The expression of two isoforms of matrix metalloproteinase-2 in aged mouse models of diabetes mellitus and chronic kidney disease

Abstract

Background: This study was undertaken to explore the effects of aging on the kidneys in mouse models of diabetes and chronic kidney disease (CKD), and to compare the expression of two isoforms of matrix metalloproteinase-2 (MMP-2)-secretory full-length MMP-2 and intracellular N-terminal truncated MMP-2 (NTT-MMP-2)-in these models.

Methods: Two experimental ICR mouse models were used: a streptozotocin (STZ)-induced type 1 diabetes mellitus model and a 5/6 nephrectomized (5/6Nx) CKD model. The abundance of each isoform of MMP-2 was determined by quantitative polymerase chain reaction (qPCR), and functional analyses were conducted. Moreover, the protein levels of the two MMP-2 isoforms were determined semi-quantitatively by immunohistochemical staining, and their association with tissue damage was assessed.

Results: Both isoforms of MMP-2 were upregulated in the kidney tissues of STZ-induced diabetic mice and 5/6Nx mice, irrespective of age. Characteristically, NTT-MMP-2 protein expression was elevated in old control mice, in line with the qPCR results. NTT-MMP-2 expression was limited to the renal cortex, and to the tubulointerstitial area rather than the glomerular area. In terms of tissue damage, tubulointerstitial fibrosis was more severe in old 5/6Nx mice than in their young counterparts, whereas glomerulosclerosis was comparable in old and young 5/6Nx mice.

Conclusion: The intracellular isoform of MMP-2 was induced by ageing, irrespective of the presence of diabetes or CKD, and its induction may be related to tubulointerstitial fibrosis in chronic kidney disease.

Keywords: Aging; Chronic renal insufficiency; Diabetes mellitus; Matrix metalloproteinase-2.

Figure 1. Renal function and qPCR measurement of FL-MMP-2 and NTT-MMP-2 transcript levels in streptozotocin-induced diabetic mice and 5/6 nephrectomized (5/6Nx) mice

The serum creatinine levels and urinary albumin to creatinine ratios were evaluated in the four groups of mice in each experiment, and qPCR was performed on transcripts isolated from whole kidneys in each group. (A–D) Group I, young control group (Y-C); Group II, old control group (O-C); Group III, young diabetes mellitus group (Y-DM); Group IV, old diabetes mellitus group (O-DM). (E–H) Group I, young control group (Y-C); Group II, old control group (O-C); Group III, young 5/6 nephrectomy group (Y-5/6Nx); Group IV, old 5/6 nephrectomy group (O-5/6Nx). n = 5 for each group; *P < 0.05 compared with young controls. MMP-2, matrix metalloproteinase-2; FL-MMP-2, full-length MMP-2; NTT-MMP-2, N-terminal truncated MMP-2; qPCR, quantitative polymerase chain reaction.

Figure 2. Histological assessment of kidneys in the streptozotocin model of type 1 diabetes mellitus

(A–J) Immunohistochemical staining for FL-MMP-2 and NTT-MMP-2: (A–D) FL-MMP-2 was faintly detectable in young and old control mice, and its expression increased with the induction of diabetes (×10); (E–H) NTT-MMP-2 was faintly detectable in old control mice, and its expression increased with the induction of diabetes; (I, J) semi-quantitative scoring of IHC staining (n = 5, *P < 0.05 compared with young controls). (K) Detailed microscopic findings (×200). The first row presents that interstitial fibrosis was observed in old control mice and diabetic mice in Masson’s trichrome (MT) staining. The second row presents that tubular injury was noted in old mice. In young diabetic mice, tubular injuries including tubular cast (black arrow) and dilatation (yellow arrow) were observed, and these findings were aggravated in old diabetic mice in periodic acid-Schiff (PAS) staining. The third row presents that FL-MMP-2 expression was stronger in diabetic mice than in control mice, irrespective of ageing status. The last row presents that NTT-MMP-2 expression was stronger in diabetic mice than in young control mice. Interestingly, in old control mice, NTT-MMP-2 was expressed focally and at a higher intensity than in young control mice (black arrow). MMP-2, matrix metalloproteinase-2; FL-MMP-2, full-length MMP-2; NTT-MMP-2, N-terminal truncated MMP-2.

Figure 3. Histological assessment of kidneys in 5/6 nephrectomized (5/6Nx) mice

(A–H) Immunohistochemical staining of the kidneys for FL-MMP-2 and NTT-MMP-2 in the 5/6Nx mouse model: (A–D) The levels of FL-MMP-2 were comparable in young and old 5/6Nx mice. There were stained areas in the glomeruli and renal tubules (×100 and ×400); (E–H) The levels of NTT-MMP-2 were comparable in young and old 5/6Nx mice. The principal stained areas were in the renal tubules (×100 and ×400). (I) Glomerular and tubulointerstitial damage in young and old 5/6Nx mice (×400). Upper panel: periodic acid-Schiff (PAS) and Masson’s trichrome (MT) staining in young 5/6Nx mice. There were segmental sclerotic areas in some glomeruli, and there was inflammation with fibrosis and tubular injury in the tubulointerstitial area. Lower panel: PAS and MT staining in old 5/6Nx mice. There were segmental sclerotic areas in the glomeruli, as in the young 5/6Nx model. However, tubulointerstitial fibrosis was more prominent in old 5/6Nx mice (yellow arrow). (J) Comparison of the characteristics between young and old 5/6Nx mice. There were no differences in the remnant percent of the renal parenchyma, the percent growth rate for 1 month and the glomerulosclerosis index between the groups. The only significant finding was that old 5/6Nx mice had more fibrotic areas in the tubulointerstitial area than young 5/6Nx mice (n = 5, *P < 0.05). MMP-2, matrix metalloproteinase-2; FL-MMP-2, full-length MMP-2; NTT-MMP-2, N-terminal truncated MMP-2.