Renal Protective Effect And Mechanism Of Matrix Metalloproteinase 12 Expression On Diabetic Nephropathy And High-fat-diet Induced Kidney Injury

The global incidence of obesity is increasing; currently, there are > 1billion adults who are overweight, of which at least 300 million are clinically obese(i.e., body mass index > 30 kg/m2). As an inflammatory disease, obesity is a major contributing factor in metabolic syndromes, cardiovascular disease,and diabetes. More than 130 million people struggle with diabetes in China, as one of the major diabetic microvascular complications, about one third of patients with diabetes will develop diabetic nephropathy(DN), which is the first cause of end stage renal disease(ESRD) closely associated with mortality.Clinical and experimental studies have shown that obesity is an independent risk factor for chronic kidney disease(CKD) and end-stage renal disease.Hyperglycemia, dyslipidemia, and oxidative stress-all pathological processes in obesity-induced kidney disease—contribute to albuminuria and progressive decline in renal function that ultimately lead to glomerulosclerosis and renal fibrosis. The latter is characterized by excessive extracellular matrix(ECM)accumulation and occurs in virtually every type of CKD. The current model of renal fibrogenesis describes a process analogous to the wound-healing response to injury. In response to renal injury and inflammation, resident kidney cells and infiltrating inflammatory monocytes/macrophages and T cells activate and produce toxic molecules such as reactive oxygen species(ROS)in addition to fibrogenic and inflammatory cytokines. These in turn activate mesangial cells, fibroblasts, and tubular epithelial cells that secrete ECM components. Excessive fibrosis results in the permanent loss of normal kidney function.Objective: Matrix metalloproteinase 12(MMP-12), also named macrophage metalloelastase, was first identified as an elastolytic metalloproteinase secreted by inflammatory macrophages; it is now known to be expressed in hypertrophic osteoclasts, vascular smooth muscle cells, and some cancer cell types. MMP-12 can degrade not only its major substrate elastin, but also targets other ECM components such as collagen IV,fibronectin(FN), and laminin(LN), vitronectin, proteoglycan, chondroitin sulfate, and myelin basic protein. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema. But MMP-12 function in diabetic nephropathy or in the high-fat diet induced renal fibrosis and inflammation has not been reported.Methods:In the current study, cultured rat glomerular mesangial cells(GMCs) were used to observe the effects of high glucose on MMP-12 gene expression in GMC, to detect the effect of high glucose on profibrotic, proinflammatory and oxidative stress-related gene and protein expression, and to detect the TGF-bsignaling pathway while MMP-12 si RNA knockdown in GMC. To illuminate the mechanism underlying renal protective of MMP-12, two animal models were be used in this study:(1) Streptozotocin(STZ) induced diabetic nephropathy animal model using MMP-12 knockout mice;(2) high-fat diet(HFD) induced fibrosis and glomerular inflammation animal model using apolipoprotein E(apo E) and MMP-12 double knockout mice. For animal study, real-time quantitative reverse transcription PCR(real-time q PCR) and Western blot were be used to detect the ECM compounds, profibrotic,proinflammatory and oxidative stress-related gene expression;immunofluorescence staining to determine the interaction of related proteins;immunohistochemistry staining for ECM accumulation, inflammatory cell infiltration, renal fibrosis and oxidative stress in glomeruli of DN and HFD induced renal dysfunction.Results:1 Effect of MMP-12 expression on high glucose induced oxidative stress and ECM deposition in rats GMC.1.1 Rat GMC isolation and culture: The cultured rat GMC randomly divided into three different groups, normal control group(D-glucose, 5.5mmol/L, NG group), mannitol group(mannitol, 20mmol/L, MG group) and high glucose group(D-glucose, 30 mmol/L, HG group). Collected cells at different time points from each group after treatment, extracted protein or total RNA,Western blot and Real Time-q PCR analysis were used to detect MMP-2,MMP-9, MMP-12, TGF-b, FN, LN and other major ECM compounds expression; detect the affection of the TGF-b signaling pathway while si RNA knockdown of MMP-12 expression in GMC.1.2 High glucose upregulates ECM-related genes expression in GMC: Real time q PCR results show that, compared with NG group, HG treatment increased MMP-2, MMP-9, MMP-12, FN and LN gene expression