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NDT Advance Access originally published online on October 2, 2007
Nephrology Dialysis Transplantation 2008 23(3):919-926; doi:10.1093/ndt/gfm674
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-{alpha} and interleukin-6 in type 2 diabetic patients

Juan F. Navarro1,2,3,, Carmen Mora2, Marina Gómez4, Mercedes Muros4, Celeste López-Aguilar2 and Javier García1

1 Nephrology Service, University Hospital Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain 2 Research Unit, University Hospital Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain 3 Spanish National Research Council, Madrid, Spain 4 Clinical Biochemistry Service, University Hospital Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain

Juan F. Navarro, Servicio de Nefrología, Hospital Universitario Nuestra Señora de Candelaria, Carretera del Rosario, 145,38010 Santa Cruz de Tenerife. Tel: +34-922-602061; Fax: +34-922-602349; E-mail: jnavgon{at}gobiernodecanarias.org



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Type 2 diabetes is associated with a high cardiovascular risk, which is even increased if renal damage is superimposed. Peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokines are key factors linking type 2 diabetes and atherosclerosis. We investigated the influence of renal damage on serum, urinary and PBMCs expression behavior of TNF-{alpha} and IL-6 in these patients.

Methods. PBMCs were isolated by density gradient centrifugation (Ficoll–Paque method) from fasting blood samples of 22 non-diabetic control subjects and 78 diabetic patients with normal renal function and different stages of diabetic nephropathy (18 with normoalbuminuria, 29 with microalbuminuria and 31 with macroalbuminuria). Expression levels of TNF-{alpha} and IL-6 were analyzed by real-time quantitative RT-PCR. Serum and urinary TNF-{alpha} and IL-6 concentrations were measured by a solid-phase, chemiluminescent immunometric assay.

Results. The mean percent increases in the serum and urinary levels of TNF-{alpha} and IL-6 in diabetic patients with respect to control subjects were 176% (P < 0.0001), 250% (P < 0.0001), 114% (P < 0.0001) and 39.6% (P = 0.01), respectively. The mRNA expression level of TNF-{alpha} was higher by 68.8% (P < 0.001) and IL-6 mRNA levels were higher by 64.1% (P < 0.001) with respect to non-diabetic controls. TNF-{alpha} mRNA expression in patients with macroalbuminuria was higher by 84.8% with respect to subjects with normalbuminuria (P < 0.001) and by 29% with respect to individuals with microalbuminuria (P < 0.05). Likewise, microalbuminuric patients showed a 44.5% increase in TNF-{alpha} mRNA expression compared to subjects with normoalbuminuria (P < 0.05). Concerning IL-6, the mRNA expression levels of this cytokine was higher by 63.1% with respect to normoalbuminuric subjects (P < 0.01), and by 23.1% with respect to patients with microalbuminuria (P < 0.05). However, with respect to controls, diabetic patients with normoalbuminuria had similar serum TNF-{alpha} and urinary excretion of IL-6, without any differences in the mRNA expression levels of these cytokines in PBMCs. Partial correlation and multiple regression analysis using TNF-{alpha} and IL-6 mRNA levels as the dependent variables showed that urinary albumin excretion (UAE) was direct and independently associated with the expression profile of these pro-inflammatory cytokines in PBMCs.

Conclusions. These data show for the first time the relationship between inflammatory activation of PBMCs (reflected by enhanced mRNA expression of TNF-{alpha} and IL-6) and renal involvement (reflected by increased UAE) in type 2 diabetic patients. These results provide potential insights for the increased inflammation, accelerated atherosclerosis and cardiovascular risk associated with nephropathy in type 2 diabetes.

Keywords: cytokines; diabetes mellitus; diabetic nephropathy; inflammation; mononuclear cells



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Diabetes mellitus is a major public health problem. A large body of epidemiological and pathological data documents that diabetes is a critical risk factor for the development of cardiovascular disease (CVD) [1–3], and furthermore, when patients with diabetes develop clinical CVD, they sustain a worse prognosis for survival than do CVD patients without diabetes [4–6]. This excess risk for CVD in diabetes cannot be explained solely by conventional risk factors. Therefore, the diabetic state, per se, confers an increased propensity to accelerated atherogenesis. However, the intimate mechanisms that participate in this process remain to be elucidated.

