Journal of Clinical Medicine Review Matrix Metalloproteinases in Diabetic Kidney Disease Nuria Garcia-Fernandez 1,2, * , Conxita Jacobs-Cachá 3,4 , José María Mora-Gutiérrez 1 , Ander Vergara 3,4 , Josune Orbe 2,5 and María José Soler 3,4, * 1 Nephrology Department, Clinica Universidad de Navarra, 31008 Pamplona, Spain; jmora@unav.es 2 Instituto de Investigación Sanitaria de Navarra (IdiSNA), 31008 Pamplona, Spain; josuneor@unav.es 3 Nephrology Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, 08007 Barcelona, Spain; conxita.jacobs@vhir.org (C.J.-C.); vergara.ander@gmail.com (A.V.) 4 Nephrology Research Group, Vall d’Hebron Research Institute (VHIR), 08035 Barcelona, Spain 5 Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima-Universidad de Navarra, CIBERCV, 31008 Pamplona, Spain * Correspondence: nrgarcia@unav.es (N.G.-F.); mjsoler01@gmail.com (M.J.S.)  Received: 24 December 2019; Accepted: 1 February 2020; Published: 8 February 2020  Abstract: Around the world diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD), which is characterized by mesangial expansion, glomerulosclerosis, tubular atrophy, and interstitial fibrosis. The hallmark of the pathogenesis of DKD is an increased extracellular matrix (ECM) accumulation causing thickening of the glomerular and tubular basement membranes, mesangial expansion, sclerosis, and tubulointerstitial fibrosis. The matrix metalloproteases (MMPs) family are composed of zinc-dependent enzymes involved in the degradation and hydrolysis of ECM components. Several MMPs are expressed in the kidney; nephron compartments, vasculature and connective tissue. Given their important role in DKD, several studies have been performed in patients with DKD proposing that the measurement of their activity in serum or in urine may become in the future markers of early DKD. Studies from diabetic nephropathy experimental models suggest that a balance between MMPs levels and their inhibitors is needed to maintain renal homeostasis. This review focuses in the importance of the MMPs within the kidney and their modifications at the circulation, kidney and urine in patients with DKD. We also cover the most important studies performed in experimental models of diabetes in terms of MMPs levels, renal expression and its down-regulation effect. Keywords: matrix metalloproteinases; diabetic kidney disease; experimental models of diabetes; tissue inhibitors of metalloproteinases 1. Introduction Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) which is characterized by mesangial expansion, glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Serveral mechanisms such as renin-angiotensin system disbalance, inflammation, proteinuria, and hypertension have been related to DKD progression from the early to the advanced stages of the disease [1]. The trademark of the pathogenesis of DKD is an increased extracellular matrix (ECM) accumulation causing thickening of the glomerular and tubular basement membranes, followed by mesangial expansion, sclerosis, and tubulointerstitial fibrosis. The ECM levels are regulated by a homeostatic balance between deposition and degradation of ECM components [2]. The matrix metalloproteases (MMPs) family are composed of zinc-dependent enzymes that are involved in the degradation and J. Clin. Med. 2020, 9, 472; doi:10.3390/jcm9020472 www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 472 2 of 17 hydrolysis of ECM components [3]. The MMPs were discovered in the early 60s and are involved in the collagen degradation processes during the tadpole tail reabsorption [4]. Since then this family of enzymes and their roles have been expanded. Several MMPs are expressed in the kidney; in the nephron compartments, the vasculature and the connective tissue [5,6]. MMPs can contribute to diverse physiologic and pathological pathways in the kidney. Whereas the gelatinases, MMP-2 and MMP-9, have the ability to degrade type IV collagen, the major component of the glomerular basement membrane; the interstitial collagenases, MMP-1, MMP-8 and MMP-13, hydrolyze collagen I and III. Considering their important role in DKD, several studies have been performed in patients with DKD proposing that the measurement of their activity in serum or urine may become in the future markers of early DKD. Studies from diabetic nephropathy (DN) experimental models suggest that a balance between MMPs levels and their inhibitors is needed to maintain renal function. In this review, we describe the importance of the MMPs family within the kidney and their modifications at the circulation, kidney and urine in patients with DKD. We also cover the most important studies performed in experimental models of diabetes in terms of MMPs levels, renal expression and its inhibition effects. 2. Matrix Metalloproteinases Family MMPs are zinc-dependent proteases that degrade components of the extracellular matrix (ECM). MMPs were initially described as ECM regulatory proteins because of their ability to degrade ECM proteins like collagen, gelatin, laminin, aggrecan, fibronectin, elastin, and proteoglycans [7]. It was later observed that they can also process growth factors, cell-surface receptors, cytokines, and chemokines, as well as other MMPs, and proteases [8]. The activation or inactivation of biomolecules by MMPs cleavage points towards possible unexpected functions of these proteases beyond ECM remodeling [9]. Therefore, MMPs have a relevant physiological function and an altered expression or dysregulation leads to the development of several diseases such as chronic inflammatory diseases, vascular and renal diseases, diabetes, neurological disorders, and cancer [2,10,11]. All MMPs are multidomain enzymes that share a highly conserved core structure. They contain a signal peptide, which directs them to the secretory pathway; a prodomain with a cysteine residue to stabilize and inhibit a conserved HEXGHXXGXXH motif in the catalytic domain; a hinge or linker region; and a hemopexin-like domain that mediate protein-protein interactions and contributes to substrate specificity [7,12]. MMPs are classified into six groups based upon substrate and sequence homology: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and “other MMPs” [13–15]. Collagenases (MMP-1, -8, -13, and -18) cleave native collagens I, II, and III at a specific site. Gelatinases (MMP-2 and -9) cleave the denatured collagens (gelatins) and laminin, as well as some chemokines. Stromelysins (MMP-3, -10, -11, and -19) degrade a variety of substrates, including fibronectin, laminin, gelatin, and casein, and can cleave the prodomain of other MMPs activating them [13]. Matrilysins (MMP-7 and -26) are characterized by the lack of a hemopexin domain [15]. MMP-7 degrades not only ECM components such as laminin but also a large number of cell surface molecules, such as E-cadherin, Fas ligand and pro-α-defensin. Membrane-type MMPs (MT-MMPs; MMP-14, -15, -16, -17, -24, and -25) are structurally similar to the other classes of MMPs but they are anchored to the outside of the cell membrane by a transmembrane domain, or by a glycosylphosphatidylinositol residue [15] (Table 1). The remaining MMPs, MMP- 12, -20, -21, -23, -27, and -28, which do not fit into any of the above categories, are currently referred as “other MMPs”. J. Clin. Med. 2020, 9, 472 3 of 17 Table 1. Different matrix metalloproteinases (MMPs) implied in diabetic kidney disease (DKD) and their inhibitors. MMPs and tissue inhibitors of metalloproteinases (TIMPs) expression and characterization is still complex and vary between species. Group MMP Other Names Molecular Weight Substrates Renal Location 54 kDa. Interstitial Cleaved in different Type I, II, III, VII and X MMP-1 Rats: glomeruli collagenase isoforms that range from collagens. Collagenases 22 to 27 kDa. Type I, II, III, IV, X and XIV MMP-13 Collagenase 3 54 kDa. Rats: glomeruli. collagens and fibronectin. Rats/Mice: glomeruli, and proximal and distal Type IV, V, VII and X tubules. Range from 65 to 72 kDa. MMP-2 Gelatinase A collagens, type I gelatin and Monkey: proximal and Different isoforms. elastin. distal tubules. Gelatinases Human: tubules (in pathological conditions). Rats/Mice: glomeruli and Type IV and V collagens and tubular cells. MMP-9 Gelatinase B 92 kDa. type I and V gelatins. Monkey: proximal and distal tubules. Rats: glomeruli. Type III, IV, IX and X Monkey: proximal and collagens, type I, III, IV and MMP-3 Stromelysin-1 54 kDa. distal tubules. V gelatins, proteoglycans, Human: glomeruli and Stromelysins fibronectic and laminin. tubules. Type III, IV and V collagens, gelatin and elastin. Mice: glomeruli and MMP-10 Stromelysin-2 62 kDa Enhances tissue juxtaglomerular apparatus. plasminogen activator. Type I, III, IV and V gelatins, Rats: glomeruli. proteoglycans, fibronectin, Human: distal tubules and Matrilysins MMP-7 Matrilysin 30 kDa. laminin, E-cadherin and collecting duct (in entactin. pathological conditions). Range from 40 to 80 kDa. Progelatinase A MMP-14 MT1-MMP There are membrane and Rats/mice: glomeruli. (pro-MMP-2) and MMP-15. soluble forms. Membrane-type MMPs N-cadherin (CDH2), Human: proximal and distal progelatinase A MMP-24 MT5-MMP 64 kDa. tubules, collecting duct and (pro-MMP-2), proteoglycans loop of Henle. and fibronectin. pro-TNF-α, pro-HER Human: proximal and distal ADAM17 TACE 93 kDa. ligands and other proligand tubules (in pathological substrates. conditions). Other MMPs Type I and III procollagens, Meprin A FGF-19, VEGF-A, IL-1β, Mice: proximal tubule and MEP1B 80 kDa. subunit beta IL-18, ADAM10, E-cadherin juxtamedullary region. and others Inhibits MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, Rats/mice: glomeruli. TIMP-1 None 23 kDa. MMP-9, MMP-10, MMP-11, Human: glomeruli and MMP-12, MMP-13 and tubules. MMP-16. Tissue inhibitors of Inhibits MMP-1, MMP-2, MMPs MMP-3, MMP-7, MMP-8, TIMP-2 None 24 KDa. MMP-9, MMP-10, MMP-13, Not clearly defined. MMP-14, MMP-15, MMP-16 and MMP-19 Inhibits MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, TIMP-3 None 24 KDa. Not clearly defined. MMP-13, MMP-14 and MMP-15. MMP: metalloproteinase, MT-MMP: membrane-type metalloproteinase, TACE: tumor necrosis factor-alpha converting enzyme, ADAM: a disintegrin and metalloproteinase, HER: human epidermal growth factor receptor, FGF-19: fibroblast growth factor 19, MEP1B: Meprin A subunit beta, VEGF-A: vascular endothelial growth factor A, IL-1β: interleukin 1 beta, IL-18: interleukin 18, TIMP: tissue inhibitor of metalloproteinases. The role of MMPs in multiple physiological processes is controlled by different factors. In fact, dysregulation of MMPs has been associated to the development of pathological conditions. Regulation of MMP activity depends on transcriptional, epigenetic, and post-transcriptional mechanisms, proteolytic activation, post-translational modifications and extracellular inhibition [9]. MMPs expression is regulated at the transcriptional level, keeping these enzymes at low levels in normal physiological state. However, MMPs expression is induced in response to different stimuli J. Clin. Med. 2020, 9, 472 4 of 17 when ECM remodeling is required. In addition, DNA methylation or histone acetylation are epigenetic mechanisms of MMP genes transcription. At the post-transcriptional level, MMP expression can be regulated by modulation of messenger RNA (mRNA) stability and micro RNA (miRNA)-based or maintaining the pro-enzymes in an inactive state [12]. MMPs normally remain inactive due to a “cysteine switch” motif located in the propeptide region that blocks the Zn2+ dependent catalytic region. Diverse proteolytic enzymes (serine proteases, furin, plasmin or even other MMPs) can activate the MMPs [14]. Oxidative stress or nitric oxide (NO) can also activate MMPs by destabilization of the “cysteine switch” resulting in active proMMPs [16]. Finally, the MMPs activity is also controlled by specific endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs). TIMPs share a similar structure to MMPs that fits into the active site of the MMP catalytic domain in 1:1 stoichiometric ratio [11]. Overall, the proteolytic activity of TIMPs depends on the relative concentration of the active enzymes and their inhibitors [17]. 3. Matrix Metalloproteinases Pathways in the Kidney Several MMPs are expressed in the kidney; in the nephron compartments, the vasculature and the connective tissue [5,6]. MMPs can contribute to diverse physiologic and pathological pathways in the kidney. Chronic kidney disease (CKD) is characterized by a progressive decline in renal function that end up with fibrosis at a histological level [18,19]. The pathological mechanism of fibrosis is complex as a series of molecular events lead to an excess of ECM deposition. In response to noxious stimuli, renal cells (glomerular, tubular, vascular or pre-existing infiltrated cells) secrete profibrotic and proinflammatory molecules that will promote the recruitment of inflammatory cells in a positive feedback mechanism. This inflammatory state leads to epithelial-to-mesenchymal transition (EMT) of the tubular cells, recruitment of fibrocytes and proliferation and dedifferentiation of fibroblasts to myofibroblasts. All these cell types produce and deposit ECM thus contributing actively to fibrosis progression [20–22]. The primary function of the MMPs is to degrade ECM components thus it seems obvious that MMP activation or decrease of TIMPs in the kidney would be beneficial but this relation is not that straightforward. Some studies sustain that down-regulation of MMPs activity or upregulation of TIMPs in the kidney could contribute to fibrosis [23,24]. MMP knockout mice models have shown increased fibrosis and accelerated kidney disease progression [25]. In contrast, several reports have shown that upregulation of MMPs promote fibrosis [5,26–28] maybe due to interaction with overexpressed TIMPs [27,29] or due to the capacity of MMP degradation products to induce EMT [5,30]. In a study performed by Tan et al., they exposed murine tubular epithelial cells to an activated macrophage conditioned medium. They demonstrated that exposure to this medium induced EMT in tubular epithelial cells, and that the use of MMP broad spectrum inhibitor reversed these changes [31]. They also identified that macrophage conditioned medium was rich in MMP-2 and MMP-9. Moreover, MMPs actively contribute to inflammation promoting proinflammatory pathways activation mediated by cytokines cleavage [32] and also MMP degradation products induce infiltration of immune cells [5]. In summary, under physiological conditions a regulated MMP activity is crucial for ECM turnover and tissue homeostasis but in a pathological context most MMPs are upregulated contributing actively to all stages of renal fibrosis progression: inflammation, fibrotic type cells recruitment, stimulation of tubular EMT and ECM production and deposition (Figure 1). beneficial but this relation is not that straightforward. Some studies sustain that down-regulation of MMPs activity or upregulation of TIMPs in the kidney could contribute to fibrosis [23,24]. MMP knockout mice models have shown increased fibrosis and accelerated kidney disease progression [25]. In contrast, several reports have shown that upregulation of MMPs promote fibrosis [5,26–28] maybe due J. Clin. Med. to9,interaction 2020, 472 with overexpressed TIMPs [27,29] or due to the capacity of MMP 5 of 17 degradation products to induce EMT [5,30]. Figure Figure 1.1. Matrix Matrix metalloproteinases/Tissue metalloproteinases/Tissue inhibitors inhibitors of of metalloproteinases metalloproteinases (MMPs/TIMP) (MMPs/TIMP) activity activity and and function in the extracellular matrix turnover. In diabetes the majority of MMPs are function in the extracellular matrix turnover. In diabetes the majority of MMPs are upregulated upregulated contributing actively to contributing actively toall allstages stagesofofrenal renal fibrosis fibrosis formation formation andand progression: progression: inflammation, inflammation, fibrotic fibrotic type type cell recruitment, cell recruitment, stimulation stimulation of tubular of epithelial-to-mesenchymal tubular epithelial-to-mesenchymal transition transition (EMT) (EMT) and and extracellular extracellular matrix (ECM)matrix (ECM)and production production deposition.and deposition. 4. MMPs in Human In a study Diabetic performed Kidney by Tan et al.,Disease they exposed murine tubular epithelial cells to an activated macrophage conditioned The activity medium. of several They MMPs is demonstrated altered that exposure in human diabetic kidneytodisease this medium (DKD).induced EMT As explained before MMPs are not only involved in ECM remodeling, but also in the release of different growth factors such as tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), and others [33]. Therefore, this protein group dysregulation interferes with normal ECM turnover and may stimulate EMT and fibrosis by increasing the availability of previously mentioned growth factors [34]. In addition, the expression of these endopeptidases has not been well established in human kidney and there are species-dependent differences that hinder the characterization of these proteins (Table 1). MMP-2 and MMP-9 are two of the proteinases that have been widely studied in human DKD. The concentrations and activity of both proteins are increased in urine of type 1 and 2 diabetic patients [35–37]. The increase in these MMPs is especially frequent in patients with albuminuria, and has been correlated with an established renal injury. However, other studies also demonstrated an increase in MMPs in normoalbuminuric patients that could indicate early renal involvement before the development of proteinuria [36,38]. The significance of the increase in plasmatic concentrations of these proteinases is controversial and may be more related to diabetic vascular damage and endothelial dysfunction than to kidney disease. Recently, two different isoforms of MMP-2 have been identified: the full length MMP-2 (FL-MMP-2) and the N-terminal truncated MMP-2 (NTT-MMP-2). The latter is an intracellular isoform and appears to be induced by an alternate promoter located in the first intron that is activated by oxidative stress. It has been related to the activation of the innate immune response and the subsequent inflammation. Expression of these two MMP-2 isoforms was increased in 25 kidney biopsies of type 2 diabetic patients as compared to healthy controls. Moreover, both proteinases co-localized in affected tubules and have been associated to mononuclear cell infiltration [39]. These findings support previous data where MMP-2 seemed to facilitate EMT and fibrosis by interfering with intercellular connections and releasing TGF-β1 [34]. Similarly, another group of MMPs, the stromelysins, have been related to tubular atrophy, interstitial infiltration and fibrosis in DKD. MMP-3 was found inversely correlated with mesangial expansion and glomerular damage, whereas it was increased in tubular atrophy and interstitial lesions [40]. Another proteinase, the MMP-7, is not expressed nor detected in healthy renal tubular epithelium. However, its upregulation has been described in different renal pathological states such as autosomal-dominant polycystic kidney disease and hydronephrosis [41,42]. Interestingly, in a study performed in 121 patients with type 2 diabetes, increased urinary MMP-7 levels were associated with all-cause mortality. However, no clear connection was found between J. Clin. Med. 2020, 9, 472 6 of 17 these levels and end-stage renal disease (ESRD) development [43]. Other metalloproteinases have also been related to tubular lesions and interstitial fibrosis by releasing profibrotic factors. Among them, enhanced tubular expression of a disintegrin and metalloproteinase protein 17 (ADAM17) has been demonstrated in DN. This proteinase is responsible for TNF-α and amphiregulin (an epidermal growth factor receptor ligand) activation from its proligand substrates [44]. Moreover, the increase in ADAMs serum activity has been independently associated with CKD progression in males and an increased risk of cardiovascular events in CKD patients [45]. Specific MMPs are additionally responsible for other metalloproteinases activation. For instance, pro-MMP-2 is converted into its active form by MMP-24, a membrane-type metalloproteinase also known as MT5-MMP. In human DKD, there is an increased expression of this protein in the tubular epithelium, which correlates with a consequent MMP-2 elevation. Furthermore, proximal, distal and collecting tubules that where positive to MT5-MMP usually showed tubular atrophy as a probable result of DN progression [46]. An additional endopeptidase responsible for pro-MMP-2 activation is the MMP-14, of which membrane and soluble forms had been identified. In patients with type 1 and 2 diabetes these soluble forms are also increased in the urine [37]. TIMPs have also proven to be altered in human DKD. In some studies, circulating concentrations of TIMP-1 and TIMP-2 are decreased in patients with type 2 diabetes and diabetic nephropathy when compared to non-diabetic CKD or patients with diabetes alone [47]. In contrast, increases of serum and urinary concentrations of TIMP-1 were reported in patients with diabetes, and this increase was associated with greater glomerular lesions [48]. The same study that demonstrated increased MMP-3 expression also showed a higher TIMP-1 expression in tubules with atrophy, but both increases inversely correlated with stablished glomerular mesangial expansion [40]. Altogether, interstitial and tubular lesions, which usually correlate with glomerular lesions in DKD, seem more likely to be the source of urinary TIMP-1, as the inhibitor expression decreases in damaged glomeruli. A greater understanding of kidney MMPs is still needed to better delineate their pathophysiological role in DKD. Although most of these endopeptidases appear to be increased in human DN and apparently related to tubular atrophy and interstitial fibrosis, it is not certain if the activity and concentrations measured in the urine reflect what is really happening in renal tissue or are the consequence of an increased systemic activity [49]. Moreover, it is not yet established if metalloproteinases upregulation precedes fibrosis or if it is a consequence of this lesion. Some studies suggest that MMPs increases antedates the development of albuminuria and, therefore, a more established renal lesion [38,50]. Probably multiple pathological pathways that meet in diabetic nephropathy such as hyperglycemia, advance glycation end products (AGEs), oxidative stress or renin angiotensin system (RAAS) activation are involved in MMPs imbalance. Nevertheless, further MMPs characterization is needed to suggest the use of these proteinases as a possible DKD biomarker or therapeutic target. 5. MMPs in Experimental Diabetic Nephropathy Experimental studies have shown valuable insights into the potential role of MMPs in DN, demonstrating altered expression and enzyme activity in DN rodent models. Evidence supports that MMPs may have both a pathogenic and renoprotective effects. Moreover, the same MMP can play both roles, which underlines the complexity of MMP-system pathobiology [51]. Table 2 summarizes some of the most important experimental studies related to MMP-system performed in experimental models (in vivo and in vitro) of DKD. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 17 BN rats In vivo (STZ)/Cortex & Gene expr. MMP-2 6 wks [52] In vitro medulla Enz. activity Podocytes * J. Clin. Med. 2020, 9, 472 Gene & Protein 7 of 17 C57BL/6J (STZ)/ expression compensatory MMP-2 In vivo 16 wks [25] Renal J. Clin. Med. 2020, 9, x FOR PEER cortex REVIEW Enz. activity KO-Mmp2 = renal injury 7 of 17 J. Clin. Med. 2020, 9, x FOR PEER REVIEW KO-Mmp2 7 of 17 Table 2. Experimental murine J. Clin. Med. 2020, 9, x FORstudies In vivo/ S-D involving different metalloproteinases. PEER REVIEW BN rats 2 days 7 of 17 MMP-2 In vivo BN(STZ)/Glomerulirats (STZ)/Cortex & Protein expr. Gene expr. [53] MMP-2In vivo In vitro (STZ)/Cortex & Gene expr. 6 wks [52] J. In xvitro &medulla Tubules Enz. activity 4 wks MMP-2 J. Clin. Clin. Med. Med. 2020, 2020, 9, 9, x FORFOR PEER PEER REVIEW BN REVIEW rats Age/Exposure 6 wks [52] 77 of of 17 17 MMP In Vivo/in Vitro Species (Model)/CellIn vitro In Type Condition Studied Outcome Ref vivo medulla S-DPodocytes (STZ)/Cortexrats (HG) *&Enz. activity Gene Enz. expr. &Time activity MMP-2 In vitroPodocytes * 5 6 days wks [52] [54] In vitro medulla Mesangial Gene Enz.&activity Protein BN rats (STZ)/Cortex & BN BN ratsratscellsGene Protein expr. In vivo In vivo vivo C57BL/6J Podocytes ICER (STZ)/Cortex Gene(STZ)/ *& & Protein Iγ expr. expression Gene compensatory MMP-2 MMP-2 medulla MMP-2 In In vivo C57BL/6J (STZ)/Cortex (STZ)/ &expression Gene expr. Protein expr. expr. 6 wks16 40 wks wks 6 wks [25] [52] [55] [52] In vitro MMP-2 Renal cortex Enz. activity Gene Enz. activity 16 wks 6 wks compensatory [52] MMP-2Podocytes In vivo In vitro * In vitroRenal cortex mice/Glomeruli medulla medulla Enz. Enz.&activity Enz. activity Protein activity KO-Mmp2 = renal injury [25] C57BL/6J Podocytes (STZ)/ ** KO-Mmp2 expression Gene & Protein KO-Mmp2 = renal injury MMP-2 In vivo In vivo/ Podocytes OLETF 16 wks 40–50 compensatory [25] Gene Renal & Protein KO-Mmp2 S-D cortex rats Enz.&activity Geneexpr. Gene & Protein Protein 2 days KO-Mmp2 = renal injury [56] MT1-MMP Invivo/ In vitro Rats/Glomeruli wks C57BL/6JMMP-2 (STZ)/Renal S-D(STZ)/Glomeruli rats expression C57BL/6J (STZ)/ KO-Mmp2 Enz. Protein expr.2 days activitiy expression compensatory [53] MMP-2 In vivo MMP-2 In vivo/ In In vitro vivo C57BL/6J (STZ)/ expression 16 wks 16 wks compensatory compensatory [25] [25] MMP-2 J.J.Clin. MMP-2Med. cortex 2020, 9, In x FOR vivo PEER (STZ)/Glomeruli REVIEW Enz. activity Protein expr. 16 wks [53] 77ofof1717 [25] Clin. Med. 2020, 9, x FOR PEER In vitro & Renal Renal Tubules REVIEW S-D cortex rats cortex Enz. Enz. activity activity 4 wks 2 days KO-Mmp2 KO-Mmp2 Induced by KO-Mmp2 == renalinjury = renal AGE renal &injury Ang- injury In vivo/& Tubules KO-Mmp2 Gene expr. 4 wks (STZ)/Glomeruli KO-Mmp2 Protein expr.& [53] J. Clin. Med. MMP-2 2020, MMP-2 9, x FOR PEER In vitro REVIEW (STZ)/Proximal S-D rats (HG) Enz. activity KO-Mmp2 Enz.&activity 32 wks 5 days II 7 of 17 [57] J. Clin. Med. 2020, 9, x FOR PEER REVIEW S-D ratsMesangial (HG) & BN BNrats Tubules tubular c. cells rats Enz. activity Protein expr.5 days 224 days wks 7 of 17 [54] S-D S-D rats rats Attenuated by Ramipril [54] In vivo/ MMP-2 S-D rats (STZ)/GlomeruliIn vitro In InInvivo/ In vivo vivo/& Mesangial (STZ)/Cortex cells &Protein Gene expr. expr. 2 days days MMP-2 MMP-2 MMP-2 vivo S-D Protein (STZ)/Cortex ICER (STZ)/Glomeruli rats (STZ)/Glomeruli Iγ expr. (HG) & Gene Protein Enz. expr.& expr. activity Protein expr. 6 wks [53] [53] [52] [53] In vitro Tubules MMP-2 In vitro In vivo BN rats medulla Protein Enz. expr. 4 wks 5 6days 40 wks wks [52] [55] MMP-2 In Invitro BNICER vitro ratsmice/Glomeruli Iγ & Mesangial && medulla Tubules Tubules cells Enz.activity Protein activity expr.40 wks 44 wks wks Mmp-2, Mmp- [54] MMP-2 MMP-2 InInvivo vivo (STZ)/Cortex Podocytes Protein Gene * * expr. expr. expr. 9 [55] MMP-2S-D rats In vivo (STZ)/Cortex mice/Glomeruli &Podocytes Gene Gene & expr. Protein 6 wks [52] J. MMP-2 Clin. Med. 2020, MMP-2 In 9, x (HG) vitro FOR PEER In vivo/ medulla REVIEW Enz. S-D S-D activity ICER rats rats OLETF Iγ (HG) (HG) & Protein Enz. Gene Enz. activity Enz. activity activity6 wks & & 40–50 [52] 7 of 17 MMP-2 In vitro MMP-2 In vitro vivo medullaS-D rats (STZ) & Gene &&Protein Protein expr. 5 days24 40 wks [54] [58] [55] MMP-2 MMP-2 Mesangial In vitro cellsInIn vitro mice/Glomeruli Mesangial expr.Enz. Gene cells activityProtein Gene Enz.expr. Protein Protein activity expr. 55 days days [54] [54] [56] MMP-9In vivo/ In vitro MT1-MMP Podocytes Podocytes OLETF Mesangial * Rats/Glomeruli C57BL/6J * cells (STZ)/ Protein expr.40–50 wks expression compensatory MMP-2 InInvivo C57BL/6J ICER (STZ)/ expr. Iγ Geneexpression Enz. &activitiy Proteinwks 1616wks MMP-2 compensatory activity [56] [25] MT1-MMP MMP-2 In vitro In Rats/Glomeruli vivo Renal ICER Iγ cortexGene & Protein Enz. activity wks [25] MMP-2 In vivo ICER Iγ mice/Glomeruli MMP-2 MMP-2 In vivoBN rats In vivo/ vivo OLETF Protein Renal Gene cortex expr.& Protein Enz. Protein activitiy Enz. Protein expr.40 wks40 expr. activity expr. 4040–50 wks wks KO-Mmp2 Attenuated KO-Mmp2 by= renal renalinjury perindropil [55] [55] [55] [56] In C57BL/6J mice/Glomeruli (STZ)/ S-D & rats expression mice/Glomeruli expression KO-Mmp2 Induced by=AGE compensatory &injury Ang- MMP-2MT1-MMP MMP-2 Invivo vivo In In (STZ)/Cortex vitro (STZ)/ vivo/ C57BL/6J S-D Rats/Glomeruli C57/BL6 rats (STZ) Gene expr.Gene KO-Mmp2 Enz. expr. activitiy16 6 wks wks wks compensatory [25] [52] MMP-2 In vivo/ MMP-2 MMP-2 InInvivo Renal (STZ)/Proximal Gene cortex & Protein Enz. Gene expr. activity Gene & & 16 wks Protein Protein 32 wks Induced=by AGE &II Ang- [25] [57] J. Clin. Med. 2020, MMP-2 9,Invitro x vivo/ FOR In PEER vitro Renal vivo/ medulla REVIEW cortex OLETF S-D rats Enz. Enz. Gene activityexpr. activity Enz.& activity 240–50 KO-Mmp2 renal injury 7[57] of 17 [56] MT1-MMP In vitro OLETF MMP-2 Rats/Glomeruli MMP-2 In In vivo/ (STZ)/Proximal vivo/ db/db OLETF mice/ S-Drats tubular Enz. rats c. KO-Mmp2 activitiy Gene Protein expr. 40–5032 wkswks 2days KO-Mmp2 40–50 4–24 days = renal injury II NTT-MMP-2 [56] MMP-2 MT1-MMP MMP-2 In vitro In In In vivo Invivo/ Podocytes vivo/ vitro *S-D Rats/Glomeruli (STZ)/Glomeruli Enz. activity KO-Mmp2 expr. Protein Gene expr. expr. wks Attenuated Induced byby AGERamipril & Ang- [56] [59] [53] MT1-MMP MMP-2 In vitro InInvitro tubular Rats/Glomeruli Cortex c. (STZ)/Glomeruli & expr. Protein Enz. expr. activitiy wks Attenuated by FL-MMP-2 Ramipril [53] MMP-2 S-D (STZ)/Proximal rats Enz. activitiy 2 days 32 wks [57] In vivo/ In vitro vitro S-D BNrats rats &medulla & Tubules Tubules Gene & Protein Enz. activity 2 days 44wks wks Mmp-2,II by AGE Induced Mmp-& In vivo/ S-D rats MMP-2 In vivo/ (STZ)/Proximal (STZ)/Glomeruli C57BL/6J (STZ)/tubular S-D Gene rats c.expr. Protein expression expr. Attenuated by Ramipril [53] MMP-2 MMP-2 MMP-2 In vivo vivo In (STZ)/Cortex (STZ)/Glomeruli S-D & (HG)rats ProteinGeneexpr. expr. 32 Mmp-2, Induced compensatory Induced by by AGE Mmp- & 9 AGE & Ang- Ang- [53] & wks32 [57] vivo/ S-D Gene expr. B6 mice: In vitro MMP-2 MMP-2 tubular In InInvitro vitro c. In vivo/ S-Drats ratsactivity (HG) Enz. Gene Gene Enz. activity expr. expr.16 activity 64& wks wks Ang-II [25] [52] S-DEnz. MMP-2 In vitro &medulla Renal Tubules (STZ)/Proximal cortex Enz. activity wks wks 55days [57] [54] J. Clin. Med. MMP-2 MMP-2 MMP-2 2020, In 9, vitro x FORIn In InPEERvivo Invitro vitro & vitro Tubules REVIEW (STZ)/Proximal Mesangial rats (STZ) Enz. cells Gene activity Enz. Protein Enz. expr. 4 wks activity expr. activity 32 24 wks KO-Mmp2 = renal wks days Attenuated 9Mmp-2, injury II IIby RamiprilMmp- [57] 7 of 17 [58][54] In vivo S-D rats Mesangial tubular tubular (STZ) c. c.cellsKO-Mmp2 Enz. activity Protein expr. 24 wks Attenuated Hyperglycemia by Ramipril [58] MMP-9 S-D rats Podocytes (HG) * Enz. activity & Attenuated by Ramipril MMP-2 MMP-2 In vitro S-D rats (HG) ICER ICER Iγ Enz. Iγ activity Enz. & activity Gene expr. 5 days MMP-2 9 activity [54] MMP-9 MMP-2 MMP-2 In vitro InInvivo Mesangial S-D cells Protein Protein Proteinexpr. expr. 5expr. 2days 4040wks [54] [55] MMP-2 In vivo/ InMesangial vivo vivo BNrats S-D rats (STZ) mice/Glomeruli rats cells mice/Glomeruli Gene Protein &expr. Protein days 24 wks MMP-2 wks Attenuated Mmp-2, activity Mmp-2, Mmp-2, Mmp-9 by perindropil Mmp- Mmp- [55] [58] MMP-2 MMP-2 S-D (STZ)/Glomeruli C57BL/6J ICERB6 Iγ& ROP (STZ)/ Genemice/expr. Protein expression Enz. activity expr. [53] In vivo MMP-2 MMP-9 MMP-2rats MMP-2 In In (STZ) Invitro vivo vivo (STZ)/Cortex ICER Iγ C57/BL6 & (STZ) Gene expr. Protein Gene expr. Gene Gene &&expr. Protein40 16 Protein 24 wks 5–10Attenuated wks compensatorybyNormoglycemia perindropil MMP-2 99 activity [55] [25] [58] MMP-9 J. Clin. J. MMP-2 Clin. MMP-2 Med. Med. MMP-2 2020, 2020, In9,vivo9, x FOR x FOR InIn PEER vivo/ PEER &REVIEW Renal REVIEW Tubules mice/Glomeruli cortex Enz. OLETF activity Protein Enz. expr.Gene 4046wks expr. wks 40–50 wks MMP-2 activity [55]7[52] of 7 of 1717 [60] J. Clin. Med. MT1-MMP MMP-2 MMP-2 2020, 9, In x FOR In vitro PEERIn In vitro vivo/ vivo medulla C57/BL6 mice/Glomeruli vivoREVIEW Mesangial S-D (STZ) S-Ddb/db OLETF rats cells (STZ) ratsmice/ (STZ) Enz.activity Gene activity Gene Enz. expr. expr. &activity Protein expr. 24 24 40–50 KO-Mmp2 wks 4–24 wks wks = renal Attenuated injury NTT-MMP-2 by perindropil 7 of 17 [58] [56] [58] [56] MMP-2 InInvivo In vitro Rats/Glomeruli Enz. activity wks Attenuated ROPby perindropil J. Clin. Med. MT1-MMP MMP-2 2020, MMP-9 MMP-9 9, xvivo/ In FOR PEER vitro S-D rats (HG) REVIEW Podocytes db/db OLETF mice/ *(HG)Gene Rats/Glomeruli Cortex Gene &Enz.GeneKO-Mmp2 &&Protein &activity Enz. Enz. Protein ProteinEnz. activity 4–24 &expr. activitiy activitiy 40–50 wks wks NTT-MMP-2 FL-MMP-2 mice: 7 of 17 [59] MMP-2 MMP-2 MMP-2 C57/BL6 In In In vivo/ vitro vivo OLETF BN BN rats C57/BL6 rats (STZ) 5 40–50 days MMP-2 activity MMP-2 activity [56] [54][59] (STZ) Mesangial S-D Cortex rats cells & Protein Gene expr. & expr. Protein expr. 2 days wks FL-MMP-2 MT1-MMP Invivo/ vitro Rats/Glomeruli rats db/db medulla S-D rats mice/ expr. Gene & Protein wks 4–24 Induced Hyperglycemia NTT-MMP-2 NTT-MMP-2 [56] MMP-2 In vivo MT1-MMP db/db MMP-2 MMP-2 MMP-2J. Clin. MMP-2 Med.In InIn vitro mice/CortexIn vivo vivo In 2020, & 9,In FOR vivo BN PEER Rats/Glomeruli (STZ)/Cortex (STZ)/Cortex xvivo/ (STZ)/Glomeruli ICER C57BL/6J vivo/ BNmedulla Gene Iγ rats (STZ)/ &S-D REVIEW& ratsEnz. & Protein Enz. Gene Gene Protein expr. expr. expression expr. Gene activitiy expr. activitiy Gene expr.wks expr.64–24 wks 6 wkswks wks Inducedby Attenuated Attenuated by bybyAGEAGE&&Ang- perindropil perindropil Ang- [53] [52][52] [59] 7 of 17 [59] MMP-2 MMP-2 MMP-2 In MMP-2 InIn In vivo In vitro In(STZ)/Cortex vitro vivo vivo vitro (STZ)/Proximal Cortex(STZ) & C57/BL6 (STZ)/Proximal & Gene Protein expr. expr. expr. 40 16wks wks 3232 wks wks compensatory B6 mice: FL-MMP-2 FL-MMP-2 IIII [55] [25] [57] [57] MMP-2 medulla In vivo vitro &medulla vitro medulla Inmice/Glomeruli Tubules S-D Renal (STZ)/Cortex ratsC57/BL6 cortex & tubular medulla (STZ)Enz. c.Enz. Enz. Enz. Gene activity activity Enz. activity Enz. expr. activity 6 activity 4 wks wks B6 InducedAttenuated KO-Mmp2 by mice: AGE = renal & Ang- injury [52] J. Clin.MMP-2 Med. 2020, In 9, Inxvitro vivo/ FOR PEER S-D medulla Podocytesrats db/db Podocytes REVIEW * db/db tubular * mice/ mice/ c. Gene activity expr. Gene & Protein Gene & Protein 6 wks 4–24 4–24Induced by AGE NTT-MMP-2 & NTT-MMP-2Ang- Normoglycemia Hyperglycemia by Ramipril 7[52] of 17 MMP-2 MMP-2 InIn vivo/ vitro In (STZ)/Proximal vivo medulla BN rats Gene Gene expr. KO-Mmp2 Enz. & Protein activity 32 wks Attenuated by Ramipril [57] [59] MMP-2 MMP-2 In vivo S-D (STZ)/Proximal rats (HG) Enz. Enz.activity &expr.3240–50 II B6 mice: mice: [59] MMP-2 MMP-2 InIn Invivo/ In vitro vitro vitro In vivo Podocytes vivo OLETF tubular Podocytes *Cortex c.Cortex (STZ)/Cortex * Kkay & &Gene Enz.Gene &activity & & activity expr. Gene Protein Protein Gene & Protein expr. expr. 5wks days wks wks Hyperglycemia II Mmp-2, B6 FL-MMP-2 FL-MMP-2 [57] Mmp-[54] [56] MT1-MMP MMP-9 MMP-2 In In vitro In Mesangial tubularS-D rats Rats/Glomeruli c. cellsmedulla mice/Glomeruli Gene Protein expr. & Protein 2wksdays 16 wks 6 wks Attenuated by Attenuated Ramipril Mmp-2, Hyperglycemia by Ramipril Mmp- [61] [52] MMP-2 MMP-2 MMP-2 MMP-2 MMP-2 InIn vivo/ In C57BL/6J vivo vivo C57BL/6J C57BL/6J vitro BN rats (STZ)/Glomeruli ICER(STZ)/ (STZ)/ B6 (STZ)/ medulla Iγ &medullaROP mice/ Enz. Gene expression expression Protein& Enz.expr. activitiy Protein expr. activity 16 16wks wks compensatory compensatory Hyperglycemia Normoglycemia 99 [25][25] [53] In vitro Renal Renal cortex cortex expression Enz. Enz. activityGene Gene activity Gene expr. expr. expr. 5–10 Mmp-2, compensatory B6 B6 mice: Mmp- mice: MMP-2 B6 & ROP MMP-2 MMP-2 InIn vivo vivo mice/Mesangial In vivo In (STZ)/Cortex InInvitro vivo B6 C57BL/6J &Tubules ROP S-D & mice/ rats Podocytes Mesangial (STZ)/ Gene(STZ) Protein Gene expr. cells *expression expr. expr. 1640 wkswks 2424wks 2–3 KO-Mmp2 daysKO-Mmp2 = renal Normoglycemia Mmp-2, =MMP-9, renal injury injury Normoglycemia Mmp- [55] [25] [58] [60] MMP-2 In vitro MMP-2 vivo& mice/Glomeruli S-D rats S-D rats (STZ) Enz. 6 activity 4 wks 5–10 wks wks compensatory Ø MMP-2 [52] [58] MMP-2 MMP-2 MMP-2 InIn vivo Renal cortex Podocytes Enz. *Gene Gene KO-Mmp2 KO-Mmp2 expr. activityEnz. Gene Enz. activity expr. 16 wkswkswks 5–10 Induced =byrenal KO-Mmp2 9 AGE & Ang- injury [25] [60] MMP-2 MMP-9 cells In MMP-9 In vivo/ vitro (HG) vitro In vitro medulla Mesangial Renal cortex Enz. cells Enz. (HG)activity Enz. Gene expr. activity Gene activity expr. &activity Protein 5–10 KO-Mmp29II ROP Hyperglycemia ROP Hyperglycemia Normoglycemia mice: mice: [60] [62] MMP-2 MMP-2 MMP-9 InIn In vivo vitro vivo/ S-DOLETF S-D ratsB6 (STZ)/Proximal rats Podocytes (HG) (STZ) & * ROP (HG) mice/ Gene Enz. Gene Enz. Enz. KO-Mmp2 & Protein activity activity expr. Enz. activity &activity Gene expr. 32 wks 24 40–50 wks wks 5–10 = renal MMP-2 injury MMP-2 activity activity [57] [58] MMP-2 MMP-2 InInInvivovitro InS-D S-D S-D rats (HG) rats (STZ) rats C57BL/6J c.cells (STZ)/ KO-Mmp2 Enz. expression activity 2 24 5wks days 2 days days days ROP mice: Hyperglycemia compensatory [58] [54] MMP-2 In vivo/ vitro vivo/ tubular Mesangial cells expr. Attenuated by Øperindropil [56] [60] MMP-9 MT1-MMP MMP-2 MMP-9MMP-2 In vitro In(STZ)/Glomeruli vivo Mesangial S-D rats Renal Rats/Glomeruli (STZ)/Glomeruli Enz. Gene cortex Protein Protein activity & Protein expr. Enz. Enz. activity Protein expr. expr. 2 days activity wks 16wkswks Attenuated MMP-2 by Attenuated Ramipril Hyperglycemia MMP-9, activity by MMP-2 perindropil[53][53] [25] InInvivo/ S-D ratsC57/BL6 (HG) Enz. activitiy 2 days KO-Mmp2 Hyperglycemia ROP=mice: renal injury MMP-2 J. Clin. Med. In Invitro vitro 9,(STZ)/Glomeruli 2020, vivo/ x FOR C57BL/6J& &ICERPEER Tubules B6 B6 Iγ (STZ)/ Tubules & C57BL/6 & ROP(STZ) ROP REVIEW C57/BL6 mice/ mice mice/ Protein (STZ)expression expr.Gene KO-Mmp2 expr. 4 wks 4 wks 5–10 MMP-2 Mmp-2, Normoglycemia Ø,activity Normoglycemia Minimal Normoglycemia Mmp- Interstitial[53] 7 of 17 MMP-2 MMP-2 MMP-2 MMP-2 MMP-2 In In In vitro vivovivo (STZ)/Glomeruli db/db Protein Protein expr. expr. Gene &&expr.16 40 wks wks 214–24 5–10 compensatory Attenuated by perindropil [25][55] [53] MMP-2 MMP-2 vitro In In vivo/ In vitromice/Glomeruli vivo InInvitro S-Dcortex & Tubules Renal vivo rats db/dbmice/ Mesangial (High-fat Mesangial cells high cells mice/ Enz. Gene activityProtein Gene Enz. Protein expr. 4 wks Protein Attenuated wks 4–24 by Inducedlesions=by NTT-MMP-2 perindropil AGE None Hyperglycemia NTT-MMP-2 & Ang- glomerular [60] [63] [60] [59] MMP-9 In vivo MMP-2 Kkay MMP-9 MMP-2 In mice/Glomeruli C57/BL6 S-D S-D vivo & ratsrats Tubules (STZ) (HG) Gene (HG) S-D Cortex & (HG)rats Enz. Protein & Gene Enz. Gene expr. activity expr. Enz. activity expr. &activity activity &expr. 4 16 wkswks 2wks wksKO-Mmp2 days wks 9renalNormoglycemia injury FL-MMP-2 ROP mice: [59] [61] MMP-2 MMP-2 MMP-2 In Invitro vitro In C57/BL6 (STZ)/Proximal (STZ) sucrose (HG) Cortex diet)Gene &KO-Mmp2 & Protein 32 5 days wks 5wks days ROPlesions mice: [57] [54][54] MMP-2J. Clin. MMP-2 In Med. Invivo2020, vitro vivo/ vivo/ 9, xS-DS-D FOR db/db Mesangial rats rats OLETF Mesangial (STZ) (HG) PEERmice/cells BN REVIEW Kkay cells (STZ)/Glomeruli ratsEnz. Enz. Gene Protein activity & & &expr. Gene activity Protein Protein expr. expr. Protein 24 Protein expr. 40–50 4–24 wks NTT-MMP-2 II Normoglycemia FL-MMP-2 [58] 7 of 17 [53] MMP-2 MMP-2 MMP-9 InIn vitro vivo In In vivo db/db S-D vitro InMesangial vivo tubular mice/ rats Kkay c. medulla (HG) medulla (STZ)/Cortex Gene Enz. Enz. Gene activity & &expr. Protein expr. activity & & expr. Gene Protein 54–24days 16 wks NTT-MMP-2 Attenuated byNormoglycemia Ramipril Hyperglycemia [54] [56] [59] [61] MMP-9 MMP-2 MT1-MMP MMP-2 In Invivo In vitro vitro Rats/Glomeruli S-D Cortex ICER rats ICER cells mice/Glomeruli Iγ &Iγ & Tubules Protein expr. expr. 2 5 dayswks days wks 4 wks FL-MMP-2 Hyperglycemia [59] [54] MMP-2 MMP-9 MMP-2 In vivo 2–3 16 days6 wks MMP-2MMP-9, B6 Ø MMP-2[55] [61] [52] In vitro MMP-2 MMP-2 InInvivo/ In vivo vivo MMP-2Podocytes * (HG)In(STZ)/Glomeruli CortexGene Mesangial vitro mice/Glomeruli ICER & cells Iγ medulla expr.Enz. Protein expr. Enz. Protein Protein activitiy Protein activity expr.expr. expr. expr. Enz. activity expr. wks 40 40wks wks FL-MMP-2 activity B6mice: mice: [53] [55] [62] MMP-9 medulla mice/Glomeruli mice/Glomeruli BN Kkayrats Gene & Protein days Mmp-2, Mmp- MMP-2J. Clin. MMP-9 Med. vivo In vitro 2020, 9, xvivo FOR medulla ICER Inmice/Glomeruli S-D PEER S-D Iγ rats rats REVIEW Podocytes (HG) Protein *& Enz. activity expr. 405–10 & days wks 2–3 16 Attenuated wks byMMP-9, perindropil MMP-9, Hyperglycemia Ø Ø MMP-2 MMP-2 [55] 7 of 17 [61] MMP-2 MMP-2 In In vivo vivo/ In In vitro & vivo Tubules Podocytes (STZ)/Cortex mice/Glomeruli *Protein Gene Gene Gene& & Gene Gene expr. Protein expr. expr. expr. Protein expr. 4 40 2–3 wks wks days 5 days Induced B6 MMP-9, by mice: 9 AGE & Hyperglycemia Normoglycemia Ø MMP-2 Ang- [55] [54] C57/BL6 In vitro (STZ) Mesangial cells Protein expr. 5–10 B6 mice:Normoglycemia [57] [62] MMP-2 MMP-2 MMP-2 MMP-2 MMP-9 In In vivo/ vivo/ mice/Glomeruli In vitro OLETFOLETF Podocytes (STZ)/Proximal * (HG)Gene medulla Gene Gene & expr. expr. Gene Enz. Protein Enz. & Protein activity activity 40–50 32 40–50 wks 6 wks Ø, II Minimal Interstitial [52] In InIn vivo vitro vitro S-D S-Ddb/db rats rats (STZ) (HG) mice/ICER Kkay Enz. IγGene Enz.expr. expr. Gene activity activity & & Protein & 24 Protein 4–24 wks 5–10 [56][56] [58] [62] MMP-2 MMP-2 C57BL/6 MT1-MMP MT1-MMP MMP-9 In mice Invivo/ In (High-fat vitro vitro In OLETF Rats/Glomeruli vivoRats/Glomeruli c. BN Kkay rats Gene Enz. & Gene Protein activity & Protein 540–50 wks wks 2–3 daysdays NTT-MMP-2 Hyperglycemia MMP-9, Ø Ø MMP-2 MMP-2[54] In vivo MMP-9 MMP-2 MMP-2 MMP-2 MMP-2 MMP-9 MMP-9 InIn vitro vivo InInvitro vivo/ In InMesangial vivo (HG) tubular OLETF C57BL/6J Protein Podocytes &Podocytes (STZ)/ Enz. *expr. expr. * Enz. Enz. activity expression Protein Gene activitiy activitiy expr. expr. days21 40–50 wks16 40 wks 16 wks Hyperglycemia Attenuated lesions MMP-9, by compensatory Ramipril None glomerular Normoglycemia [59] [56] [61] [55] [61] [63] MMP-9 MT1-MMP highMMP-2sucrose diet) vivoCortexB6 vivo InRats/Glomeruli In vitro cells&& mice/Glomeruli (STZ)/Cortex B6 ROP mice/GlomeruliROP mice/ Protein mice/ & expr. expr. expr. Geneexpr. expr. expr. wks wksdays 16 wks 5–10 FL-MMP-2 Normoglycemia [25] [62] MT1-MMPMMP-9 MMP-2 In vitro In vitro Rats/Glomeruli ICER Iγ Renal C57BL/6 Mesangial (HG)cortex mice Enz. activitiy cells Enz. GeneGene Enz. activity expr.wks &activity Protein 6 wks 5–10 MMP-2 MMP-9, activity Ø Ø, Minimal KO-Mmp2 MMP-2 injury[56] Interstitial = renal lesions [52] [60] MMP-2 MMP-2 In Invitro medulla vitroS-D S-D rats rats medulla Mesangial cells Enz. Gene Enz. activitiy &activity Protein days wks Mmp-2, Attenuated Induced Induced by byby perindropil AGEAGE & & Mmp- Ang-Ang- [60] MMP-2 MMP-2 In In vivo In vivo/ vivo/ In vivo C57BL/6 B6 S-D vivo/ & ROP mice (High-fat mice/ OLETF Protein high Gene Gene expr. expr.KO-Mmp2 Protein expr. 40 expr. Enz. activity wks 2–3 21 2–3 days wks days 40–50 Ø, Minimallesions Normoglycemia wks Normoglycemia Interstitial MMP-9,None MMP-9, MMP-9, Ø Ø MMP-2 glomerular MMP-2 Ø MMP-2 [55] [63] MMP-2 MMP-2 B6mice/Glomeruli & ROP ratsC57BL/6J mice/ Podocytes(HG) (STZ)/***Gene expression Gene expr. ROP compensatory mice: MMP-2 MMP-2 MMP-2 MMP-2 C57BL6J MMP-9 MMP-2 & S-D MMP-2 J. MT1-MMP Clin. Med. InIn In(STZ)/Urine Invivo/2020, vivo In In 9, (STZ)/Proximal vivo(STZ)/Proximal C57/BL6 xvitro FOR (High-fat S-D PEER Podocytes Urinary (STZ) rats Podocytes REVIEW high sucrose Enz. (HG)diet) Gene Gene Activity Protein expr. expr. Gene expr. expr. expr. expr. 325–10 5–10 32 21wks wks 165–10 wks wks lesionsB6 Induced bymice: None IIAGE & Ang- 9II glomerular ROP mice: [57][57] [63] [56] [25] 7 of 17 [62] In vivo MMP-2 MMP-9 MMP-2 MMP-2 MMP-9 MMP-9In In Invitro vitro In vitro vitro vivo/ vivo In vitro Mesangial In(STZ)/Proximal vitro Mesangial S-Dtubular tubular rats cells Rats/Glomeruli C57BL/6 Renal cells c. (STZ) c. S-D(HG)rats (HG) mice cortex Gene Enz. Enz. Gene Gene Enz. & activity activity expr. Enz. Protein expr. Enz. activity activity 32 activity 24 wks wks wks25–10 16wks wks days InducedKO-Mmp2 Ø, II AGElesions by Minimal &= Ang- Interstitial renal injury [60] [57] [60] [58] [62] [59] MMP-9 In vivo/ Enz. activitiy MMP-2 &MMP-2 MMP-2 MMP-2 Glomeruli MMP-2 In vitro Invivo/ vivo In vitro db/db sucrose OLETF (STZ)/Proximal vivotubular (HG) mice/ diet) ProteinEnz. (STZ)/Glomeruli (High-fat c. high Expr. Gene Enz. Enz. Gene & Protein &activity Protein activity activity Protein KO-Mmp2 40–50 wks expr. 32 4–24 wks 21 days daysAttenuated Attenuated wks ROP NTT-MMP-2 ROP byby lesions HyperglycemiaIImice: lesions Hyperglycemia Ramipril MMP-9, Ramipril Hyperglycemia None Øglomerular MMP-2 [57] [59] [53] [63] MMP-9 In vitro (HG) C57BL/6J Enz. expr. activity Attenuated mice: by MMP-9, Ramipril Ø MMP-2[56] MT1-MMP MMP-9 MMP-2 In vitro In Cortex InRats/Glomeruli vivotubularUrinary &c.&S-D BN sucrose C57BL/6 Tubules (STZ)/ rats levels diet) mice expr. expression Gene Gene expr. wks wks 164 days wks wksAttenuated FL-MMP-2 MMP-2 Mmp-2, Positive Mmp-2, Induced Ø, by compensatory activity correlation by lesions Ramipril Minimal Mmp- AGE & with Mmp- Ang- Interstitial [25] MMP-9 In vivo Wistar rats (STZ) In vivo/ C57BL/6 Renal S-D rats Enz. mice cortex activitiyEnz. activity 8 wks 2 Ø, Hyperglycemia Minimal Interstitial [64] MMP-2 In In vivo/ vivo medulla (STZ)/Proximal (STZ)/Cortex & Gene expr. 32 wks Hyperglycemia Attenuated KO-Mmp2 =IIrenal injury [57] MMP-2 MMP-2MMP-2 MMP-2 In In vivo vitro B6vivo & ROP S-D ratsexpr. (High-fat mice/ (STZ)/Glomeruli (High-fat high (HG) high Protein Enz. Enz. expr.& activity activity Protein expr. 21 21 wks 6 wks wks Mmp-2, Normoglycemia9by9 Normoglycemia lesions lesions perindropil None None Mmp- albuminuria Normoglycemia glomerular glomerular [63] [53] [52] [63] MMP-2 MMP-9 MMP-2 MMP-9 In vitro S-D C57/BL6 rats (STZ)tubular medulla c. Gene Gene expr.KO-Mmp2 expr. Enz.&activity 5–10 5 days Mmp-2, Induced by B6 9 mice: AGE & Ang-Mmp- [54] InIn vivo/ In vivo vivo S-D S-D ratsrats (STZ) (STZ) sucrose Mesangial Kkay diet) Gene cells Gene expr.Protein Protein 24 24wks wks Attenuated lesions by Ramipril [58][58] MMP-2 MMP-2 MMP-9 vitro In(STZ)/Proximal Mesangial vivo cells & Protein sucrose Tubules Kkay diet)Gene expr. expr. Gene & expr. Protein 4 wks II9 lesions [60] MMP-2 MMP-9 MMP-9 In vitro MMP-9 In vivo InS-D rats db/db vivo (HG) (STZ) mice/ S-D ratsEnz. Podocytes mice/Glomeruli ICER Enz. Gene Enz. *Gene activity &activity Protein expr. activity expr. 32wks 24 4–24 16 wks wks 216 wks days Normoglycemia NTT-MMP-2 wksNormoglycemia Hyperglycemia ROP mice: [57] [58] [61] [61] J. Clin. Med. 2020, MMP-2 Zucker 9, Inxvivo diabetic In FOR vivo In PEER fatty vivo/ S-D REVIEW rats Enz. Iγ mice/Glomeruli (STZ) S-D Enz. activity activity expr. 24& wks 40 wks MMP-2 MMP-2 Mmp-2, by activity activity Mmp- 7 of 17 [59] [58] In vivo MMP-9 MMP-2 MMP-2 In vitro In tubular vivo Kkay Cortex Kkay c. & rats (HG) (STZ)/Glomeruli mice/Glomeruli Gene Gene Enz. & expr. Enz. Gene &activity Protein Protein activity Protein expr. & Protein 20–28 wks wks 5 Attenuated days FL-MMP-2 Correlated Ramipril with podocituria [55] [53] [54] MMP-9 MMP-9 MMP-2 MMP-9rats/Glomeruli In vivo In vitro Urinary Mesangial activity cells Protein expr. 16 wks 2–3 Attenuated MMP-2 Attenuated days by Hyperglycemiaactivity byperindropil perindropil MMP-9, 9Ø MMP-2[61] [61] [65] In vitro MMP-9 MMP-2In vivo mice/Glomeruli medulla & C57BL/6J Tubules Podocytes (STZ)/* * expr. expression expr. Gene Gene 16 wks 4 wks expr. 24–48 h2–3 days MMP-2 MMP-9, activity Ø MMP-2 MMP-2 MMP-2 S-D rats/Podocytes In (HG) In mice/Glomeruli vivo B6 &BN C57/BL6 vitro ROP C57/BL6 ratsS-D mice/ (STZ) rats (STZ) Podocytes (STZ) Protein ICER Iγ expr. Gene Gene& expr. Protein expr. 16 wks Mmp-2, Attenuated 245–10 by and Normoglycemia albuminuria compensatory perindropil Mmp- [25] [58] [62] MMP-2 In InIn vivo/ vitro Renal OLETF cortex Enz.activity activity 40–50 5–10 Attenuated B6 mice: = renal injury [62] MMP-2 MMP-2 MMP-9 MMP-2 MMP-9 MMP-9 MMP-2 Invitro In vivo In vivo C57/BL6 (STZ)/Cortex Mesangial db/db vitro db/db (STZ) mice/ S-D cells mice/ (HG) rats &(HG) Enz. mice/Glomeruli (HG)Gene expr. activity Gene Gene Gene Enz. Enz. Protein Enz. expr. &expr. &Protein Protein activity expr.expr. 2–3 activity 5–10 &days 40 4–244–24 5days wks days MMP-9, NTT-MMP-2 9byØperindropil KO-Mmp2 NTT-MMP-2 MMP-2 [60] [55] [56] [54] MMP-2 MMP-2 MMP-2 MMP-2 MT1-MMP In In vivo vivo S-DIn vitro Podocytes C57/BL6 Rats/Glomeruli (STZ) * Mesangial GeneGene Enz. cells expr. KO-Mmp2 activity Protein 2–3 expr. days 65–10 wks wks wks days MMP-9, Ø MMP-2 MMP-2 MMP-9, activity ØØMMP-2 [52] [59][59] MMP-2 InIn vivo vitro Podocytes db/db rats (STZ) mice/ * Gene Gene & expr. Enz. Protein 24 activitiy 4–24 wks Normoglycemia NTT-MMP-2 Hyperglycemia MMP-9, MMP-2 [58] [62] MMP-9 MMP-2 MMP-2 In In In vitro vitro vivo medulla Cortex(HG) Cortex (HG) && Enz. Enz. Enz. activity expr. expr. Gene & Protein activity activity 5–10 wks wks ROP FL-MMP-2 FL-MMP-2mice: by perindropil Attenuated [62] [59] db/db mice/ Gene & Protein 4–24 NTT-MMP-2Ø, Minimal MMP-9 MMP-9 MMP-2 MMP-2 MMP-9 In vivo In Invivo In vivo/ In vivo/ vivo Kkay&C57BL/6 (HG) Cortex Podocytes medulla medulla S-D ICER OLETF C57BL/6 rats * ratsmice S-D mice IγGene Enz.expr. & Protein activity Protein expr.expr.wks 16 days days 240–50 days FL-MMP-2 wks 40 wks MMP-9, MMP-2 MinimalInterstitial Ø,activity Interstitial [59] [61] [55] [56] MMP-2 Cortex In mice/Glomeruli vivo C57/BL6 & (High-fat (STZ) expr. expr. Protein wks Hyperglycemia FL-MMP-2 Ø MMP-2 Induced by glomerular AGE & Ang- MMP-2 MT1-MMP MMP-9 MMP-2 In vivo/ vitro InInvitro medulla vivo (High-fathigh mice/Glomeruli (STZ)/Glomeruli Rats/Glomeruli high Gene & Protein Gene Protein expr. expr. expr. 21wks 3221 wks wks MMP-9, lesions Ø MMP-2 Co-stained lesions None None wih nephrin glomerular [63] [53] [63] MMP-9 In B6C57BL/6 vitro & ROP(STZ)/Proximal medulla mice mice/ db/db sucrose & mice/ Tubulesdiet) Enz. Gene Enz. &activitiy Protein activity 4 wks Attenuated 4–24 wks Ø, B6 Minimal B6 by mice: Normoglycemia mice: perindropil Interstitial NTT-MMP-2 lesionsII [57] MMP-2 MMP-2 MMP-2 In C57BL/6 vivo C57BL/6J mice Protein sucrose (STZ)/tubular diet) c. expr. Gene expr. expression Gene & Protein 5–10 Ø, Minimal B6 KO-Mmp9 Interstitial mice: = lesionsFewer renal [59] MMP-2 FVB In MMP-2 mice vivo vitro In vivo/ C57/BL6 (High-fat Mesangial (STZ)high cellsOLETF Cortex S-D rats& Protein expr. expr. expr. 2–321 days wks 40–50 wks MMP-9, lesions compensatory None Ø Attenuated MMP-2 glomerular FL-MMP-2 by Ramipril [63] [60] In vivo MMP-2 MMP-2 MMP-9MT1-MMP In In vivo vivo Podocytes (High-fat * S-DEnz. high activity Protein rats (HG) Gene expr. expr. Enz. activity activity Enz. 16 21 wks activity & wks 24 wks lesions Normoglycemia wks B6 mice: Hyperglycemia Hyperglycemia Induced None glomerular by AGE & Ang- [63][25] [56] MMP-9 MMP-2 In vitro In (STZ)/glomeruliPodocytes* MMP-9 Invivo/ In Renal vitro db/db sucrose vitro (HG)cortex mice/Rats/Glomeruli diet) medulla (STZ)/Proximal Gene Enz.& Protein Gene expr. 4–245–10 5wks days NTT-MMP-2 KO-Mmp2 ROP renal lesion = mice: lesions Hyperglycemia injury [62] [66] [54] In vitro J. Clin.MMP-2MMP-9 Med. 2020, 9, In x FORvivo PEER sucrose REVIEW In vitro (HG) Kkay diet) Mesangial KO-Mmp9 Enz. & cells activity Enz. Enz. activitiy Protein expr. activity 14 days32 wks lesionsMmp-2, Podocyte II dedifferentiation 7[59] of 17 Mmp- [57] MMP-9 MMP-2 (#) In vivo Cortex & tubular c.Gene expr.Protein KO-Mmp2 16 days wks wks Hyperglycemia FL-MMP-2 MMP-9, Ø B6 mice: MMP-2 [61] mice/Glomeruli B6 B6 & &ROP MMP-9 ROP mice/ S-D mice/ overexpression ICERrats Iγ expr. Gene expr.2 days Attenuated Hyperglycemia Interrupt Normoglycemia Normoglycemia by 9 cells Ramipril junction MMP-2 In vivo/ In vivo medulla S-D rats Gene Gene Protein expr. expr. expr. Gene expr. 5–105–10 2440 wks Induced by AGE & Ang- [55] C57BL/6 mice Ø, Minimal Interstitial MMP-2 MMP-2 MMP-2 InInIn vitro InB6 vivo/ vitro vivo BN& MesangialROP rats mice/ Mesangial S-D rats (STZ) mice/Glomeruli (STZ)/Proximal cellscells Enz. 32 wks wks Normoglycemia Hyperglycemia Mmp-2,integrityII Mmp-[60][60] [58] [57] MMP-2 In vivo (STZ)/Glomeruli In vitro B6 & ROPcells (High-fat mice/ high Protein Gene Enz. Enz. expr. Enz. activity expr. activity activity activity5–10 2–3 wks days wks B6 MMP-9, mice: Normoglycemia Ø MMP-2 [53] MMP-2 MMP-2 MMP-2 MMP-9 MMP-9 In InInvivo vitro (STZ)/Cortex vitro Mesangial Podocytes (HG)(HG) &tubular * c.Protein Gene Geneexpr. expr. expr. Gene & Protein 21 wks 5–10 lesions None Stimulate ROP ROP glomerular mice: Attenuated Normoglycemia mice: MMP-2 GBM 9 by synthesis Ramipril activity [63] [60] MMP-2 MMP-2 MMP-2 In vitro In vivo/ & Mesangial sucrose Tubules cells diet)OLETF Enz. activity Gene 6 wkswks expr.5–10 4 wks 40–50 Hyperglycemialesions [52] [60] [62] MMP-9 Kkay/glomeruli In vitro In & tubular medulla In vivo (HG) (HG) Kkay B6S-D & rats Enz. ROP(STZ) Enz. Gene mice/ activity activity & Proteinexpr. wks days 24wkswks ROP mice: Attenuated Negatively ROP Hyperglycemiamice: Hyperglycemia by perindropil Normoglycemia correlatedwith [56] [58] MMP-9 MT1-MMP In vitro vivo S-D (HG) vitro rats Rats/Glomeruli (HG) Enz. activity Enz.&activity Gene expr.16 wkswks5–10 MMP-9, ØMmp-2, MMP-2 Mmp- [61] MMP-9 In vivo MMP-2 MMP-9 MMP-2 In In Podocytes mice/Glomeruli vitro Gene * C57/BL6 Mesangial & Protein (STZ) cells expr.Enz. expr. activitiy 58–20days Hyperglycemia [54] [67] [60] MMP-2cells Mesangialmice cells Protein expr. Enz. activity wks Minimal MMP-2miR-21 9 activity Hyperglycemia MMP-2 MMP-2 B6C57BL/6 & ROP mice/ ICER Iγdb/dbS-D (HG)Gene & Protein rats mice/ Gene Gene & expr. Protein 4–24Ø, Normoglycemia Interstitial Induced Attenuated NTT-MMP-2 ROP by by mice: AGE & Ang- perindropil In vivo In vivo/ In vivo (High-fat S-D high rats (STZ) Gene Protein expr. Gene expr. expr. 5–10 2–3 21 days 24 wks MMP-9, None Ø Normoglycemia Normoglycemia MMP-2 [58] [59] MMP-2 MMP-2 MMP-2 InIn In vivo C57BL/6J vitro Mesangial (STZ)/ cells * Gene (STZ)/Proximal expression expr. Protein expr. Enz.expr. activity 40 wks wks lesions compensatory glomerular [63] [55] [60] MMP-7 In vivo MMP-2 MMP-9 S-D ratsMMP-9 vivo (STZ)/Glomeruli In vitro InRenal vitro Podocytes mice/Glomeruli sucroseKkayKkay Cortex C57/BL6 diet) & (STZ) Enz. Gene Gene expr. Gene activity 16 wks Enz. activity &activity &Protein Protein wks 32 wks32wks 5–10 wks Normoglycemia FL-MMP-2 exacerbated II Hyperglycemia by [25] [62] [57] [68] MMP-9 MMP-9 MMP-9 InIn vivo vivo cortex (HG)(HG) Enz. Enz. tubular medulla activity c. activity Enz. 16 16wks wks KO-Mmp2 ROP=lesionsmice: renal Normoglycemia MMP-2 injury aminoguanidine Attenuated by activity+ [61] Ramipril [61] Kkay db/db mice/ Gene Gene & & Gene Protein Protein & Protein days 4–24 NTT-MMP-2 MMP-2 MMP-2 MMP-9 InInvivo vivo/ In mice/Glomeruli vivomice/Glomeruli OLETF Kkay Cortex &Gene KO-Mmp2 expr. expr. & Protein 16 40–50 wks MMP-9,Attenuated Ø MMP-2 by B6 mice: perindropil[61] [59] MMP-2 MMP-9 In vivo mice/Glomeruli expr. expr. expr. 16 wks wks Hyperglycemia FL-MMP-2 Mmp-2, Mmp- [56] [61] MT1-MMP Goto-Kakizaki vitro/cortex Inrats C57BL/6rats C57/BL6 Rats/Glomeruli S-D mice/Glomeruli mice Protein (STZ) expr. expr. 2 2–3 days 2–3 wks days days Ø, Minimal Normoglycemia ØInterstitial MMP-9 In vivo MMP-2 MMP-2 MMP-2 MMP-2 In vivo/ Podocytes Podocytes *medulla db/db *Kkaymice/ Enz. Gene Geneactivitiy expr. expr. Gene & Protein 18 wks 4–24 MMP-9, MMP-9, ØMMP-2 preceding MMP-2 Hyperglycemia NTT-MMP-2 9 glomerular [69] MMP-2 & medullaIn InIn vivo vitro (STZ)/Glomeruli vitro In vivo (High-fat high Enz. Protein Protein activity expr. expr. 2–3 Gene expr.5–10 21 days wks 5–10 16 wks lesions MMP-9, None Ø glomerular MMP-2 lesions [53] [63] [62][62] MT1-MMP MMP-9 MMP-2 MMP-2 MMP-9 MMP-9 In vitro MMP-9 InInvitro In& Podocytes vivo (HG) S-D Tubules sucrose (HG) rats S-D * rats (STZ) diet)Cortex mice/Glomeruli Gene & Enz. Enz. expr. activity activityexpr. 2–3 4 wks5–10 days 24wks MMP-9, wksInduced Normoglycemia byØAGE lesions B6 MMP-2 FL-MMP-2 mice: & Ang- [62] [59] [61] [58] MMP-2 MMP-9 vivo/ Podocytes (HG) * Gene Gene Enz. activity expr. expr. Enz. activity days days MMP-2 MMP-9 In vitro (STZ)/Proximal Kkay B6 &medulla ROP Gene mice/ & activity Protein 32 5–10 wks MMP-9, MMP-9, ØØ II MMP-2 MMP-2 Hyperglycemia Normoglycemia [62] [57] MMP-9 Invivo vitro S-D rats (HG) (HG) Enz. Enz. Enz. activityactivity &Gene expr. days MMP-2Øactivity MMP-9 MMP-2 In In vitro C57BL/6 C57BL/6 tubular micemice c. 16 days 5 days wks 2–3 5–10 days Ø, MMP-9, Ø, Minimal Minimal ØInterstitial MMP-2 MMP-9, Interstitial MMP-2 [61] [54] MMP-2 MMP-2 MMP-2 Inmice/Glomeruli vitro Mesangial Mesangial cells Podocytes * expr. cells Protein expr. Gene expr. Attenuated MMP-9, Attenuatedby Ø MMP-2 B6 by Ramipril mice:perindropil [60] InIn vivo In C57BL/6 vivo vitro (High-fat (High-fat micehighhigh Protein Protein Enz. activity expr. expr. 2121 wks 5–10 wks wks Ø,lesions Minimal lesions NoneNone Interstitial glomerular glomerular [63][63] [62] MMP-2 MMP-9 MMP-9 MMP-9 C57BL/6 ICER Iγ B6 mice C57/BL6 (HG)(STZ) Enz. activity Ø,MMP-9, Minimal ROP mice: Interstitial Normoglycemia MMP-2 In vivo (High-fat sucrose sucrose high diet) & ROP diet) mice/ Protein expr. 2–3 21 days wks lesions days Mmp-2, None lesionsØ Hyperglycemia MMP-2 glomerular lesions Mmp- [63] MMP-2 MMP-2 MMP-9 MMP-2 In vivo Invitro Podocytes vivo mice/Glomeruli (High-fat * high Protein Gene Protein expr. expr. Gene expr. 40 expr.wks 21 wks 5–10lesions None MMP-9, glomerular [55] Ø MMP-2 [63] MMP-2 In In vitro sucrose diet) db/db Mesangial mice/ cells Gene & Protein 5–10 4–24 lesions 9 NTT-MMP-2 Hyperglycemia [62] [60] MMP-9 MMP-2 MMP-9 In vivo (HG) diet) Enz. activity wks [59] MMP-2 In vivo sucrose S-D rats (STZ) C57BL/6 Cortex (HG) &Enz. mice Gene Geneactivity expr. & Protein expr. days 24 wks wks Ø, Minimal lesions FL-MMP-2 ROP Interstitial mice: [58] MMP-2 In vivo/ In vivo OLETF B6 &medulla (High-fat ROP mice/ high Enz. activityProtein 40–50 expr. wksMMP-9, 215–10 lesions Normoglycemia Ø MMP-2 None glomerular [63] MMP-9 MMP-9 expr. Gene expr. [56] MT1-MMP MMP-2 In vitro In C57BL/6 vitro Rats/Glomeruli Mesangial micesucrose Enz. cells diet) wks Ø, MinimalMMP-2 Hyperglycemia Normoglycemia activity Interstitial lesions [60] MMP-2 activitiyEnz. activity wks B6 mice: In vivo (High-fat high Kkay (HG) Protein expr. Gene & Protein21 wks Attenuated lesions Noneby ROP mice: perindropil glomerular [63] MMP-9 MMP-9 In vivoS-D ratsdiet) 16 wks Hyperglycemia [61] In vivo/ sucrose C57/BL6 (STZ) mice/Glomeruli Gene expr. expr. Induced by AGENormoglycemia lesions & Ang- MMP-2 (STZ)/Proximal 32 wks Hyperglycemia [57] In vitro db/db mice/KkayEnz. Gene & Protein activity 4–24 II NTT-MMP-2 MMP-2 MMP-9 In vivo In tubular c. & Gene & Protein 2–3 wksdays MMP-9, Ø MMP-2 [59] MMP-2 vivoCortex Podocytes * expr. Gene expr. wks 16Attenuated by Ramipril FL-MMP-2 Normoglycemia [61] In vitro B6 & ROP mice/ mice/Glomeruli expr. 5–10 [62] MMP-9 (HG) cells Geneactivity Enz. expr. 5–10 Normoglycemia MMP-2 In vitromedulla Mesangial days Mmp-2, Mmp- [60] Kkay Enz.&activity Gene Protein 2–3wks days B6 mice: MMP-9, Ø MMP-2 MMP-2 MMP-2 MMP-9 In vivo Podocytes(HG) * Gene expr. 16 wks 9 ROP mice: [61] In vitro mice/Glomeruli C57BL/6 mice Gene expr. expr. 5–10 Ø, Minimal Interstitial [62] MMP-9 In vivo MMP-2 S-D rats (STZ) (HG) Enz. activity 24 wks Hyperglycemia Hyperglycemia [58] In vivo (High-fatEnz. highactivityProtein expr. 21days wks lesions None glomerular [63] MMP-9 MMP-9 2–3 days MMP-9, MMP-9, Ø Ø MMP-2 MMP-2 MMP-2 Podocytes sucrose diet) * Gene expr. MMP-2 activitylesions In vitro C57BL/6 mice 5–10 Ø, Minimal Interstitial [62] MMP-2 MMP-9 B6 & ROP mice/ (HG) Enz. activity Attenuated by perindropil Normoglycemia Normoglycemia In vivo (High-fat highGene expr. Protein expr.5–10 21days wks lesions None glomerular [63] MMP-2 MMP-9 In vitro C57/BL6 Mesangial (STZ)cells MMP-9, Ø MMP-2 [60] sucrose Kkaydiet)Enz. activity Gene & Protein wks 16 wks lesions MMP-9 Indb/db vivo mice/ (HG) C57BL/6 mice Gene & Protein expr.4–24 NTT-MMP-2 Ø, ROP mice: Minimal Interstitial [61] MMP-2 MMP-2 In vivo mice/Glomeruli [59] In vivo Cortex & (High-fat high expr. Protein expr. wks 21 wks FL-MMP-2 lesions None glomerular [63] MMP-9 2–3 days Hyperglycemia MMP-9, Ø MMP-2 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 17 J. Clin. Med. 2020, 9, x FOR PEER REVIEW BN rats 7 of 17 In vivo (STZ)/Cortex & Gene expr. MMP-2 6 wks [52] In vitro medulla BN rats Enz. activity In vivo Podocytes *& (STZ)/Cortex Gene expr. MMP-2 6 wks [52] J. Clin. Med. 2020, 9, 472 In vitro medulla Gene Enz.&activity Protein 8 of 17 C57BL/6J (STZ)/ Podocytes * expression compensatory MMP-2 In vivo 16 wks [25] Renal cortex Enz.&activity Gene Protein KO-Mmp2 = renal injury C57BL/6J (STZ)/ KO-Mmp2 expression MMP-2 In vivo 16 wks compensatory [25] Renal cortex Enz. activity J. Clin. Med.Med. J. Clin. 2020,2020, 9, x FOR PEER 9, x FOR REVIEW InPEER vivo/ REVIEWS-D rats Table 2. Cont. 2 days KO-Mmp2 = renal injury 7 of 717of 17 MMP-2 (STZ)/Glomeruli KO-Mmp2 Protein expr. [53] In vitro &S-D Tubules rats 24 days wks J. Clin. Med. 2020, In BN FORrats 9, xvivo/ BN ratsREVIEW PEER 7 of 17 PEER REVIEW 7 expr. of 17 Age/Exposure MMP In Vivo/in Vitro Species MMP-2 (Model)/Cell In vivo In vivo InType (STZ)/Cortex (STZ)/Glomeruli (STZ)/Cortex vitro S-D & rats& (HG) Condition Studied Gene expr. Gene Protein Enz. expr. activity & Outcome [53] Ref MMP-2 MMP-2 MMP-2 & Tubules Mesangial cells 6 wks Protein expr. 6 Time wks 5 days 4 wks [52] [52] [54] In vitro In vitro medullamedulla Enz. activity Enz. activity J. Clin. Med. 2020, 9, Podocytes x FOR PEER BN rats REVIEW 7 of 17 *S-D Podocytes Protein ICER Iγ expr. Enz. activity & *rats (HG) MT1-MMP In vivo S-D ratsMMP-2 (STZ)/Glomeruli In vitro In vivo (STZ)/Cortex & Protein Gene expr. 4–48 wks expr. 40 wks 5 days [70] [55] [54] BN rats MMP-2 Inmunogold mice/Glomeruli Mesangial cells Gene & ProteinProtein expr. 6 wks [52] In vitro medulla Gene & Protein Enz. activity (STZ)/Cortex & Gene expr. C57BL/6J C57BL/6J(STZ)/ BN rats ICER (STZ)/ Iγexpression Gene & Protein In vivo In In vivo/ Gene OLETF Podocytes * expression & Proteinexpr. 12 wks40 wks compensatory 40–50 compensatory Meprin In vivo6 wks MMP-2 MMP-2 MMP-2 In vivo C57BL/6 MMP-2 (STZ) In vitro vivo InRenal vivo cortex Renal (STZ)/Cortex mice/Glomeruli cortex Enz.& [52] activity Enz. Protein16 Gene activityexpr. wks expr. expr.16 wks KO-Mmp2 = renal injury KO-Mmp2 = renal injury [25] [25] [55] [56] [71] medulla Enz. activity MT1-MMP In vitro Rats/Glomeruli KO-Meprin Gene medulla Enz. Enz. & Protein activity activitiy18 wks6wks wks KO-Meprin = renal injury [52] KO-Mmp2KO-Mmp2 Gene & Protein MMP-2 In vivo/ C57BL/6J OLETF (STZ)/ expression 40–50 compensatory Podocytes * MMP-2 (db/db)/podocytes In vivo S-D S-D &vivo/ rats rats Podocytes S-D cortex Renal rats * Enz. expr.2 days 16 2 days wks wks Induced by AGEinjury &byAng- [25] [56] MMP-10 In vivo MT1-MMP In 7 In vivo/ vivo/ In In vitro of(STZ)/Glomeruli 17 Rats/Glomeruli Gene expr.expr. Geneactivity Gene expr. 8–16 wks KO-Mmp2 , attenuated = renal [72] MMP-2 MMP-2 MMP-2 juxtaglomerular (STZ)/Proximal (STZ)/Glomeruli Protein Enz. & Protein expr. Protein activitiy 32 wks [53] [53] [57] Gene & Protein In vitro In vitro In vitro C57BL/6J (STZ)/ KO-Mmp2 Enz. activity expression II telmisartan MMP-2 In & vivoTubules & Tubules tubular S-D ratsc. 4 wks4 wks 16 wks compensatory Attenuated by Ramipril [25] S-D cortex rats 2 days Induced by AGE & Ang- C57BL/6J (STZ)/ expression In vivo/ compensatory Renal Geneactivity Enz. expr. KO-Mmp2 ==renal KO-Mmp10 Fewer renal injury MMP-10 16 wksMMP-2 In vivo MMP-2 C57BL/6 (STZ) InS-D vivo/rats rats S-D (HG)(STZ)/Proximal (HG) Enz.Enz. KO-Mmp10 activity [25] & activity &activity 17 wks32 wks II [57] [73] MMP-2 MMP-2 In vitro In In vitro vitro In vitro (STZ)/Glomeruli Protein Enz. KO-Mmp2 5expr. days 5 days Mmp-2, lesion Mmp- [54] [54] [53] Renal cortex Enz. activity KO-Mmp2 MMP-2 Mesangial = renal injury Mesangialcells tubularProtein cells & Tubules c. Protein expr.expr. 4 wks Attenuated 9by Ramipril Gene expr. MMP-3 ICERICERIγ S-D S-D rats Gene expr. 2 days KO-Mmp2 In In vivo/ vivo Iγ rats (STZ) 24 wks [58] 6 wks MMP-1 In vivo MMP-2 S-D ratsMMP-2 MMP-2 MMP-2In [52] (STZ)/glomeruli In vivo vivo In vitro mice/Glomeruli S-D rats Gene (HG) (STZ)/Glomeruli expr. Protein expr. Protein Enz. expr.activity Protein 40 & wks 4–24 wks 40 expr. Enz. activity wks 5 days Mmp-2, by Insulin , Prevented Mmp- [55] [55] [74,75] [53] [54] Enz. activity MMP-9 MMP-2 mice/Glomeruli In vitro Mesangial & Tubules cells Protein expr. 4 wks MMP-2 9 activity S-D rats 2 daysMMP-2 KunmingInmice MMP-2 vivo/ (alloxan) In vivo/ In vivo OLETF S-D Gene ICER GeneGene & & Protein Iγ Protein rats (STZ) & Gene expr. Protein 40–50 24 wks [58] MMP-1 In vivo MMP-2 In vivo OLETF S-D rats (HG)expr.expr. Enz. Enz.activity Protein activity 40–50 expr.& 8 wks40 wks Attenuated by perindropil [56] [56] [76] [55] (STZ)/Glomeruli Protein expr. MT1-MMP MT1-MMP MMP-2 (BGF) MMP-9 In vitro In vitro In vitro Rats/Glomeruli Rats/Glomeruli C57/BL6expression mice/Glomeruli Mesangial (STZ) cells [53]Protein expr.wkswks 5 days MMP-2 activity [54] ene & Protein Enz. activitiy Enz. activitiy Gene & Protein & Tubules MMP-1 In vivo 4 wks Albino MMP-2 rats In vivo/ S-D S-D db/db ICER rats rats OLETF mice/ Serum& Iγ levels Gene & Protein 14–42 days 4–24 40–50 Attenuated NTT-MMP-2 by perindropil [59] [77] expression compensatory MMP-2 In vivo/ In vivo In vivo/ levels (alloxan)/circulating Cortex mice/Glomeruli C57/BL6 Gene (STZ) Protein expr.expr. Gene expr. expr. expr. 40wks wksInduced Induced by AGEby AGE & Ang- & Ang- FL-MMP-2 [55] [56] MMP-2MT1-MMP In vitro Rats/Glomeruli 32 wks wks Enz.S-D rats (HG) 16 wks activity Enz. activity & MMP-2 In vitro [25] (STZ)/Proximal In vitro (STZ)/Proximal medulla db/db Enz.Enz. mice/ activity activity Gene & 32 wks Enz. activitiy Protein 4–24 II II NTT-MMP-2 [57] [57] KO-Mmp2 = renal 5 daysinjury MMP-2 In tubular vivo tubular In vivo/ c. c.OLETF [54] expr. 28 days40–50 Attenuated Prevented by Ramipril diabetic renal [59] Mesangial cells MMP-1 expr. Protein In vivo C57BL/6 (STZ)/renal cortex S-D Mmp1 Cortexrats& DNA wksAttenuated by FL-MMP-2 Induced Ramipril B6bymice: AGE & Ang- [78] [56] KO-Mmp2 MT1-MMP MMP-2 In Invivo/ vitro Rats/Glomeruli (STZ)/Proximal medulla Gene expr. Enz. activitiy 32wks wks fibrosis II [57] In vitro Enz. activity Mmp-2, Mmp-2, Mmp- Mmp- Hyperglycemia ICER Iγ tubular c. 9 B6 mice: 9Attenuated by Ramipril 2 daysProtein expr. Abbreviations: wksMMP-2 MMP-2 40increase/upregulation, S-D ratsGeneGene decrease/down-regulation, [55] no changes, BN:Induced Brown by AGE & Ang- STZ: Norway, 24Ø expr.expr. mice/Glomeruli MMP-2 In vivo In In vivo S-Dvivo/ rats rats S-D (STZ) (STZ) (STZ)/Proximal Gene expr. wks 24 wks 32 wks Hyperglycemia [58] [58] [57] Protein expr. [53] B6 & ROP Enz. mice/ activity Enz. activity Mmp-2, II Normoglycemia Mmp- streptozotocin-induced diabetes,MMP-9 MMP-9 HG: hyperglycaemia tubular In vitro exposure, c. B6: B6SJLF1/J Enz. Gene activity expr. sclerosis-resistant 5–10 mice, ROP: ROP/Le- Gene & Protein MMP-2 MMP-2 In vitro Mesangial cells MMP-2MMP-2 activity Attenuated activity9by Ramipril OLETF 4 wks Es1b/ES1a sclerosis-prone 40–50 mice, Alloxan: alloxan-inducedIn vivo S-D rats diabetes, Gene Enz.S-D:expr.Sprague-Dawley activity 24wks Attenuated rats, by perindropil ICER Iγ: ICER Iγ[60] expr. B6 & ROP (HG)(STZ) mice/ [56]Enz. activity wks Attenuated byNormoglycemia perindropil ROP mice: Mmp- Mmp-2, [58] nz. Rats/Glomeruli activity & transgenic mice, OLETF: wks Otsuka Long-Evans MMP-9 MMP-2 Tokushima C57/BL6 In vitro (STZ) C57/BL6Mesangial (STZ) Fattycells rats, C57BL/6: Gene expr. C57 black 5–10 6 strain, KO-Mmp2: MMP-2 9 activity MMP-2[60] Enz. activitiy Enz. activity wks Hyperglycemia 5 days knockout mice, FVB: friend MMP-2 leukemia MMP-2 In vivo In [54] virus vivo In B vivo strain, db/db mice/ Goto-Kakizaki db/db mice/ S-D Gene (HG)(STZ) rats & Protein Gene Gene & Protein expr. rats: non-hypertensive 4–24 4–24 24 wks model NTT-MMP-2 NTT-MMP-2 Attenuated ROPofbymice: type 2[59] diabetes, perindropil [59] [58] Protein expr. Cortex & & Cortex expr.expr.Enz. activity wkswks FL-MMP-2 FL-MMP-2 S-D rats (*) immortalized mouse podocyte cell line, Ang-II: MMP-9 Angiotensin C57/BL6 (STZ) II, gene expr.: gene expression Hyperglycemia determination, enz. Gene expr. Induced by AGE medulla & medulla Ang- + aminoguanidine: db/db mice/ Gene & ProteinAGE4–24 MMP-2 activity Normoglycemia NTT-MMP-2BGF: blood Protein expr. (STZ)/Proximal 40 wks activity: enzymatic activity determination, 32 wks MMP-2[55] Inwks: vivo weeks, IIIn vivotreated Kkay & Cortex [57]Geneexpr. & Protein formation wks B6 mice: inhibitor, Attenuated B6 mice: by perindropil FL-MMP-2 [59] Enz. activity glucose fluctuation model, #: podocytes MMP-9 were with C57/BL6 D-glucose, (STZ) advanced 16 wks glycoprotein end-product bovine[61] mice/Glomeruli expr. Normoglycemia tubular c. medulla Hyperglycemia Hyperglycemia ene & Protein serum albumin, angiotensin II,Attenuated VEGF, MMP-2 TNF-alfa by Ramipril db/db mice/ In vivo and TGF-beta1. Kkay Gene & Protein Gene & Protein 2–3 4–24 days NTT-MMP-2 MMP-9, B6 mice: Ø MMP-2 [59] 40–50 MMP-9 MMP-2 In vivo Cortex & * Podocytes mice/Glomeruli expr. Gene expr. expr. 16wks wks FL-MMP-2 [61] expr. MMP-9 [56] In & Mmp-2, B6 & ROPMmp- B6 vitroROP mice/ medulla mice/(HG) GeneGene Enz. activity 5–10 Normoglycemia Hyperglycemia Normoglycemia [62] wks expr.expr. 5–105–10 2–3days days MMP-9, Ø MMP-2 MMP-2 Enz. activitiy MMP-2 MMP-2 MMP-2 In vitro In vitro MesangialMesangial 9In vitro cellscells Podocytes Enz.* activity Enz. Gene activity expr. wks wks MMP-9, B6 mice: Ø [60] [60] Gelatinases (MMP-2 and -9) are the most widely studied MMPs in DN. Early compensatory Gene expr. MMP-9 (HG)(HG) B6C57BL/6(HG)mice & ROP mice/ Enz. activity 5–10 ROPROP mice:mice: Ø, Minimal Interstitial Normoglycemia Hyperglycemia [62] S-D rats (STZ) Induced by 24 wks& Ang- MMP-2 AGE In vitro vivo (High-fat high [58]Protein Gene expr. expr. days 215–10 wks Hyperglycemia lesions MMP-9,NoneØglomerular MMP-2 [63] Gene expr. increases and late decreases in MMP-2 expression and enzymatic activity have been reported in 32 wks Enz. activity MMP-2 MMP-9 [57] In Mesangial sucrose C57BL/6 cells (HG)diet) mice Enz. activity wks Hyperglycemia Ø, Minimal lesions ROP Interstitial mice: [60] Enz. activity II MMP-2 MMP-2 activity B6 & ROP mice/ Normoglycemia streptozotocin (STZ) diabetic rats [52]. Consistently, Mmp2 null mice worsened renal lesions namely Attenuated by Ramipril Attenuated MMP-9 MMP-2 In vivo In vitro (High-fat high Mesangial cells Protein expr. Gene expr. 215–10 wks lesions None glomerular Normoglycemia Normoglycemia Hyperglycemia [63] [60] by perindropil Kkay sucrose diet) Kkay (HG) GeneGene & Protein Enz. activity & Protein wks lesions ROP mice: C57/BL6 (STZ) urinary albumin excretion, accumulation of ECM in the glomeruli, atrophy and fibrosis in the MMP-9 MMP-9 In vivo In vivo mice/Glomeruli mice/Glomeruli expr.expr. 16 wks16 wks [61] [61] Mmp-2, Mmp- Normoglycemia Hyperglycemia db/db mice/ tubulointerstitium of STZ-diabetic mice [25]. Han et al. observed an increase in MMP2 expression Gene & Protein 9 4–24 NTT-MMP-2 Kkay Gene & 2–3Protein days 2–3 days MMP-9, MMP-9, Ø MMP-2 Ø MMP-2 MMP-2 MMP-9 InPodocytes vivoPodocytes 16 wks [61] mice/Glomeruli [59] expr.5–105–10 MMP-2 * * GeneGeneexpr.expr. Gene expr. In vitro In vitro Normoglycemia [62] [62] Cortex & after 2 days of hyperglycaemia, although after 4 weeks MMP2 expression decreased as a response to 24 wks expr. wks MMP-9 MMP-9 FL-MMP-2 [58] (HG)(HG) Enz.Enz. activity activity daysdays Enz. activity medulla MMP-9 MMP-2 In vivo Kkay Podocytes * Gene & Protein Gene expr. 2–3 days 16 wks MMP-9, MMP-9, Ø MMP-2 MMP-9, Ø MMP-2 Ø MMP-2 [61] the expression of TIMP-2 in glomeruli from Sprague-Dawley (S-D) rats [53]. These results suggest MMP-2 activity In vitro C57BL/6 C57BL/6 mice/Glomeruli mice mice expr. 5–10 Ø, Minimal Ø, MinimalInterstitial Interstitial [62] MMP-2 MMP-2 MMP-9 B6 mice: (HG) Enz. activity In vivo In vivo (High-fat highhigh Protein (High-fat expr.expr. 21 wks Protein dayslesions 21 wks lesions None Noneglomerular glomerular [63] [63] that an imbalance within the MMP-2 and TIMP-2 system, plays an important role in the pathogenesis Attenuated by perindropil MMP-9 MMP-9 MMP-2 sucrose Podocytes * diet)diet) sucrose Gene expr. 2–3 days lesions MMP-9, Ø MMP-2 lesions In vitro C57BL/6 mice 5–10 Ø, Minimal Interstitial [62] MMP-9 MMP-2 Hyperglycemia of diabetic nephropathy. Singh et al. observed, after 5 days of hyperglycaemia, that mesangial In vivo (HG) (High-fat high Enz. activity Protein expr. days 21 wks lesions None glomerular [63] MMP-9 MMP-9, Ø MMP-2 ene & Protein 4–24 NTT-MMP-2 sucrose diet) lesions cells presented a 25% reduction in MMP-2 activity, resulting in mesangial matrix accumulation MMP-2[59] C57BL/6 mice Ø, Minimal Interstitial expr. B6 & ROP mice/ wks FL-MMP-2 MMP-9Normoglycemia In vivo (High-fat high Protein expr. 21 wks lesions None glomerular [63] mediated by angiotensin II type 1 (AT1) receptors via TGF-β1 [54]. Decreased glomerular MMP-2 Gene expr. 5–10 sucrose diet) lesions Mesangial cells [60] Enz. activity expression accompaniedwks augmented (HG) B6 mice: ROP collagen-IV mice: expression and glomerulosclerosis in advanced DN [55]. In Hyperglycemia contrast, increased expression and activity was found in glomeruli of diabetic rats in Hyperglycemia association with mesangial expansion [56]. Proximal tubular cells also showed enhanced MMP-2 activity induced by advanced glycation Normoglycemia end products (AGE) and angiotensin-II [57]. McLennan et al. Normoglycemia Gene expr. 5–10 Kkay analysed renal gene expression and activity [60] of MMP-2 and MMP-9 at 24 weeks, observing increased Enz. activity wksGene & Protein 16 wks [61] mice/Glomeruli Mmp2expr. expressionROP but mice: decreased MMP-2 enzymatic activity and Mmp9 down-regulation. These changes were attenuated by 2–3renin-angiotensin Hyperglycemia days MMP-9,system Ø MMP-2 (RAS) inhibition [58]. Recently, two different isoforms Podocytes * Gene expr. of MMP-2 were identified 5–10 in diabetic models showing [62] differentially distribution in renal tissue [59]. (HG) Enz. activity days An interesting finding on geneticMMP-9, susceptibility Normoglycemia Ø MMP-2to DKD was described by Fornoni et al., who analysed ene & Proteinmice mesangial cells from murine strains C57BL/6 16 wks Ø, [61] Interstitial(B6) and susceptible (ROP) to sclerosis, mimicking Minimalresistant expr. (High-fat high Proteinwho expr. develop21 wks lesions an None glomerular [63]control. ROP-mice presented basal elevated patients DKD despite adequate glycaemic sucrose diet) 2–3 days MMP-9, Ø MMP-2 lesions Gene expr. levels of MMP-2, which increased even more in the presence of hyperglycaemia, although remained 5–10 [62] Enz. activity elevated when glycaemia was reduced. B6-mice presented an increment on MMP-2 when exposed days MMP-9, Ø MMP-2 to hyperglycaemia but returned to baseline levels when glycaemia was normalized. These findings Ø, Minimal Interstitial Protein expr. suggest alesions 21 wks potential role of MMP-2 in[63] None glomerular the genetic susceptibility to develop DN [60,79]. Analysing a type 2 diabetes rodent model, lesions it has been shown that renal Mmp9 expression was augmented compared with non-diabetic mice [61]. Bai et al. reported that short- or long-term exposure to hyperglycaemia J. Clin. Med. 2020, 9, 472 9 of 17 may produce diverse effects on MMP-9. After 2-3 days of hyperglycaemia, an enhanced expression and activity was related with decreased collagen-IV synthesis, while hyperglycaemic-exposure for more than 5 days revealed a down-regulation on MMP-9 expression and activity in podocytes [62]. A recent work, found no differences on MMP-2 and MMP-9 expression in high-fat high sucrose diet-induced diabetes, which presented only minimal interstitial alteration [63]. Urinary MMP-2 and MMP-9 enzymatic activities were increased in type 1 diabetic rodents, and urinary neutrophil gelatinase-associated lipocalin (NGAL)/MMP-9 ratio and MMP-9 activities augmented before the onset of albuminuria [50]. Furthermore, urinary MMP-9 excretion showed positive correlation with albuminuria in diabetic rats, and down-regulation was observed after simvastatin treatment, together with attenuation of glomerular disease [64]. Increased MMP9 staining was observed in glomerular parietal epithelial cells from Zucker diabetic Fatty rats, which was also associated with podocyturia [65]. In fact, increased MMP-9 overexpression and activity induced podocyte dedifferentiation and disruption of junction integrity [66]. MMP-9 has also been inversely correlated with miR-21 expression which plays a role on renal fibrosis on DKD [67]. Referring to matrilysins, McLennan et al. analysed mesangial cells of STZ-induced diabetic rats, observing a down-regulation on MMP-7 expression and increased fibronectin accumulation [68]. Membrane-type MMPs may also play a role in DN. Besides to modulate other MMPs, MT1-MMP (MMP-14) has shown to be up-regulated preceding the manifestation of glomerular pathologic lesions in type 2 diabetic rats [69]; although decreased expression has been reported in glomeruli of long-term diabetic mice [70]. Meprin also seems to have a protective effect on DKD [71]. Relative to stromelysins, our group recently demonstrated glomerular overexpression of MMP10, in podocytes and juxtaglomerular apparatus, in early stages of DN. Interestingly, MMP10 overexpression was prevented with telmisartan administration. These changes were observed despite the absence of significant histological lesions, moreover a correlation was observed between glomerular MMP10 and albuminuria [72]. Consistently, Mmp-10 null diabetic mice presented fewer mesangial expansion, renal macrophage infiltration and renal function impairment [73]. These findings may support a deleterious glomerular effect of MMP-10 on DN, which may be related with the RAS pathway. Another study observed decreased glomerular expression of MMP-3 and MMP-1 in diabetic rats [74]. Unlikely to enalapril [74], insulin treatment ameliorated these changes [75]. MMP-1 expression and serum levels were also down-regulated in alloxan induced-diabetic mice [76,77], while local delivery of Mmp-1 into mice kidneys prevented diabetic renal fibrosis [78]. Analysing the mechanisms by which MMPs may be altered in DN, data suggest that inflammatory pathways may regulate MMPs expression, however some MMPs may also modulate the expression of inflammatory mediators [80]. It has been reported that insulin-like growth factor 1 (ILGF-1) decreases MMP expression [81–83]. TGF-β has shown to upregulate MMP-2 [84]. Endothelin also produces an inhibitory effect on MMP-2 and MT1-MMP [85]. Podocyte Vegf164 overexpression down-regulated MMP-2 expression resulting in advanced DN [86]. Hydrogen sulphide enzymes can attenuate MMP-9 expression in the diabetic kidney and mitigate adverse ECM accumulation [87]. IL-20 may play a pathologic role in early DN through upregulation of MMP-9 [88]. Experimental studies have shown that dysregulation of MMPs expression/activity may play a role in DKD through different pathways, underlying the high complexity of the MMP-system. Therefore, in order to understand diverse outcomes from different studies, it is important to take into account: the high homology among MMPs, their diverse locations, diverse substrate specificity, regulators, the discrepancies between in vitro and in vivo models, as well as the different roles they play according to the time of evolution of the diabetes (Table 3). Taken together, MMPs expression and activity may vary in DN, and their pathobiological system is still very confounding. However, an important role seems to be played by both MMPs and their inhibitors in the hallmark of DKD. J. Clin. Med. 2020, 9, 472 10 of 17 Table 3. Crucial considerations to understand contradictory results when mimicking the experimental outcomes to human studies. Insulin [89], RAS inhibition [74,81,90], oral antidiabetics [91,92] and diverse pharmacological drugs [63,93–95] may have an effect on MMPs expression/activity. Murine models based on STZ or Alloxan-induced diabetes imitate a hypoinsulinemic state (T1D), while those which T2D (db/db, I. Kkay, OLETF) are characterized by hyperinsulinism. Moreover, the RAS inhibition is commonly used in diabetic patients with early DKD. Renin-angiotensin activation could be influenced according to the murine model employed in the study (hypertension, obesity, salt-intake). Macrophages infiltration could precede the appearance of DN signs [96]. But also, during diabetes exists a greater predisposition to urinary tract infections, which may also stimulate macrophage II. migration and could be unnoticed if screening analysis or meticulous histological inspection is not performed. The presence of macrophages due to renal infection may modify the MMPs outcomes of the study. MMPs expression/activity may vary depending on the type of the renal cell evaluated (i.e., proximal/distal tubule, mesangial, podocyte cells) [6]. Also, different conditions and time of III. evolution of disease [57] are presented according to in vitro or in vivo models. Moreover, it has been reported that murine strains may also show certain discrepancies in MMPs outcomes according to the genetic background [59]. Some MMPs share a great homogeneity, therefore certain molecular techniques may interfere in the results due to different sensitivities of the assay systems employed [97,98]. For example, MMP-3 IV. shares 82% homology with MMP-10 at the protein level, which may result in the recognition of both proteins. Clinical studies, as consequence of limitations for in vivo renal histological analysis, usually analyse the urinary activity/levels rather than renal expression. However these conclusions could present a V. wide variety of pathogenic meanings (i.e., enhanced intrarenal production vs increased tubular shedding of the protein). 6. Tissue Inhibitors of Metalloproteinases and Modulators in the Kidney TIMPs are specific endogenous inhibitors of metalloproteinases and frequently, their transcriptional regulation is related to MMPs. In fact, MMPs may modulate TIMP signaling by sequestration. Four TIMPs have been identified (TIMP-1, TIMP-2, TIMP-3 and TIMP-4). Although MMPs inhibition is the main role of TIMPs, they can also participate in metalloproteinase activation and, together with MMPs, in other biological processes such as cytokine production, inflammation, migration, cell proliferation and apoptosis [7]. All of these processes have been shown to have potential pathogenic pathways in tissue damage [99]. The role of TIMP in ECM turnover regulation can be different depending on the specific metalloproteinase inhibited and local tissue factors [17]. These multiple functions and complex interactions between TIMPs and MMPs explain how difficult is to define their pathogenic role in different pathologies and diseases [5]. Although renal expression TIMPs has not been completely characterized, all TIMPs, apart from TIMP-4, are expressed in healthy kidney. Human glomeruli express TIMP-1 and TIMP-2, and the upregulation of both has been demonstrated in glomerulosclerosis [100]. Distal convoluted tubular (DCT) expression of TIMP-2 and TIMP-3 has been described in normal kidney [5]. Disregulation of MMPs/TIMPs is implicated in excessive accumulation of ECM in CKD. In fact, suppression of MMP activity and enhanced TIMP expression are associated to fibrosis progression in CKD. Although TIMP-1 overexpression occurs in fibrosis and can promote it independently of MMP inhibition, TIMP-1 deficiency cannot prevent fibrosis due probably to other TIMP compensatory upregulation [101]. TIMPs deletion has suggested a possible protective role of TIMP-3 and fibrotic role of TIMP-2 in renal fibrosis mice models [102]. Dysregulation of MMP/TIMP has been described in clinical studies performed in patients with DKD. In patients with DKD, decreases in serum TIMP-1 and TIMP-2 levels, and increases in serum and urine TIMP-1 levels have been described in association with worsening glomerular lesions [47,72]. In contrast, in experimental DN models, decreases in some MMPs such as MMP-2 [58] and increase J. Clin. Med. 2020, 9, 472 11 of 17 of TIMP-1 [58] and TIMP-2 expression [53] have been associated to DN progression. TIMP-3 has been found to be down-regulated in diabetic nephropathy and increased TIMP-3 expression shows a renoprotective role for DN progression [103]. Furthermore, its down-regulation is associated with increased renal fibrosis [6,104]. Potential interventions to attenuate DKD involve increasing MMP-2 activity and TIMP-3 expression and/or inhibiting MMP-9, TIMP-1 and TIMP-2 expression. It has been described the positive effect of the angiotensin-converting enzyme (ACE) inhibitors, estrogens and the peroxisome proliferator-activated receptor-γ agonist in TIMP and MMPs in different models of experimental DN. ACE inhibition attenuated MMP-9, decreased TIMP-1 mRNA, and increased TIMP-2 expression in STZ-diabetic rats [58,105]. Hepatocyte growth factor (HGF) gene therapy also inhibits TIMP-1 expression in STZ-diabetic rats [106]. Fluorofenidone inhibited TIMP-1 expression in the renal cortex from db/db mice (type 2 diabetes) [107]. Dencichine and hyperoside are traditional herbal medicines that ameliorated the renal dysfunction in DN experimental models. Specifically, they increased the MMP-9/TIMP-1 ratio that is exacerbated after the oral dencichine, and subsequently decreases the degradation of ECM [108]. Hyperoside decreased fibrosis by suppressing TIMP-1 expression and promoting MMP-9 expression [109]. Iron chelation by oral deferiprone promoted MMP-9 expression and decreased TIMP-1 in diabetic rats [110]. In addition, berberine decreased MMP-9 and TIMP1/2 levels and increased MMP2 expression in diabetic rats [111]. miR-21 depletion inhibited the progression of DN by promoting TIMP-3 overexpression and inhibiting TIMP-1 expression in STZ-diabetic rats [6,89]. 7. Conclusions Taken together MMPs and TIMPs are involved in the development and progression of DN. In patients with diabetes different MMPs such as MMP-2 and MMP-9 are increased in urine, especially in patients with albuminuria and established renal injury. Moreover, it has been speculated that their upregulation may precede early diabetic nephropathy and albuminuria. Research in animal models suggested that the equilibrium between MMPs and its inhibitors is altered in DN. However, experimental studies have also shown contradictory results. These results manifest the complex regulations that exist between different MMPs, TIMPs, and other proinflammatory and profibrotic factors. MMPs and TIMPs probably modify their expression and localization throughout the renal diabetic involvement, and other elements such as drugs or associated pathologies influence these changes, which may help to explain the variability of the observed results to date. Therefore, further studies are needed to improve the characterization and knowledge of MMPs imbalance in diabetic kidney disease. Author Contributions: N.G.-F., J.M.M.-G., J.O., A.V. and M.J.S. wrote the manuscript and designed the tables. C.J.-C. wrote the manuscript and designed the figure. M.J.S., N.G.-F., A.V. and J.O. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The authors are current recipients of research grants from the FONDO DE INVESTIGACIÓN SANITARIA-FEDER, ISCIII, PI17/00257, REDINREN, RD16/0009/0030, the Integrated in the State Plan of R + D + I 2016-2018, co-financed by ISCIII, PI 15/02111 and the European Regional Development Fund (FEDER), as well as by the Plan de Investigación Universidad de Navarra (PIUNA) (2014-5). Acknowledgments: A.V. performed this work within the basis of his thesis at Department de Medicina of Universitat Autònoma de Barcelona (UAB). 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