Hippokratia 2018, 22(3) 99-104
Sampieri CL1, Orozco-Ortega RA2
1Instituto de Salud Pública, Universidad Veracruzana, Xalapa, Veracruz, México, 2Facultad de Bioanálisis, Universidad Veracruzana, Xalapa, Veracruz, México
Introduction: Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in remodeling the extracellular matrix. Tissue inhibitors of metalloproteinases (TIMPs) are a family of four proteins that act to limit the degradative actions of MMPs. Chronic kidney disease (CKD) and acute kidney injury (AKI) are public health problems worldwide, the prevalence of which has been increasing. Recent concept considers MMPs and TIMPs as critical factors before the onset of microalbuminuria, as well as accelerating factors associated with the breakdown of the glomerular basement membrane, renal scarring, and fibrosis during the progression of kidney diseases. Here we reviewed studies of the expression of MMPs and TIMPs in humans, using as clinical samples serum, plasma, and urine, with a focus on their potential role as molecular markers in CKD and AKI, as non-invasive markers.
Material and methods: We used as data sources, studies at Medline database using combinations of the following keywords: CKD, AKI, MMP, TIMP, serum, plasma, and urine.
Results: Evidence suggests that MMPs/TIMPs could be potential targets for therapeutic intervention in kidney diseases; future studies should attempt to improve the diagnostic or prognostic power of these families.
Discussion: Considering published guides, such as biospecimen reporting for improved study quality (BRISQ), strengthening the reporting of observational studies in epidemiology (STROBE), an updated list of essential items for reporting diagnostic accuracy studies (STARD), transparent reporting of a multivariate prediction model for individual prognosis or diagnosis (TRIPOD), and on the studies reviewed here, we have adapted published recommendations and proposed other news in order to enhance the transparency and quality of MMPs/TIMPs research in CKD and AKI. This review reinforces the complexities of MMPs/TIMPs in the pathobiology of the kidney and the need for well-designed and transparent biomedical studies. HIPPOKRATIA 2018, 22(3): 99-104.
Keywords: Matrix metalloproteinases, tissue inhibitors of metalloproteinases, chronic kidney disease, acute kidney injury
Corresponding author: Clara Luz Sampieri, Instituto de Salud Pública, Universidad Veracruzana, Av. Luis Castelazo Ayala S/N, Col. Industrial Ánimas 91190, Xalapa, Veracruz, México, tel.: +522288418900, fax: +522288418935, e-mail: email@example.com
Chronic kidney disease (CKD) is defined as abnormalities in the kidney structure or function, present for three or more months, with implications for health. It is classified based on its cause, glomerular filtration rate (GFR) category, and albuminuria category1. The GFR is widely accepted as the best overall index of the kidney’s function in terms of health and disease; however, it is difficult to measure and is commonly estimated from the serum creatinine (SCr)1,2.
The development of CKD eventually progresses to end-stage renal disease and leads to irreversible loss of renal function1. Most patients with reduced renal function are not identified in the stages at which it is possible to slow down, or even prevent, the progression of CKD1. Chronicity is not synonymous of irreversibility; in some cases, CKD can be reversible1.
Acute kidney injury (AKI) is defined as an increase in SCr by ≥0.3 mg/dl within a period of 48 hours or an increase in SCr to ≥1.5 times the baseline, that is known or presumed to have occurred within the previous seven days, or a urine volume <0.5 ml/kg/h for six hours2. AKI is a predictor of immediate and long-term adverse outcomes and is a significant risk factor for CKD2. As with CKD, AKI is amenable to early detection and possible prevention1,2.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are involved in the remodeling of the extracellular matrix (ECM). MMPs are multidomain enzymes, generally consisting of a pro-domain, a catalytic domain, a hinge region, and a hemopexin-like domain3. To date, over 20 mammalian MMPs have been described and are subdivided into collagenases, gelatinases, stromelysins, matrilysins, membrane type, and “other MMPs”4. MMPs are traditionally conceived as antifibrotic players in the conventional view of progression; however, recent concept considers MMPs as compensatory factors before the onset of microalbuminuria and as accelerating factors associated with the breakdown of the glomerular basement membrane (GBM), renal scarring, and fibrosis during the progression of kidney diseases (KD)5,6.
Tissue inhibitors of metalloproteinases (TIMPs) are a family of four proteins that their action limits the degradative actions of MMPs. TIMPs interact with MMP active sites to block reversible their proteolytic activity7. TIMPs have activities independent of MMPs, including cell growth, migration, and differentiation8. Here, we review MMPs and TIMPs expression studies in serum, plasma, and urine, with a focus on their potential role as molecular markers in CKD and AKI. We included diabetes mellitus (DM) and hypertension studies since these diseases are among the most frequent causes of CKD1.
The Medline database was searched on the 28 February 2018, using combinations of the following key words: CKD, AKI, MMP, TIMP, serum, plasma, and urine. A total of 284 articles were obtained. The recommendations of the PRISMA group were followed in terms of identification, screening, eligibility, and inclusion criteria9.