Nowadays, type 2 diabetes is considered an inflammatory CVD [7] with accumulating evidence indicating that inflammation, a central event in all stages of atherosclerosis, is the bridging link between type 2 diabetes and atherosclerosis [8–10]. Monocytes and macrophages are key cells in inflammation and they are the main cells responsible for innate immunity [11]. Infiltration of monocytes into the intima of the artery wall is an early hallmark of atherosclerosis, with activation of these cells resulting in the release of mediators of inflammation, including pro-inflammatory cytokines [12]. There is now evidence on the relevant contribution of pro-inflammatory cytokines, such as TNF-{alpha} and IL-6, to pathogenic (innate and adaptive) and regulatory immunity in the context of atherosclerosis [13].

Renal disease is a frequent and severe complication of diabetes [14]. Diabetic nephropathy is nowadays the principal cause of end-stage renal failure in the western world [15]. However, the importance of this complication is not only related to renal insufficiency. In addition, the development of nephropathy greatly increases cardiovascular risk, morbidity and mortality in patients with diabetes [16,17].

In spite of the role of pro-inflammatory cytokines in atherosclerosis, less is known about the serum, urinary and peripheral blood mononuclear cell (PBMC) expression profile of these molecules in type 2 diabetic patients with renal injury. The present study was undertaken to analyze in these patients with normal renal function and different grades of renal damage according to albuminuria status the serum concentrations and urinary excretion of TNF-{alpha} and IL-6, as well as the mRNA expression profile of these pro-inflammatory cytokines in PBMCs.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study subjects and characteristics
A sample size calculation to detect a 30% relative difference in the mRNA levels of TNF-{alpha} in PBMCs from type 2 diabetic patients with respect to non-diabetic subjects with an {alpha} value of 0.05 and a β value of 0.80 showed a need for a minimum of 17 diabetic subjects and 19 controls. Since no previous studies have been performed to assess the influence of renal involvement on cytokine gene expression among diabetic patients, we perform a sample size calculation to detect a 30% relative difference in the serum levels of TNF-{alpha} between type 2 diabetic subjects with and without nephropathy. This calculation (an {alpha} value of 0.05 and a β value of 0.80) showed a need for a minimum of 21 diabetic patients without nephropathy and 19 with microalbuminuria. Finally, 78 diabetic patients (40 males and 38 females; 18 normoalbuminuric, 29 microalbuminuric and 31 macroalbuminuric) and 22 age-, sex- and BMI-matched control non-diabetic individuals (13 males and 9 females) were included in the study. The protocol was in accordance with the Declaration of Helsinki and was approved by the committee, and all subjects gave their informed consent. Patients demographic characteristics are summarized in Table 1.


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  		Table 1 Demographic data of control subjects and diabetic patients (total group and classified according to urinary albumin excretion)

 
All individuals participating in the study were non-smokers between 43 and 82 years of age. All diabetic patients were insulin dependent and received treatment with acetylsalicylic acid and statins. Normoalbuminuric diabetic patients did not receive blockers of the renin–angiotensin system, whereas all patients with micro- or macroalbuminuria received treatment with blockers of the renin–angiotensin system for more than 6 months. All subjects had normal renal function, defined as a glomerular filtration rate higher than 60 ml/min, calculated using the Modification of Diet in Renal Disease study equation [18].

Before the definitive inclusion, the existence of immunologic diseases, malignancy and infections was investigated. White blood cell count was lower than 10 000/mm3 in all cases. Determination of tumoural markers including carcino-embryonic antigen, {alpha}-fetoprotein, cancer antigen 125 and prostate-specific antigen was negative. Serologic tests for antinuclear antibodies, antineutrophil cytoplasmic antibodies, rheumatoid factor, inmunoglobulins and complement were negative or within the normal range. Urine cultures and serology to hepatitis B, hepatitis C and human immunodeficiency virus were also negative.

General parameters
BMI was calculated as weight (kg)/height (m2). Arterial BP was measured by a mercury sphygmomanometer by the same observer with the patient in the sitting position after 5 min of rest. Three readings separated by 2 min were taken, and the average was used for calculation. The first appearance of sound (phase 1) was used to define systolic BP, and the disappearance of sound (phase 5) was used to define diastolic BP.

