Acute-Phase Markers of Inflammation and Glomerular Structure in Patients with Type 2 Diabetes

Patients

Seventy-four patients with type 2 diabetes were studied. Exclusion criteria were serum creatinine >180 μmol/L, presence of obvious nondiabetic renal diseases, and secondary causes of hypertension, including known renal artery stenosis and endocrinopathies. Patients were defined as microalbuminuric when albumin excretion rate (AER) was between 20 and 200 μg/min in at least two of three sterile 24-h urine collections and as proteinuric when AER was >200 μg/min. Patients were defined as being hypertensive when BP values were >130/85 mmHg or when antihypertensive therapy was used (angiotensin-converting enzyme inhibitors or angiotensin receptors blockers alone or associated with dihydropyridine calcium antagonists and/or diuretics and α- and β-blockers). Patients were admitted to the Department of Medical and Surgical Sciences at the University of Padova, where baseline evaluation were performed (medical history, physical examination, renal functional studies, and kidney biopsy). All patients gave their written informed consent before the study. This study protocol was approved by the Ethics Committee of the University of Padova.

Methods

In all patients, venous blood samples were collected in the fasting state for determination of fibrinogen, leukocytes, and erythrocyte sedimentation rate (ESR); sera of 55 patients were available for SAA, CRP, and IL-6 determination.

AER was measured by an immunoturbidimetric method on three sterile 24-h urine collections. GFR was measured by modeling analysis of plasma decay of ⁵¹Cr-EDTA as described elsewhere (16). The normal range in a group of 19 age and gender-matched normal control subjects was 85 to 135 ml/min per 1.73 m². BP was measured at least 10 times with the patients in the supine position, and the values provided the mean of these repeated measurements. HbA_1c was measured by HPLC (Variant II Analyzer; Bio-Rad, Hercules, CA).

Plasma fibrinogen levels were measured using a commercially available assay kit based on clotting method of Von Clauss. Serum SAA and CRP were measured by a particle-enhanced nephelometric immunoassay on a fully automated BN II nephelometer system (Dade Behring, Milan, Italy) (17). Both assays are based on an immune reaction involving specific antibodies, covalently coated with core shell-type particles. For SAA, the coefficient of variation ranges from 3.3 to 5.0% (within-run) and 3.1 to 6.9% (between-run). The reference limit is 6.8 mg/L. ESR was measured by the capillary photometric-kinetic technology on the Microtest 1 (SIRE Analytical System, Udine, Italy). Reference limits for men and women are as follows: from 15 to 50 yr of age, 28 and 38 mm, respectively; from 51 to 70 yr, 37 and 39 mm, respectively (18). Quantitative measurement of IL-6 was done by a solid-phase, enzyme-labeled, chemiluminescent sequential immunometric assay on a Immulite 1000 analyzer (Medical System, Genova, Italy). The coefficient of variation within and between run ranges from 5.1 to 7.5%.

A percutaneous kidney biopsy was performed under ultrasound guidance in all patients. Tissue was immediately processed for light, electron, and immunofluorescence microscopy. Electron microscopic examination was conducted on tissue that was fixed in 2.5% glutaraldehyde in Millonig buffer and fixed in Polybed (19). At least three glomeruli for each biopsy were examined. Glomeruli were photographed at a magnification of ×3900 to produce photomontages of the entire glomerular profile. The montages were used to estimate mesangial fractional volume [Vv(mes/glom)] by point counting (normal values, 0.19 ± 0.03) (19). Another set of micrographs, obtained at ×12,000 magnification, by systematically sampling approximately 20% of the glomerular profile, was used to estimate glomerular basement membrane (GBM) width by the orthogonal intercept method (19) (normal values, 320 ± 38 nm in 26 age- and gender-matched normal kidney donors). None of these patients had light, immunofluorescent, or electron microscopy findings of any definable renal disease other than diabetic nephropathy.

Statistical Analyses

Data are expressed as mean ± 1 SD or as median and interquintile range. For checking the Gaussian distribution, data were preliminary evaluated by applying the Kolgomorov-Smirnov test, taking P < 0.001 as significant. AER values, not normally distributed, were logarithmically transformed before analyses. Data were analyzed using the SPSS statistical package for Macintosh. One-way ANOVA was used to compare mean values among groups, followed by least significant difference tests for multiple comparisons. Univariate and multivariate regression analysis was used to evaluate the relationships between the variables studied. Values for P < 0.05 were considered significant.

Patients were divided into two groups on the basis of their GBM width: those with GBM width in the normal range (≤396 nm = mean + 2 SD in normal controls) and those above. Mann-Whitney U test and Kruskal-Wallis test were used to compare the results in the two groups.

We divided the distributions of variables into quintiles and used regression models to estimate the significance of trend in relative risk (RR) across increasing quintiles and to estimate risk for having GBM width above normal in each quintile relative to the lowest quintile. Relative risk is defined as the increased risk in one study group compared with the risk in another group. The formula to obtain relative risk is as follows: RR = [TP/(TP+FN)]/[FP/(FP+TN)], where TP is true positive, FN is false negative, FP is false positive, and TN is true negative.

Relationships between Renal Function and Inflammatory Markers

Twenty-three patients were normoalbuminuric (AER, 6 μg/min [median, range, 2 to 18 μg/min]), 30 were microalbuminuric (AER, 76 μg/min [range, 20 to 175 μg/min]), and 21 were proteinuric (AER, 646 μg/min [range, 215 to 3958 μg/min]). Table 1 summarizes the clinical features of patients. Age, diabetes duration, GFR, and BP were similar in the three groups; in contrast, HbA_1c was higher in proteinuric than in normoalbuminuric patients. GBM width and Vv(mes/glom) increased from normoalbuminuric to microalbuminuric and proteinuric patients [P < 0.005 normoalbuminuric and microalbuminuric versus proteinuric for GBM width and P < 0.01 normoalbuminuric versus proteinuric for Vv(mes/glom)].

In Table 2, the inflammatory markers are summarized: ESR, CRP, fibrinogen, SAA, and IL-6 differed among groups, with proteinuric patients having the highest levels. In contrast, white blood cell count was not significantly different in the three groups.

AER was directly correlated to both GBM width (r = 0.48, P < 0.001) and Vv(mes/glom) (r = 0.39, P < 0.005); AER was also positively correlated with fibrinogen (r = 0.47, P < 0.001), SAA (r = 0.28, P < 0.03), and HbA_1c (r = 0.26, P < 0.05). When HbA_1c and inflammatory markers were included in a multiple regression model, only fibrinogen and HbA_1c explained AER (r = 0.57, P < 0.02, β 0.32 for fibrinogen; P < 0.05, β 0.24 for HbA_1c). GFR was not significantly correlated to any inflammation marker.

Relationships between Glomerular Structure and Inflammatory Markers

Patients were divided into two groups on the basis of their GBM width: those with GBM width in the normal range (≤396 nm) and those above. AER was significantly higher in patients with GBM above normal (99 μg/min [14 to 518 μg/min] versus 37 μg/min [12 to 104 μg/min]; P < 0.05), whereas GFR was not significantly different (92 versus 108 ml/min per 1.73 m²). Patients with GBM >396 nm, compared with patients with GBM in the normal range, had higher levels of CRP (4.6 mg/L [3.1 to 11.5 mg/L] versus 3.1 mg/L [3.0 to 5.6 mg/L]; P < 0.003), SAA (5.4 mg/L [1.5 to 12 mg/L] versus 2.3 mg/L [1.0 to 8.1 mg/L]; P < 0.004), and IL-6 (3.7 ng/L [2.0 to 8.0 ng/L] versus 2.0 ng/L [2.0 to 4.4 ng/L]; P < 0.0004). The RR to have abnormal GBM width was in the highest quintile 2.96 for IL-6, 2.97 for SAA, and 2 for CRP.

Linear regression analysis showed that GBM width was related to diabetes duration (r = 0.38, P < 0.002), fibrinogen (r = 0.33, P < 0.002), and IL-6 (r = 0.25, P < 0.05). Vv(mes/glom) was not significantly related to any acute-phase marker.