Eligibility, inclusion, and exclusion criteria
The abstract of each article was carefully studied to verify the following eligibility criteria: i) English or Spanish language, ii) original or primary research concerning human renal function, iii) expression of MMPs/TIMPs families, and iv) CKD, AKI, DM or hypertension. The criteria for exclusion from consideration were: i) number of subjects in the group(s) of nine or less cases, ii) DNA sequencing study only, iii) renal transplant study only, and iv) studies performed in patients with a mean or median age under 18 years. Applying these criteria, 247 studies were discarded, and 37 were reviewed to verify the following inclusion criteria: i) reference to the sex and age of the groups, ii) agreement of data in the text and tables. Exclusion criteria were featuring data that, in our judgment, were duplicated. After applying these criteria, 17 studies were included, and a further 37 studies were incorporated into the introduction and conclusions. The description and discussion of these studies include the original name of the study groups, according to their authors.
MMPs and TIMPs in CKD and AKI
While the activity and the spatial and temporal expression of MMP/TIMP families in the human kidney have not been thoroughly characterized, the observational studies reviewed here demonstrated dysregulation of these families in a wide variety of kidney disorders in different fluids (Table 1).
Most of the studies focused on the levels of MMP-2 and MMP-9 quantified using enzyme-linked immunosorbent assay (ELISA) (Table 1); however, for KD type, fluid analyzed [in this case serum (S)] and formula used to calculate the GFR (Modification of Diet in Renal Disease), only three studies were comparable: Peiskerova et al14 analyzed SMMP-2 and SMMP-9 in non-dialyzed patients with CKD at stages 1-5; Smith et al19 investigated SMMP-2 in predominantly male and hypertensive pre-dialysis CKD patients with stages 3 and 4; and Gluba-Brzozka et al25 determined SMMP-2 and SMMP-9 in CKD patients with stages 1-5, where patients at stage 5 had mean dialysis time of 27.9 months. These studies also quantified levels of gelatinase, compared to those of healthy subjects14,19 or volunteers without CKD25, noting a consistent increase in the levels of SMMP-2 in CKD, compared to the reference group, while for SMMP-9 they report no differences. These data are of great interest since they are the product of studies conducted in different countries and patients diagnosed with CKD through diverse etiologies, at different stages of the disease, with a wide variety of comorbidities and under different schemes of treatment14,19,25.
Moreover, other studies report that plasma (P) MMP-2 (PMMP-2) is upregulated in CKD10, type 1 DM (T1DM)11 and end-stage kidney disease18, compared to control subjects10 or healthy controls (HC)11,18. Upregulation of PMMP-2 is also observed in normoalbuminuric hypertensive patients, compared to albuminuric resistant hypertensive patients24. On the other hand, urinary (U) MMP-2 (UMMP-2) is proposed as a marker for elevated risk of hyperglycemia, hyperfiltration, and microalbuminuria in patients with T1DM11. In subjects with renal impairment living at high altitude UMMP-2 is also associated with microalbuminuria6.
The fraction sMMP-9 associated with TIMP-1, among other findings, has been reported as a predictor of low GFR in hypertensive patients21, upregulated in diabetic nephropathy compared to T2DM12 and chronic renal failure12, but down-regulated in sepsis-associated AKI, compared to non-sepsis-associated AKI and controls23.
Data regarding UMMP-9 concentration analyzed in patients with AKI, as an absolute value or normalized to UCreatinine, indicated that the results do not markedly differ, although authors reported that normalizing to UCreatinine is less than ideal due to its non-steady state balance in those patients13. An elevated UMMP-9 level could function as a molecular marker of AKI13, T1DM15, and urinary tract infection (UTI)13. Differential levels according to gender have been reported for UMMP-9 in T1DM15 and HC15.
Different proportions have also been observed in detection of the activity of UMMP-916 and PMMP-924 according to the albuminuria category in T2DM and hypertensive patients, respectively. Most of the studies have likely focused on MMP-2 and MMP-9, due to their action on col-IV, the main ECM protein in the GBM, tubular basement membrane, and mesangium5,26. On the other hand, UMMP-8 in 24-hour collection is upregulated in T2DM and its levels depend on the albuminuria category16.
Finally, the only study in which the outcome was death states that UMMP-7 is associated with an increased risk of mortality in patients with T2DM and diabetic kidney disease22. This association remains robust after adjustment of demographic and clinical covariates, while SMMP-7 is not associated with mortality and does not attenuate the association of UMMP-722.
Since the evidence suggests that progressive glomerulosclerosis is characterized by a profound shift in ECM turnover27 and that MMPs/TIMPs could be potential targets for therapeutic intervention in KD28, future studies should attempt to improve the diagnostic or prognostic power of these genetic families through methods to optimize reproducibility, as well as increased sample sizes and greater numbers of MMPs/TIMPs analyzed.