General biochemical variables and cytokines
Blood samples were drawn before breakfast in the morning (between 8 and 11 a.m.), after an 8- to 12-h overnight fast. Samples were collected in sterile tubes, centrifuged at 3000 g for 10 min at 4°C, and then stored at –80°C until assayed. The plasma glucose level was measured by an automated enzymatic method. The HbA1c concentration was measured by HPLC. The concentration of high-sensitive C-reactive protein (CRP) was measured by an ultra-sensitive competitive immunoassay (interassay coefficient of variation of 8.4%) (Calbiochem, La Jolla, CA, USA). Serum and urinary concentrations of TNF-{alpha} and IL-6 were measured by a solid-phase, chemiluminescent immunometric assay. The analytical sensitivity for TNF-{alpha} was 1.7 pg/ml, and the intra- and interassay coefficients of variability were 3.6% and 4.4%, respectively. Regarding IL6, the analytical sensitivity was 2 pg/ml, and the intra- and interassay coefficients of variability were 6.2% and 7.5%, respectively.

Urinary albumin excretion (UAE) was determined by 24-h urine collection. The samples were centrifuged at 3000 g for 10 min and the supernatant was stored at –20°C. Urinary albumin was quantified by immunoturbidimetry (coefficient of variation 5.5%). Normoalbuminuria was considered when the UAE rate was lower than 30 mg/24 h. Microalbuminuria was defined as a UAE rate of 30–300 mg/ 24 h, whereas a level of urinary albumin higher than 300 mg/ 24 h was defined as macroalbuminuria. ELISA was used for the detection of urinary TNF-{alpha} and IL-6, which were related to the concomitant urinary creatinine content to compensate for alterations caused by varying urinary concentration and is expressed as pg/mg.

Isolation of PBMCs and RNA extraction
PBMCs were isolated from whole venous blood (20 ml) collected into heparinated tubes and diluted with an equal volume of PBS. PBMCs were isolated by density gradient centrifugation (Ficoll–Paque method). For each sample, two 20-ml centrifuge tubes were used to layer 7 ml of diluted blood onto an equal volume of Ficoll–Hypaque. The suspension was centrifuged for 30 min at 450 g and 20°C. The mononuclear cell layer was removed with manual pipetting, washed twice with PBS and centrifuged for 10 min at 10°C and 275 g after each wash. Washed cells were resuspended in 1 ml of PBS. For RNA extraction, the PBMC suspension was centrifuged for 3 min at 3000 g, and the supernatant was discarded. Total RNA was isolated by using TRIreagent (Sigma-Aldrich, Steinheim, Germany). The concentration and purity of the recovered RNA were established by measuring ultraviolet absorbance at 260 and 280 nm. Total RNA integrity was checked by agarose gel electrophoresis and ethidium bromide staining.

Primers and real-time polymerase chain reaction
Expression levels of TNF-{alpha} and IL-6 were analyzed by real-time quantitative RT-PCR. The sequence of primers was designed for an annealing temperature of 60°C using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi), except for β-actin primers (a housekeeping gene) that also anneal at 60°C. The primers were used for relative quantification of targeted gene expression as follows. For TNF-{alpha}: forward primer, 5'-GCCACCACG- CTCTTCTGT-3'; reverse primer, 5'-GGCTACGGGCTTG- TCACTC-3'. For IL-6: forward primer, 5'-GTATGAACA- GCGATGATGCAC-3'; reverse primer, 5'-GAAACGGAA- CTCCAGAAGACC-3'. For β-actin: forward primer: 5'-TCCCTGGAGAAGAGCTACGA-3'; reverse primer, 5'-ATCTGCTGGAAGGTGGACAG-3'. Reactions were optimized to a final volume of 25 µl: 1X reaction buffer, 0.2 mM of each deoxynucleotide, 2.5 mM MgCl2, 0.15 µM of each primer, 1:100 000 SYBR Green I (Molecular Probes, Leiden, The Netherlands) and 0.4 U of HotStart DNA ‘Taq’ Polimerase (Ecogen/Bioline, Madrid, Spain). Fluorescein was added in a final concentration of 1:100 000 in order to normalize the differences in the amount of intercalating dyes caused by pippetting errors. RT-PCR amplification was performed on an iCycler iQ real-time PCR system (BioRad, Hercules, CA, USA) and analyzed with the provided software (iCycler software version 3.0). A three-step run protocol was used: (i) an initial hold of 9 min at 95°C to activate Taq polymerase and a fluorescence measurement to normalize the whole plate amount of fluorescein dye, (ii) an amplification and quantification cycle with 45 repeats (denaturation 20 s at 95°C, annealing 20 s at 60°C and extension 20 s at 72°C with a fluorescence measurement), (iii) a melting curve analysis from 65°C to 95°C to check the specificity of the amplified product. Agarose gel electrophoresis was performed to confirm that there were single-product amplifications without primer dimers. Additionally, all fragments were checked for specificity by direct sequencing of both strands with an ABI PRISM 310 genetic analyzer using Big Dye Terminator kit v 3.1 (Applied Biosystems, Foster City, CA, USA). Expression of cytokine mRNA for each sample was then expressed as an arbitrary ratio of the quantity of mRNA to that of β-actin as housekeeping gene.

Statistical analysis
Statistical analysis was conducted using Statistica 7.1 (Statsoft Inc., Tulsa, OK, USA). Results were expressed as mean ± SEM. Serum and urinary levels of TNF-{alpha} and IL-6, as well as expression ratios, were logarithmically transformed for statistical analyses and then back-transformed to their natural units for presentation in tables and figures. Statistical comparisons among groups were performed by Student's t-test for independent samples or analysis of variance (ANOVA) followed by post hoc analysis using Scheffe's test when appropriate. Simple regression analysis was performed using Pearson's correlation. Partial correlation analysis was performed to determine the extent to which a relationship was altered after adjusting for the other variables. Finally, a forward stepwise multiple regression analysis was performed to determine the independent association between potential predictor variables (age, sex, duration of diabetes, BMI, systolic and diastolic blood pressure, HbA1c, CRP, UAE, and serum and urinary TNF-{alpha} and IL-6) and the mRNA levels for TNF-{alpha} and IL-6 as the dependent variables. Colinearity was excluded by the analysis of the eigenvalue. A P value < 0.05 was considered to be statistically significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Demographic characteristics
In the diabetic group, the mean duration of diabetes was 11.3 ± 3.5 years, and the mean value of HbA1c was 7.4 ± 1%. Most of the subjects presented overweight or obese: 94.6% in the diabetic group and 81.1% in the control group, without differences in BMI between groups (Table 1). After stratification by albuminuria status (Table 2), patients with micro- and macroalbuminuria had diabetes for a longer time compared with normoalbuminuric subjects, whereas metabolic control (HbA1c concentration) was similar among the groups. Likewise, both systolic and diastolic BP were significantly higher in patients with macroalbuminuria with respect to those with microalbuminuria and normal UAE, but there was no difference in BP between normo- and microalbuminuric individuals. Finally, when compared with control non-diabetic subjects, diabetic patients without clinical signs of nephropathy (normal UAE) showed higher systolic (135.3 ± 4.2 versus 126 ± 5.2 mmHg, P < 0.0001) and diastolic BP (80.5 ± 6.5 versus 70.8 ± 4.5 mmHg, P < 0.0001).


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  		Table 2 Mean percent differences (95% confidence intervals) in the serum and urinary concentration of pro-inflammatory cytokines in diabetic patients with micro- and macroalbuminuria

 
Serum and urinary CRP and cytokines
Serum levels of CRP, TNF-{alpha} and IL-6, as well as the urinary excretion of these cytokines, were significantly higher in diabetic patients with respect to control subjects (Table 1). The mean percent increases in the serum and urinary levels of TNF-{alpha} and IL-6 in diabetic patients with respect to control subjects were 176% (P < 0.0001), 250% (P < 0.0001), 114% (P < 0.0001) and 39.6% (P = 0.01), respectively.

