Fifty control subjects and 93 subjects with type 1 diabetes were evaluated. Control and type 1 diabetic groups were comparable with respect to sex (52 vs. 47% female, respectively); racial distribution (84 vs. 91% Caucasian), and baseline BMI (25.2 ± 4.8 vs. 24.8 ± 4.4 kg/m2). The diabetic subgroup was slightly younger (control 24.1 ± 6.8 years vs. type 1 diabetic 19.3 ± 6.3 years; P < 0.001). Therefore, additional analyses, as detailed below (see RELATIONSHIP OF MMP-2 TO AGE) were conducted to examine any potential effect of age on MMP-2 results.
Expected differences between the control and type 1 diabetic subgroups were confirmed by baseline measurements of FPG (81.2 ± 6.5 vs. 155.3 ± 65.1 mg/dl, respectively; P < 0.001), A1C (4.97 ± 0.3 vs. 8.49 ± 1.85%; P < 0.001), C-peptide (0.84 ± 0.49 vs. 0.14 ± 014 ng/ml; P < 0.001), and 3- to 5-day average glucose by the CGMS (88.0 ± 10.2 vs. 170.5 ± 47.7 mg/dl; P < 0.001). Of the type 1 diabetic subjects, 59% were being treated with insulin injections, and 41% were using insulin pumps.
A history of hypertension (n = 4), retinopathy (n = 5), nephropathy (n = 3), or neuropathy (n = 0) was ascertained by self-report. During the baseline physical examination and as a result of the 24-h urine collection, an additional three subjects demonstrated evidence of sensory neuropathy, and nine subjects were identified as having microalbuminuria. (Dilated funduscopic examination was not a component of the protocol.) Among the type 1 diabetic subjects, the median urinary albumin excretion (UAE) was 12.2 mg/g creatinine and microalbuminuria (30–299 mg/g creatinine) was present in 12 subjects (microalbuminuria range 30.4–280.3 mg/g creatinine). No subjects in this study displayed macroalbuminuria (≥300 mg/g creatinine).
MMP-2, TIMP-1, and TIMP-2 concentrations in biological fluids
MMP-2 concentrations were significantly elevated in the urine and plasma of type 1 diabetic subjects compared with control subjects. The increase in urine MMP-2 was apparent whether analyzed as 1) an undiluted urine concentration (control 48.4 ± 10.2 [mean ± SEM] vs. type 1 diabetic 184.9 ± 31.3; P < 0.001), 2) a urine MMP-2–to–urine creatinine ratio (), or 3) total MMP-2 excretion per day for the 135 of 143 subjects who provided a complete 24-h urine sample (). When all study subjects were analyzed together, a weak correlation between plasma MMP-2 and urine MMP-2–to–creatinine ratios was seen (). Similar to MMP-2 protein concentrations, MMP-2 activity was also significantly increased in the plasma of type 1 diabetic subjects (control 193 3 ± 163.0 vs. type 1 diabetic 292.7 ± 190.2 ng/ml; P < 0.005). Moreover, MMP-2 activity in plasma was correlated both with plasma MMP-2 concentrations (R = 0.453, P < 0.001) and urine MMP-2–to–creatinine ratios (R = 0.331, P < 0.001). No sex differences were seen for urine or plasma MMP-2 values.
Plasma and urine MMP-2 concentrations correlation with glycemic control and renal function
The activity of MMPs is tightly regulated by a family of TIMPs (TIMPs 1–4); MMP-2 preferentially binds TIMP-2, whereas MMP-9 preferentially binds TIMP-1. To determine whether the increase in plasma MMP-2 concentrations was offset by a simultaneous upregulation of inhibitor, plasma concentrations of TlMP-1 and TIMP-2 were measured. Neither TlMP-1 nor TIMP-2 differed between the type 1 diabetic and control groups (TlMP-1 88.7 ± 30.2 [mean ± SEM] vs. 93.2 ± 29.0 ng/ml respectively; TIMP-266.2 ± 25.2 vs. 66.5 ± 19.5 ng/ml, respectively).
Relationship of MMP-2 to age
When age was examined as a continuous variable, values for the urine MMP-2–to–creatinine ratio and total MMP-2/day, but not plasma MMP-2, were very weakly inversely correlated with age (MMP-2–to–creatinine ratio R = −0.181, P < 0.05; total MMP-2/day R = −0.202, P < 0.05). However, when MMP-2 values were compared in subjects who were aged ≤18 years as a group (n = 64) with those who were aged 19–40 years (n = 79), plasma MMP-2 concentrations were significantly higher among the younger cohort, whereas the urine MMP-2–to–creatinine ratio and total MMP-2/day displayed a trend toward higher values in younger subjects but did not reach statistical significance. To exclude the possibility that the younger mean age of the type 1 diabetic subgroup alone accounted for the increase in MMP-2 values seen in type 1 diabetes, comparisons were made between the subset of type 1 diabetic subjects and control subjects who were aged 19–40 years (adult subgroup: control, n = 42; age [mean ± SEM] 25.8 ± 0.9 years; type 1 diabetic, n = 37: age 24.8 ± 1.1 years; between-group age comparison, P = 0.3). A similar comparison was made between the subset of type 1 diabetic subjects and control subjects who were aged ≤18 years (adolescent subgroup: control, n = 8, age 15.5 ± 0.3 years; type 1 diabetic, n = 56, age 15.6 ± 0.2 years). Consistent with differences reported for the entire study population, for subjects aged 19–40 years, urine concentrations of MMP-2, the urine MMP-2–to–creatinine ratio, and total MMP-2/day were significantly higher among subjects with type 1 diabetes (), For subjects aged 14–18 years, a trend toward higher values in the type 1 diabetic subjects was also evident, although these differences did not attain statistical significance. Plasma MMP-2 concentrations were significantly higher in type 1 diabetic subjects compared with control subjects for the entire study population and for the adolescent subgroup ().
Relationship of MMP-2 to duration of disease
Among subjects with type 1 diabetes, values for the urine MMP-2–to–creatinine ratio and total MMP-2/day were elevated in subjects with a duration of disease of >3 years (n = 70), compared with those with a ≤3 year history of type 1 diabetes (n = 23). Specifically, for subjects with duration of diabetes of >3 vs. ≤3 years, median MMP-2–to–creatinine ratio concentrations were 113.4 vs. 29.8 pg/g, respectively (P < 0.05), and median total MMP-2/day values were 151,766 vs. 45,466 pg/day (P < 0.05), This difference was seen despite the fact that the mean age of type 1 diabetic subjects with a longer duration of disease was slightly older (20.2 ± 0.8 years) than the mean age of type 1 diabetic subjects with a ≤3 year history of type 1 diabetes (16.7 ± 0.6 years). In contrast, plasma MMP-2 concentrations were not different between those subjects with duration of type 1 diabetes of >3 vs. ≤3 years.
Relationship of MMP-2 to glycemic control
Values for the urine MMP-2–to–creatinine ratio and total MMP-2/day were correlated positively with A1C and CGMS average daily glucose and less strongly correlated with FPG (). Moreover, urine MMP-2–to–creatinine ratio and total MMP-2/day were significantly higher in type 1 diabetic subjects whose A1C was ≥8.25% vs. <8.25% (P < 0.001 for both urine values) and in subjects whose CGMS average daily glucose was ≥ 140 vs. < 140 mg/dl(P< 0.001 for both urine values). Urine MMP-2 relationships also differed as a function of sex; specifically, correlations with FPG, A1C, and CGMS average daily glucose were stronger in female subjects (). In contrast, no correlations between plasma MMP-2 concentrations and A1C, FPG, or CGMS average daily glucose were demonstrated. However, plasma MMP-2 activity was weakly correlated with A1C (R = 0.249, P = 0.004) and CGMS average daily glucose (R = 0.318, P < 0.001).
Relationship of MMP-2 to renal function
For subjects with type 1 diabetes, values For the urine MMP-2–to–creatinine ratio and total MMP-2/day were correlated positively with UAE, GFR, and CrCl (). Again, these relationships were strongest among women. Similar correlations with UAE, GFR, or CrCl were not demonstrated for plasma MMP-2 concentrations, although weak correlations were demonstrated between MMP-2 activity in plasma and UAE (R = 0.278, P = 0.001) or GFR (R = 0.278, P < 0.001).
To examine the relationship between urinary concentrations of MMP-2 and predictors of diabetic nephropathy, values for the urine MMP-2–to–creatinine ratio and total MMP-2/day were compared in diabetic subjects with UAE of <30 mg/g creatinine (n = 76) and in those with microalbuminuria (≥30 mg/g creatinine; n = 12). A statistically significant increase was seen in the urine MMP-2–to–creatinine values (UAE <30 mg/g creatinine [mean ± SEM]: 169.9 ± 23.7 pg/g; UAE ≥30 mg/g creatinine: 622.4 ± 172.3 pg/g; P = 0.02) and in total MMP-2/day (UAE <30 mg/g creatinine: 252,892 ± 41,532 pg/ml; UAE ≥30 mg/g creatinine: 678,663 ± 177,584; P = 0.003). Urine MMP-2–to–creaimine ratios were also compared by subset for both control and type 1 diabetic subjects with UAE of 1) <10 mg/g creatinine, 2) 10–30 mg/g creatinine, or 3) > 30 mg/g creatinine (type 1 diabetic subjects only, as dictated by study exclusion criteria for control subjects). MMP-2–to–creatinine ratios were as follows: 1) UAE <10 mg/g creatinine: control 31.7 ± 7.7 vs. type 1 diabetic 108.5 ± 37.6 pg/g; P = 0.01; 2) UAE 10–30 mg/g creatinine: control 81.6 ± 26.7 vs. type 1 diabetic 205.2 ± 31.1 pg/g; P = 0.01; and 3) UAE >30 mg/g creatinine: type 1 diabetic 718.4 ± 171.8 pg/g; P < 0.001 for comparison with both control subsets. A progressive increase in the urine MMP-2–to–creatinine ratio concentrations was seen among type 1 diabetic subjects, with the increase in MMP-2 concentration noted even when the UAE was not yet clinically abnormal.
Urine MMP-2–to–creatinine ratios were further examined among the subset of type 1 diabetic subjects who demonstrated evidence of hyperfiltration. Urine MMP-2–to–creatinine ratios were higher (P < 0.05) in those subjects with type 1 diabetes and GFR > 130 ml/min per 1.73 m2 (n = 25; 405.4 ± 100.0 pg/g) compared with those with GFR ≤ 130 ml/min per 1.73 m2 (n = 68; 158.4 ± 22.5 pg/g) or CrCl >130 ml/min (300.2 ± 63.8) compared with those with CrCl ≤130 ml/min (165.3 ± 29.7). Among those type 1 diabetic subjects with GFR > 130 ml/min per 1.73 m2, an increase in urine MMP-2–to–creatinine ratios was seen for those with microalbuminuria (UAE <30 mg/g creatinine: 229.7 ± 61.1 pg/g; UAE ≥30 mg/g creatinine: 853.5 ± 218.3 pg/g; P < 0.05).
Multiple regression analysis
Tree analysis demonstrated that if the urine MMP-2–to–creatinine ratio was >65 pg/g or if the urine total MMP-2/day value was >66,720 pg/ml, markers for elevated risk, such as hyperglycemia, hyperfiltration, and microalbuminuria, could be established ().