Recommendations to enhance the transparency and quality of MMPs/TIMPs research in CKD and AKI
In this sense, we make some recommendations regarding procurement, storage and quality assurance of frozen biospecimens29,30 and the guides STROBE31, STARD32, TRIPOD33, adapting these, in some cases, to studies in humans with CKD or AKI:
- Describe the study design33 and sample size calculation31,33. Some statistical methods for calculating confidence intervals for relative risk and standardized ratios are for large sample approximations and are unreliable for studies with less than 20 cases34.
- Describe the criteria of inclusion, exclusion, and elimination of all the groups and how subjects flow through the study; a diagram may be helpful33,35. Where applicable, specify whether stratification or matching was carried out. If exist indicate criteria of exclusion about habits, illnesses, and treatments. Note that PMMP-9 is up-regulated in tobacco smokers36 and that significant change in its level is observed 12 weeks after smoking cessation37.
- Specify the period of recruitment and the population base, e.g., primary care, secondary care, general, rural or urban population33.
- Indicate whether there is control of the conditions that affect pre-analytical and analytical urinary albumin to creatinine ratio, such as UTI, exercise and patients with amputations, which muscle mass could which be lower1,2.
- Indicate the formula used to calculate GFR.
- Provide minimum anthropometric data, such as body mass index and waist size, and minimum sociodemographic information, e.g., sex, age, education level, economic level, and access to health services. Note that the term “race” is controversial in biomedical studies38,39. In human genetic research, the use of biological concepts of race has been described as “problematic at best and harmful at worst”40. Smart et al argue that “it seems currently unlikely that a genetic concept of race and ethnicity will ever be portable enough to wholly supplant a socio-political one”39.
- Indicate whether there are differences between the age and sex proportions in the study groups. Note that renal MMP expression appears to be sex- and age-dependent15,41.
- Refer to the duration of the illness32 or, where appropriate, indicate that this is unknown. Refer similarly to symptoms and comorbidities32. In the case of patients undergoing dialysis treatment, indicate the type and duration.
- Above all, in patients with DM, indicate the glycemic control.
- Identify the use of certain antibiotics that alter the expression of MMPs, such as doxycycline and minocycline42,43.
- Indicate the initial process by which the biospecimens were stabilized during collection; type of long-term preservation, the constitution of preservative time or range between biospecimens acquisition and distribution or analysis and storage duration13,30,43-45. Where applicable, the number of freeze/thaw cycles of the biospecimens6,13,46,47.
- In studies with clinical blood samples, indicate the fluid type analyzed as well as the preservative, given that some reports indicate discrepancies between levels of certain MMPs/TIMPs in serum and plasma, explained by additional unspecific release during the collection of serum47,48 and/or by the additive type48-50.
- In studies with clinical urine samples, indicate the type: 24-hour collection, minuted; sample isolated by spontaneous micturition in the morning or random, mid-stream programmed sample, obtained via a probe through a supra-pubic puncture. Indicate whether biospecimens with hematuria were excluded to avoid false positives6,51. Indicate whether the analyses were with cell-free urine20.
- Indicate whether the assay used has been validated in the fluid studied11. Specify whether the assay was performed blinded. Assay methods should be reported completely and transparently with a level of detail that would enable another laboratory to reproduce the measurement technique35. It may be helpful to use supplementary material.
- Studies utilizing ELISA should include the limit of detection, the coefficients of intra- and inter-assay variation.
- Studies utilizing gel zymography should indicate the limit of detection, concentration, and type of chelant used in the control gels or, where applicable, indicate that they were not conducted52.
- Indicate whether the analyses were conducted with the absolute values of the MMPs/TIMPs or whether these were normalized.
MMPs and TIMPs are essential components in many physiological and pathological processes due to their ability to remodel ECM components53. The ECM is not a mere scaffold for cells; it is a versatile and dynamic compartment that harbors cryptic biological functions that can be revealed on proteolysis53. ECM is involved in modulating cell proliferation, migration, differentiation, and apoptosis28,46,54. MMPs have been associated with renal hypertrophy, renal scarring, tubular cell proliferation, and fibrosis4. This sheds new light on the interplay between ECM, cells, and MMPs/TIMPs in renal pathophysiology.
Finally, it is important to highlight that studies in animal models were excluded from this review due to the complexity of MMPs/TIMPs in the kidney and because the expression of these families has been proposed as likely to be species-specific3. Moreover, experimental models do not always recapitulate the clinical findings of MMPs/TIMPs4,28. Collectively, these data highlight the complexities of MMPs/TIMPs in the pathophysiology of KD and the continued need for biomedical studies. We hope that these recommendations will help the scientific community in planning future research.
Conflict of interest
Authors declare no conflict of interest.
A support was received (POA 2017) by the Instituto de Salud Pública, Universidad Veracruzana.
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