When diabetic patients were classified according to UAE, the serum levels of CRP and TNF-{alpha} were higher in micro- and macroalbuminuric subjects compared to normoalbuminuric individuals. However, normoalbuminuric and microalbuminuric patients had similar serum IL-6, whereas the concentration of this cytokine was significantly elevated in subjects with macroalbuminuria. Concerning urinary excretion of cytokines, there was no difference in the urinary level of IL-6 among the three groups. In contrast, urinary concentration of TNF-{alpha} was significantly higher in patients with macroalbuminuria with respect to subjects with microalbuminuria and normal UAE. Likewise, urinary TNF-{alpha} was also higher in subjects with microalbuminuria compared to subjects with normoalbuminuria (Table 2). The mean percent increase in serum and urinary cytokine concentrations in diabetic patients with micro- and macroalbuminuric with respect to subjects with normoalbuminuria is represented in Table 3.


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  		Table 3 Inflammatory parameters in diabetic patients stratified by urinary albumin excretion after multivariate ANCOVA analysis

 
When compared with control non-diabetic subjects, diabetic patients without clinical signs of nephropathy showed increased serum concentrations of IL-6 and PCR, as well as higher urinary excretion of TNF-{alpha}. However, serum TNF-{alpha} and urinary IL-6 were similar between groups.

RNA expression of TNF-{alpha} and IL-6
Mean levels of β-actin mRNA were similar among groups for each set of PCR reactions, confirming that starting concentrations of cDNA were similar and not subjected to systematic error.

Levels of expression of the pro-inflammatory cytokine genes were significantly increased in diabetic patients. The mRNA expression level of TNF-{alpha} was higher by 68.8% (P < 0.001) and IL-6 mRNA levels were higher by 64.1% (P < 0.001) with respect to non-diabetic controls (Figure 1). After classifying diabetic patients according to UAE, TNF-{alpha} mRNA expression in patients with macroalbuminuria was higher by 84.8% with respect to subjects with normalbuminuria (P < 0.001) and by 29% with respect to individuals with microalbuminuria (P < 0.05). Likewise, microalbuminuric patients showed a 44.5% increase in TNF-{alpha} mRNA expression compared to subjects with normoalbuminuria (P < 0.05). Concerning IL-6, the mRNA expression levels of this cytokine were higher by 63.1% with respect to normoalbuminuric subjects (P < 0.01), and by 23.1% with respect to patients with microalbuminuria (P < 0.05) (Figure 2). Diabetic patients with normal UAE had higher expression levels of TNF-{alpha} and IL-6 than control non-diabetic subjects, although the differences did not reach statistical significance.


Figure 1
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  		Fig. 1 mRNA expression of proinflammatory cytokine genes (TNF-{alpha} and Il-6) measured by RT-PCR in diabetic patients and non-diabetic control subjects (n = 22 versus 75). *P < 0.001

 

Figure 2
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  		Fig. 2 mRNA expression of proinflammatory cytokine genes (TNF-{alpha} and Il-6) measured by RT-PCR in non-diabetic control subjects (n = 22) and diabetic patients stratified according to urinary albumin excretion (normoalbuminuria = 15, microalbuminuria = 29, macroalbuminuria = 31). *P < 0.05 versus microalbuminuria; {dagger}P < 0.0001 versus normoalbuminuria and controls; {ddagger}P < 0.05 versus normoalbuminuria; §P < 0.01 versus controls; #P < 0.05 versus microalbuminuria; **P < 0.05 versus normoalbuminuria and controls.

 
Unadjusted correlation analysis
Expression level of TNF-{alpha} was significantly correlated with HbA1c, systolic BP, serum concentration of both TNF-{alpha} and IL-6, CRP, IL-6 mRNA levels, and with urinary excretion of TNF-{alpha} and albumina. Expression level of IL-6 was significantly correlated to time of diabetes, HbA1c, systolic BP, serum concentrations of CRP and TNF-{alpha}, TNF-{alpha} mRNA levels and UAE.

Partial correlation and multiple regression analysis
After adjusting for the effect of other variables by partial correlation analysis, the previous associations between TNF-{alpha} mRNA expression and HbA1c (r = 0.34, P < 0.01), UAE (r = 0.28, P < 0.05) and urinary TNF-{alpha} (r = 0.21, P < 0.05) remained significant, but not the relationship with the other variables. Likewise, the relationship between IL-6 mRNA expression and UAE (r = 0.35, P < 0.05), CRP (r = 0.32, P < 0.05), serum IL-6 (0.29, P = 0.01) and time of diabetes (r = 0.24, P < 0.01) remained significant. All correlation coefficients (r) and probability values for these correlations are summarized in Table 4.


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  		Table 4 Multivariate regression analysis of correlations between mRNA expression levels of TNF-{alpha} and IL-6 with other independent variables

 
Finally, multiple regression analysis was performed using TNF-{alpha} and IL-6 mRNA levels as the dependent variables. The result showed that from the many independent variables included in the analysis, HbA1c (P < 0.001), urinary TNF-{alpha} (P < 0.01), UAE (P < 0.01) and CRP (P < 0.05) were independently associated with TNF-{alpha} mRNA expression according to the following function (adjusted R2 = 0.55, P < 0.001): TNF-{alpha} mRNA expression = –1.41 + (0.25 x HbA1c) + (0.24 x EUA) + (0.22 x urinary TNF-{alpha}) + (0.21 x CRP). Likewise, UAE (P < 0.001), CRP (P < 0.001), serum IL-6 (P < 0.01) and time of diabetes (0.05) were the independent variables significantly associated with IL-6 mRNA expression according to the following function (adjusted R2 = 0.43, P < 0.01): IL-6 mRNA expression = 0.89 + (0.35 x UAE) + (0.32 x PCR) + (0.29 x serum IL-6) + (0.24 x time of diabetes).



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our data show that type 2 diabetes is associated with the presence of an inflammatory milieu, confirming the results from previous studies where these patients presented higher serum levels of inflammatory markers and pro-inflammatory cytokines [19–22]. Furthermore, also in accordance with previous works [23], our study shows that PBMCs from type 2 diabetic subjects show abnormal behavior, with a pro-inflammatory profile exhibiting increased mRNA expression of TNF-{alpha} and IL-6. However, there are no previous studies examining the relationship between biomarkers of inflammation and monocyte function in patients with type 2 diabetes and different stages of diabetic nephropathy. The present study shows a comprehensive report of the relevance of renal involvement in the development of the inflammatory profile in type 2 diabetic patients. When compared with healthy non-diabetic controls, diabetic patients with micro- or macroalbuminuria showed significantly elevated CRP levels, serum and urinary concentrations of TNF-{alpha} and IL-6, as well as increased expression of TNF-{alpha} and IL-6 genes in PBMCs. In contrast, and interestingly, diabetic patients without renal involvement (normal UAE) had similar serum levels of TNF-{alpha} and also a similar expression profile of TNF-{alpha} and IL-6 genes in PBMCs.

Diverse investigations have demonstrated that increased serum levels of TNF-{alpha} and IL-6 are powerful and independent predictors of CVD events and mortality [24–27]. Serum concentration of these pro-inflammatory cytokines has been reported to be elevated in diabetic subjects and even in patients with only impaired glucose tolerance, compared to healthy individuals [19–22,28]. In the present work, diabetic patients had approximately 2.8-fold higher serum TNF-{alpha} and 2.6-fold higher serum IL-6 than non-diabetic individuals. However, there were no significant differences in serum TNF-{alpha} between diabetic subjects with normoalbuminuria and non-diabetic controls. In fact, and confirming the previous results, serum TNF-{alpha} concentrations were increased only in diabetic patients with signs of nephropathy (micro- or macroalbuminuria) [21,29].

Data are scarce concerning the urinary excretion of TNF-{alpha} and IL-6 in type 2 diabetes and their potential relationship with vascular damage. In the present study, urinary concentrations of IL-6, and especially TNF-{alpha} (3.3-fold increase), were significantly elevated in diabetic patients with respect to non-diabetic subjects. In a previous study [30], the median urinary TNF-{alpha} concentration in 16 patients with DN was 14 (range 8–52) pg/ml, similar to the values found by us in the present work. When we compared the urinary level of these pro-inflammatory cytokines in patients classified according to UAE, where IL-6 concentration was similar among groups, urinary TNF-{alpha} increased as nephropathy progressed, but interestingly, without differences between diabetic patients with normoalbuminuria and control subjects. Similar results were observed in previous studies by our group [21,29]. Importantly, experimental studies have demonstrated the local production of TNF-{alpha} into the kidney [31,32], with a direct and independent association between renal TNF-{alpha} expression and urinary TNF-{alpha} excretion with the severity of microvascular renal injury in terms of UAE [21,29,31,32].