at 6-12 h with time-dependent manner in GMC, and reached to the peak at 48 h, then the expression declined slightly at 72 h after treatment, but still higher than that in MG or NG group at the same time point(P<0.05); while the expression of MMP-12 gene in HG group began to increase at 6 h with time-dependent manner, and reached to the peak at 12 h after treatment, then the expression declined slightly at 72 h after treatment, but still higher than that in NG group or MG at the same time point(P<0.05).1.3 High glucose upregulates oxidative stress-related genes expression in GMC: Western blot results showed that high glucose induced different types of NADPH oxidase(Nox) expression in GMC, compared with NG group, HG treatment increased Nox2, Nox4 and their subunits p67 phox and p22 phox expression in GMC, and reached to the peak at 48 h, then the expression declined slightly at 72 h after treatment, but still higher than that in MG or NG group at the same time point(P<0.05).1.4 MMP-12 knockdown significantly attenuated HG induced-TGF-bsignaling pathways: GMC exposure to hyperglycemia can increase the expression of TGF-b1. Western blot showed that, HG treatment enhanced TGF-b1(4 ng/m L, 10 min) induced ERK1/2 and Akt phosphorylation in GMC.MMP-12 knockdown significantly abrogated HG enhanced TGF-b-induced ERK1/2 and Akt phosphorylation.2 Renal protective effect of MMP-12 deletion in diabetic nephropathy animal model2.1 MMP-12 deficiency attenuated STZ-induced renal dysfunction: DN animal model was generated by intraperitoneal injection of STZ in MMP-12 knockout mice and the same genetic background C57BL/6J(WT) mice. Eight weeks later, body weight and blood pressure of the WT and MMP-12-/-mice increased at similar rates throughout the experimental period in each group.Compare with NC mice, blood sugar levels of WT or MMP-12-/-mice with DN showed significantly increased between those two groups(WT group, DN288.4±14.3 vs WT NC 128.6±13.6, P<0.05; MMP-12-/-group, DN 245.3±11.7vs NC 134.9±14.1, P<0.05), which the results showed that the generated DN animal model successful and stable, and MMP-12 knockout significantly inhibited STZ-induced hyperglycemia in WT DN mice(MMP-12-/-group,DN 245.3±11.7 vs WT DN 288.4±14.3, P <0.05). Compared with NC group,STZ induced urinary albumin excretion mg/24 h, serum creatinine(SCR) and blood urea nitrogen(BUN) increased significantly in DN group(P <0.05), but these values were reduced at the same time points in mice that lacked MMP-12, which it suggested that MMP-12 deficiency can partially reverse STZ-induced renal dysfunction.2.2 STZ induces morphological changes in the mesangial area of the kidney:The role of MMP-12 in STZ-induced kidney injury was investigated by characterizing morphologic changes in the mesangial area of the kidney by PAS staining. The glomerular matrix in each group was quantified and expressed as a percentage of glomerular area. Compared with NC group, DN mice showed a significant increased in glomerular matrix area, an effect that was abrogated with concurrent MMP-12 deficiency.2.3 MMP-12 deletion suppresses STZ-induced profibrotic and proinflammatory gene expression in renal glomeruli: Real time q PCR results showed that transcript levels of MMP-12, MMP-2 and MMP-9 gene were increased in mice on DN; In contrast, the m RNA level of the profibrotic marker gene TGF-β1 and its downstream FN and LN gene were increased in glomeruli of DN mice, whereas MMP-12 deficiency suppressed this increase;MMP-12 gene deletion also suppressed the expression of monocyte/macrophage marker molecules monocyte chemotactic factor-1(MCP-1) and CD11 b.2.4 MMP-12 deletion reversed STZ-induced glomerular macrophage infiltration: Significant CD68+ immunostaining was detected in the renal glomeruli of DN mice, whereas this increase in macrophage infiltration induced by STZ was abrogated by MMP-12 deletion. CD68 m RNA level increased in glomeruli showed a similar trend.2.5 MMP-12 deletion reversed STZ-induced renal oxidative stress: Western blot results showed STZ caused an increase in Nox4 and p67 phox expression in glomeruli of DN mice, an effect that was abrogated by MMP-12 deletion. The expression of MCP-1 and TGF-b usually can be detect in DN kidney, and the downstream signaling molecules of TGF-b(11) Akt/PKB and ERK1/2phosphorylation was markedly increased in glomeruli of DN mice, but this effect was abolished by loss of MMP-12.3 MMP-12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity3.1 MMP-12 deletion attenuates HFD-induced renal dysfunction: The body weights of apo E-/-and apo E-/-MMP-12-/-mice on HFD