Urinary matrix metalloproteinase -8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in patients with diabetic nephropathy

Figures & data

Table

CMT	chemically modified non-antimicrobial tetracycline
DM	diabetes mellitus
DNP	diabetic nephropathy
DU	densitometric unit
ECM	extracellular matrix
ELISA	enzyme-linked immunosorbent assay
IFMA	immunofluorometric assay
LDD	low-dose doxycycline
MMP	matrix metalloproteinase
MMP-2	matrix metalloproteinase-2
MMP-8	matrix metalloproteinase-8
MMP-9	matrix metalloproteinase-9
MMP-14	matrix metalloproteinase-14
MT-MMP	membrane-type MMP
PMN	polymorphonuclear neutrophil
RRT	renal replacement therapy
TIMP	tissue inhibitor of matrix metalloproteinases
TRY-1	trypsin-1
TRY-2	trypsin-2
TATI	tumor-associated trypsin inhibitor

Table I.  Clinical characteristics of the diabetic nephropathy patients.

Patient number	Age (years)	Sex	Type of DM	Duration of DM (year)	Anti-DM drug	S-crea (µmol/L)	dU-prot (mg)	Statin	ACEI/ARB	CCB	Number of anti-HT
1	33	male	type I	19	ins	211	<200	0	1	1	3
2	56	male	type II	3	oral	252	10,610	1	1	0	2
3	69	male	type II	20	oral	119	13,470	0	0	0	0
4	57	male	type II	3	0	180	12,000	0	0	1	1
5	30	female	type I	16	ins	131	780	0	0	1	2
6	65	male	type II	15	ins	226	8,411	0	1	0	2
7	33	male	type I	18	ins	129	4,847	0	1	0	1
8	44	male	type II	10	ins	90	6,061	0	1	0	1
9	72	male	type II	15	oral	121	3,567	0	1	0	1
10	48	Female	type II	9	ins	307	5,278	0	1	1	3

DM = diabetes mellitus; ins = insulin; oral = oral drug; 0 = no; 1 = yes; S-crea = serum creatinine concentration (reference values: male <115 µmol/L; female <100 µmol/L); dU-prot = diurnal urinary protein content; ACEI = angiotensin-converting enzyme inhibitor; ARB = angiotensin receptor blocker; CCB = calcium channel blocker; anti-HT = antihypertensive drug.

Figure 1.  Determination of total MMP-8 immunoreactivity by Western blotting in urine. The concentrations (expressed as densitometric units (DU)) were significantly higher (P < 0.001) in patients with diabetic nephropathy (DNP) than in healthy controls.

Figure 2.  The proportion of active polymorphonuclear neutrophil (PMN) type MMP-8 determined by Western blotting in urine was significantly higher (P = 0.002) in diabetic nephropathy (DNP) patients than in healthy controls.

Figure 3.  Western blotting of collagenase-2 (MMP-8) (panel A), zymography and Western blot of gelatinase (MMP- 2 and -9) (panel B) and membrane-type I matrix metalloproteinase (MMP-14, panel C) in urine from DNP patients (lanes 1–3, DNP) and healthy controls (lanes 4–6, cont). Also in DNP, but not in urine of healthy control, high molecular weight (>80 kDa) MMP-8 and (>92 kDa) MMP-9 complexes could be detected. Human PMN- and rheumatoid synovial fibroblast-culture media were used as positive control for PMN- and mesenchymal (Mes)-type MMP-8 isoforms and for MMP-9 and -2. Also pure human MMP-2, -9, and -14 were used as positive controls. Mobilities of the molecular weight markers are indicated on the left.

Figure 4.  Total immunoreactivity of urinary MMP-14 (MT1-MMP) determined by Western blotting. The concentrations in diabetic nephropathy (DNP) patients were significantly higher (P < 0.05) than in healthy controls.

Figure 5.  Concentrations of TRY-2 and TRY-1 in urine. In diabetic nephropathy (DNP) patients the concentrations of TRY-2 (panel A) and TRY-1 (panel B) were significantly higher than in healthy controls (P < 0.001 and P<0.01, respectively).

Table II.  Gelatinolytic activity, percent active MMP-9 and TATI in urine of patients with diabetic nephropathy, and of healthy controls. Data are presented as median (25%–75% percentiles).

	DM nephropathy	Controls	Significance
Gelatinolytic activity	15.1 (1.2–25.7)	0 (0–0.8)	0.01
Percent active MMP-9	12 (0–44)	0 (0–0.1)	<0.05
TATI (µg/L)	21.6 (9.3–148.6)	10.3 (7.2–14.3)	NS

DM = diabetes mellitus; MMP = matrix metalloproteinase; NS = not significant; TATI = tumor-associated trypsin inhibitor.

Table III.  Correlations between active urinary MMP forms and TRY-2, TRY-1 and TATI in urine of DNP patients (n = 10).

	TRY-2	TRY-1	TATI
% active PMN MMP-8, Spearman correlation coefficient	0.705	0.720	0.778
Significance, P value, two-tailed	<0.05	<0.05	<0.01
% active fibroblast MMP-8, Spearman correlation coefficient	−0.231	−0.202	−0.306
Significance, P value, two-tailed	NS	NS	NS
% active MMP-9, Spearman correlation coefficient	0.679	0.577	0.782
Significance, P value, two-tailed	<0.05	NS	<0.01

DNP = diabetic nephropathy; MMP = matrix metalloproteinase; NS = not significant; PMN = polymorphonuclear neutrophil; TATI = tumor-associated trypsin inhibitor; TRY = trypsin.