Previous studies examined the ex vivo responses of macrophages in db/db mice [33], showing that these cells displayed enhanced expression levels of the pro-inflammatory cytokines TNF-{alpha}, interleukin-1 (IL-1) and IL-6. Other works have shown that peripheral blood monocytes from both type 1 and type 2 diabetic patients are activated in poorly controlled diabetes, as well as in hypertension [34,35]. In the present study, the mean HbA1c concentration and BP were 7.4% and 139/85 mmHg in the whole diabetic group, values slightly higher than those recommended for adults with diabetes (7% and <130/80 mmHg, respectively) [36]. On the other hand, there were no significant differences in HbA1c values among diabetic patients classified according to UAE, whereas BP was higher only in patients with macroalbuminuria. Finally, although unadjusted correlation analysis showed that TNF-{alpha} and IL-6 mRNA expression were related to HbA1c and systolic BP, partial correlation and multiple regression analysis confirm only the association between TNF-{alpha} mRNA and HbA1c. In another study, Giulietti et al. [23] showed that circulating monocytes from type 2 diabetes patients were more prone to overexpression of TNF-{alpha} and interleukin-1 when stimulated by an immune stimulus. In those studies, only a limited number of type 2 diabetic subjects were included, and unfortunately, no data were reported about renal damage and the potential influence of this complication.

Diabetes is an independent risk factor for CVD [37]. However, the classical and known non-modifiable risk factors (age, sex, family history) cannot fully explain the excess risk for CVD in diabetes. Type 2 diabetes is now considered an inflammatory CVD [7], where activation of PBMCs is a relevant additional facet of the inflammatory profile of diabetic vascular disease [35]. Monocytes and macrophages are key cells in inflammation and atherogenesis. The activation and differentiation of these cells, with production of pro-inflammatory cytokines, are critical to develop and sustain an ongoing inflammatory process leading ultimately to lesion formation [38]. In addition, mononuclear cell activation is, among other factors, responsible for atherogenic plaque rupture [39]. In the present study, mRNA expression levels of TNF-{alpha} and IL-6 in the whole group of type 2 diabetic patients were significantly higher by 68.8% and 64.1% with respect to non-diabetic controls. However, diabetic patients without nephropathy (normal UAE) had expression levels of these pro-inflammatory cytokines similar to those in non-diabetic subjects. The significant increase in the mRNA expression of TNF-{alpha} and IL-6 was only observed in PBMCs from patients with micro- or macroalbuminuria. Furthermore, partial correlation and multiple regression analysis showed that UAE was an independent predictor of mRNA expression levels of TNF-{alpha} and IL-6.

Diabetic nephropathy is a very common complication of diabetes [14]. Microalbuminuria is the first clinical sign of diabetic damage to the kidney [40,41]. However, and importantly, albuminuria is a marker of greatly increased cardiovascular risk, morbidity and mortality, especially in type 2 diabetes [16,17]. The results of the present study suggest that inflammatory activation of PBMCs may be an important factor in the elevated cardiovascular risk associated with the development of renal damage in type 2 diabetes.

In conclusion, to the best of our knowledge, this is the first report about the association between inflammatory activation of PBMCs (reflected by enhanced mRNA expression of pro-inflammatory cytokines) and renal involvement (reflected by increased UAE) in type 2 diabetic patients. These results provide potential mechanistic insights for the increased inflammation, accelerated atherosclerosis and cardiovascular risk associated with nephropathy in type 2 diabetes.



   Acknowledgments
 
This work was supported in part by grants from the Sociedad Española de Nefrología (SEN), Asociación Científica para la Investigación Nefrológica (ACINEF) y Fundación Canaria de Investigación y Salud (FUNCIS).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 9. 4.07
Accepted in revised form: 4. 9.07


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M. A. Niewczas, L. H. Ficociello, A. C. Johnson, W. Walker, E. T. Rosolowsky, B. Roshan, J. H. Warram, and A. S. Krolewski
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