increased at similar rates throughout the experimental period. When mice were switched to HFD,there was an increase in blood glucose concentration, and total cholesterol,triglyceride, HDL, and LDL levels compared to control(apo E-/-) mice fed regular chow. Systolic blood pressure values did not differ significantly between apo E-/-and apo E-/-MMP-12-/-mice at any time point. Apo E-/-mice consuming HFD showed increases in 24-h urinary protein excretion and serum creatinine concentration at 6 and 9 months relative to control mice fed normal chow, whereas these values were reduced at the same time points in mice that also lacked MMP-12.3.2 HFD induces MMP-12 expression in renal glomeruli: Significant MMP-12 immunostaining was detected in the renal glomeruli of HFD apo E-/-mice compared to those fed normal chow at 3, 6, and 9 months, whereas MMP-12 expression was undetectable in glomeruli of apo E-/-MMP-12-/-mice. Real time q RT-PCR showed MMP-12 m RNA level increased with time; suggesting that MMP-12 expression is induced over the course of glomerular disease progression by consumption of HFD.3.3 MMP-12 deletion suppresses HFD-induced profibrotic and proinflammatory gene expression in renal glomeruli: Transcript levels of MMP-2 and MMP-9 were increased in mice on HFD, but there were no differences between apo E-/-and apo E-/-MMP-12-/-mice at any time point.In contrast, the m RNA level of the profibrotic marker gene TGF-β1 was increased in glomeruli of HFD apo E-/-mice at the 3-month time point,whereas MMP-12 deficiency suppressed this increase at 3 and 9 months. HFD is associated with macrophage recruitment in glomeruli; this was evidenced by upregulation of the monocyte/macrophage marker CD11 b and the chemokine MCP-1 in the glomeruli of HFD apo E-/-mice at 3, 6, and 9 months.Meanwhile, the concomitant deletion of MMP-12 attenuated the increases in CD11 b and MCP-1 expression at these time points. In vitro cell invasion assays in 3D collagen IV showed vertical invasion depth of macrophage cells from apo E-/-MMP-12-/-mice less than those from apo E-/-mice, which it correlated with MCP-1 protein expression.3.4 MMP-12 deletion attenuates HFD-induced macrophage infiltration in renal glomeruli: The expression of the macrophage marker CD68 was upregulated in glomeruli at 3, 6, and 9 months in HFD apo E-/-mice relative to those fed normal chow. This increase in macrophage infiltration induced by HFD was abrogated by MMP-12 deletion. CD68 m RNA level increased in glomeruli at all the time points in HFD apo E-/-but not apo E-/-MMP-12-/-mice. Most of the infiltrated(CD68+) macrophages co-expressed MMP-12 in apo E-/-samples. Glomerular mesangial cells, a type of modified smooth muscle cell,are activated by local injury and synthesize ECM, which affects the progression of renal dysfunction in humans and experimental models of renal diseases. In apo E-/-and apo E-/-MMP-12-/-glomeruli a subset of MMP-12-positive cells expressed smooth muscle α-actin(α-SMA), indicating that they were activated mesangial cells.3.5 MMP-12 deletion suppresses HFD-induced glomerular fibrosis:Glomerular matrix accumulation is the hallmark of HFD-induced CKD. To determine whether MMP-12 is involved in this process, the m RNA and protein expression of FN and collagen IV was detected in glomeruli by q RT-PCR and immunohistochemistry, respectively. Consuming HFD induced the expression of both FN and collagen IV at all the time points in apo E-/-mice, whereas the absence of MMP-12(in apo E-/-MMP-12-/-mice) was associated with the downregulation of both factors at the m RNA and protein levels.3.6 MMP-12 deletion suppresses HFD-induced oxidative stress: Oxidative stress plays a critical role in the pathophysiology of CKD; Nox4 and p67 phox expression in glomeruli was detected by western blotting. HFD caused an increase in Nox4 and p67 phox expression in glomeruli of apo E-/-but not apo E-/-MMP-12-/-mice at 3, 6, and 9 months, an effect that was abrogated by MMP-12 deletion. Akt/PKB and ERK1/2 phosphorylation was markedly increased in glomeruli of HFD apo E-/-mice at each time point, but this effect was abolished by loss of MMP-12.Conclusions:1High glucose up-regulated the expression of MMP-12 and other ECM genes, oxidative stress and inflammation-related genes in glomerular mesangial cells.2MMP-12 knockdown significantly attenuated HG induced-TGF-?signaling pathways.3MMP-12 deficiency attenuated STZ-induced profibrotic,proinflammatory and oxidative stress related-gene expression in renal glomeruli of diabetic nephropathy mice.4MMP-12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity.