell populations, triggering the loss of ability to replicate and repair. This is likely significant for the tubules, where the diminished repair ability could increase the susceptibility to develop acute renal insufficiency in response to acute stress (16). In addition, the early occurrence of senescence in podocyte could hinder their functions and contribute to loss of nephrons in patients with diabetic chronic kidney disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (FIRB 2001) and from Grants P. F. Regione Liguria 2003, AIL Cuneo, and Regione Liguria 2005 n. 1582.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Giacomo Garibotto MD, Department of Internal Medicine, Division of Nephrology, Viale Benedetto XV,6 16132 Genoa, Italy (e-mail: gari{at}unige.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Al-Douahji M, Brugarolas J, Brown PA, Stehman-Breen CO, Alpers CE, Shankland SJ. The cyclin kinase inhibitor p21WAF1/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy. Kidney Int 56: 1691–1699, 1999.[CrossRef][Web of Science][Medline]
  2. Ben-Porath I, Weinberg RA. When cells get stressed: an integrative view of cellular senescence. J Clin Invest 113: 8–13, 2004.[CrossRef][Web of Science][Medline]
  3. Blazer S, Khankin E, Segev Y, Ofir R, Yalon-Hacohen M, Kra-Oz Z, Gottfried Y, Larisch S, Skorecki KL. High glucose-induced replicative senescence: point of no return and effect of telomerase. Biochem Biophys Res Commun 296: 93–101, 2002.[CrossRef][Web of Science][Medline]
  4. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277: 831–834, 1997.[Abstract/Free Full Text]
  5. Chang E, Hartley CB. Telomere length and replicative aging in human vascular tissues. Proc Natl Acad Sci USA 92: 11190–11194, 1995.[Abstract/Free Full Text]
  6. Chen L, Wang Y, Tay YC, Harris DC. Proteinuria and tubulointerstitial injury. Kidney Int Suppl 61: S60–S62, 1997.[Medline]
  7. Dabouras V, Rothermel A, Reininger-Mack A, Wien SL, Layer PG, Robitzki AA. Exogenous application of glucose induces aging in rat cerebral oligodendrocytes as revealed by alteration in telomere length. Neurosci Lett 368: 72–78, 2004.
  8. Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P. Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes. Diabetes 52: 1031–1035, 2003.[Abstract/Free Full Text]
  9. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92: 9363–9367, 1995.[Abstract/Free Full Text]
  10. Ding G, Franki N, Rapasi AA, Reddy K, Gibbons N, Pravin Singhal PC C. Tubular cell senescence and expression of TGF-b1 and p21WAF1/CIP1 in tubulointerstitial fibrosis of aging rats. Exp Mol Pathol 70: 43–53, 2001.[CrossRef][Web of Science][Medline]
  11. Endlich N, Endlich K, Taesch N, Helwig JJ. Culture of vascular smooth muscle cells from small arteries of the rat kidney. Kidney Int 57: 2468–2475, 2000.[CrossRef][Web of Science][Medline]
  12. Ferraris M, Pujic N, Mangerini R, Rappezzi D, Gallamini A, Racchi O, Casciaro S, Gaetani GF. Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres. Br J Haematol 130: 391–393, 2005.[CrossRef][Web of Science][Medline]
  13. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 408: 239–247, 2000.[CrossRef][Web of Science][Medline]
  14. Gardner JP, Li S, Srinivasan SR, Chen W, Kimura M, Lu X, Berenson GS, Aviv A. Rise in insulin resistance is associated with escalated telomere attrition. Circulation 111: 2171–2177, 2005.[Abstract/Free Full Text]
  15. Haruna Y, Kashihara N, Satoh M, Tomita N, Namikoshi T, Sasaki T, Fujimori T, Xie P, Kanwar YS. Amelioration of progressive renal injury by genetic manipulation of Klotho gene. Proc Natl Acad Sci USA 104: 2331–2336, 2007.[Abstract/Free Full Text]
  16. Hsu CY, Ordoñez JD, Chertow GM, Fan D, McCulloch CE, Go AS. The risk of acute renal failure in patients with chronic kidney disease. Kidney Int 74: 101–107.
  17. Itahana K, Campisi J, Dimri GP. Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods Mol Biol 371: 21–31, 2007.[Medline]
  18. Melk A, Ramassar V, Helms LM, Moore R, Rayner D, Solez K, Halloran PF. Telomere shortening in kidneys with age. J Am Soc Nephrol 11: 444–453, 2000.[Abstract/Free Full Text]
  19. Melk A, Schmidt BM, Takeuchi O, Sawitzki B, Rayner DC, Halloran PF. Expression of p16INK4A and other cell cycle regulator and senescence associated genes in aging human kidney. Kidney Int 65: 510–520, 2004.[CrossRef][Web of Science][Medline]
  20. Melk A, Schmidt BM, Vongwiwatana A, Rayner DC, Halloran PF. Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney. Am J Transplant 5: 1375–1382, 2005.[CrossRef][Web of Science][Medline]
  21. Morocutti A, Earle KA, Sethi M, Piras G, Pal K, Richards D, Rodemann P, Viberti G. Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease. Kidney Int 50: 250–256, 1996.[CrossRef][Web of Science][Medline]
  22. Reddy GR, Kotlyarevska K, Ransom RF, Menon RK. The podocyte and diabetes mellitus: is the podocyte the key to the origins of diabetic nephropathy? Curr Opin Nephrol Hypertens 17: 32–36, 2008.[Web of Science][Medline]
  23. Ruggenenti P, Gambara V, Perna A, Bertani T, Remuzzi G. The nephropathy of non-insulin-dependent diabetes predictors of outcome relative to diverse patterns of renal injury. J Am Soc Nephrol 9: 2336–2343, 1998.[Abstract]
  24. Sampson and Hughes DA MJ. Chromosomal telomere attrition as a mechanism for the increased risk of epithelial cancers and senescent phenotypes in type 2 diabetes. Diabetologia 49: 1726–1731, 2006.[CrossRef][Web of Science][Medline]
  25. Sampson MJ, Winterbone M, Hughes JC. Monocyte telomere shortening and oxidative DNA damage in type 2 diabetes. Diabetes Care 29: 283–289, 2006.[Abstract/Free Full Text]
  26. Serra V, von Zglinicki T, Lorenz M, Serra V, Saretzki G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J Biol Chem 278: 6824–6830, 2003.[Abstract/Free Full Text]
  27. Shankland SJ, Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. Am J Physiol Renal Physiol 278: F515–F529, 2000.[Abstract/Free Full Text]
  28. Sis B, Tasanarong A, Khoshjou F, Dadras F, Solez K, Halloran PF. Accelerated expression of senescence associated cell cycle inhibitor p16INK4A in kidneys with glomerular disease. Kidney Int 71: 218–226, 2007.[CrossRef][Web of Science][Medline]
  29. Stein GH, Drullinger LF, Soulard A, Dulic V. Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 19: 2109–2117, 1999.[Abstract/Free Full Text]
  30. Verzola D, Bertolotto MB, Villaggio B, Ottonello L, Dallegri F, Frumento G, Berruti V, Gandolfo MT, Garibotto G, Deferrari G. Taurine prevents apoptosis induced by high ambient glucose in human tubule renal cells. J Investig Med 50: 443–451, 2002.[Web of Science][Medline]
  31. Verzola D, Bertolotto MB, Villaggio B, Ottonello L, Dallegri F, Salvatore F, Berruti V, Gandolfo MT, Garibotto G, Deferrari G. Oxidative stress mediates apoptotic changes induced by hyperglycemia in human tubular kidney cells. J Am Soc Nephrol 15: S85–S87, 2004.[Abstract/Free Full Text]
  32. Verzola D, Gandolfo MT, Ferrario F, Rastaldi MP, Villaggio B, Gianiorio F, Giannoni M, Rimoldi L, Lauria F, Mij M, Deferrari G, Garibotto G. Apoptosis in the kidneys of patients with type II diabetic nephropathy. Kidney Int 72: 1262–1272, 2007.[CrossRef][Web of Science][Medline]
  33. Verzola D, Gandolfo MT, Salvatore F, Villaggio B, Gianiorio F, Traverso P, Deferrari G, Garibotto G. Testosterone promotes apoptotic damage in human renal tubular cells. Kidney Int 65: 1252–1261, 2004.[CrossRef][Web of Science][Medline]
  34. Wolf G, Reinking R, Zahner G, Stahl RA, Shankland SJ. Erk 1, 2 phosphorylates p27(Kip1): Functional evidence for a role in high glucose-induced hypertrophy of mesangial cells. Diabetologia 46: 1090–1099, 2003.[CrossRef][Web of Science][Medline]
  35. Wolf G, Schroeder R, Zahner G, Stahl RA, Shankland SJ. High glucose-induced hypertrophy of mesangial cells requires p27Kip1, an inhibitor of cyclin-dependent kinases. Am J Pathol 158: 1091–1100, 2001.[Abstract/Free Full Text]
  36. Xu D, Finkel T. A role for mitochondria as potential regulators of cellular life span. Biochem Biophys Res Commun 294: 245–248, 2002.[CrossRef][Web of Science][Medline]
  37. Zhang X, Chen X, Wu D, Liu W, Wang J, Feng Z, Cai G, Fu B, Hong Q, Du J. Downregulation of connexin 43 expression by high glucose induces senescence in glomerular mesangial cells. J Am Soc Nephrol 17: 1532–1542, 2006.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/F1563    most recent
90302.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Verzola, D.
Right arrow Articles by Garibotto, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verzola, D.
Right arrow Articles by Garibotto, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.