Role of matrix metalloproteinase-9 in chronic kidney disease: a new biomarker of resistant albuminuria

Abstract

Resistant albuminuria, developed under adequate chronic blockade of the renin-angiotensin system, is a clinical problem present in a small number of patients with chronic kidney disease (CKD). The mechanism underlying this resistant albuminuria remains unknown. Matrix metalloproteinases (MMPs) are involved in the pathophysiology of cardiovascular and renal diseases. In the present study we tested the role of MMPs in resistant albuminuria. First we evaluated gelatinase MMP-2 and MMP-9 activity by zymography in the Munich Wistar Frömter (MWF) rat, a model of progressive albuminuria, and subsequently in patients with resistant albuminuria. Markers of oxidative stress were observed in the kidneys of MWF rats, together with a significant increase in pro-MMP-2 and active MMP-9 forms. These changes were normalized together with reduced albuminuria in consomic MWF-8(SHR) rats, in which chromosome 8 of MWF was replaced with the respective chromosome from spontaneously hypertensive rats. The MMP-2 and MMP-9 protein levels were similar in patients with normal and resistant albuminuria; however, high circulating levels of collagen IV, a specific biomarker of tissue collagen IV degradation, were observed in patients with resistant albuminuria. These patients showed a significant increase in gelatinase MMP-2 and MMP-9 activity, but only a significant increase in the active MMP-9 form quantified by ELISA, which correlated significantly with the degree of albuminuria. Although the expression of the tissue inhibitor of MMP-9 (TIMP)-1 was similar, a novel AlphaLISA assay demonstrated that the MMP-9-TIMP-1 interaction was reduced in patients with resistant albuminuria. It is of interest that oxidized TIMP-1 expression was higher in patients with resistant albuminuria. Therefore, increased circulating MMP-9 activity is associated with resistant albuminuria and a deleterious oxidative stress environment appears to be the underlying mechanism. These changes might contribute to the progression of CKD in these patients.

MMP-2 and 9 in Chronic Kidney Disease

“…OS also can activate MMP-2 and 9 [65,66,67,68], the activity of which may change in different stage of CKD, as previously mentioned. With the progression of CKD, renal interstitial fibrosis is further aggravated, which further hinders the diffusion of oxygen, finally leading to serious hypoxia.…”

Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9–Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA

“…This is because traditional estimations of the MMP-9/TIMP-1 ratio do not consider that TIMP-1 can also be subjected to posttranslational modification under specific circumstances. For example, TIMP-1 can be oxidized which, as demonstrated recently (15, 16), modifies its inhibitory capacity on MMP-9. In this type of situations, calculating the ratio between total circulating MMP-9 and TIMP-1 as a surrogate measure of MMP-9–TIMP-1 interaction would be hiding that TIMP-1 is posttranslationally modified and thus could not interact with MMP-9 in order to inhibit it.…”

“…Thus, alterations in plasma MMP-9 and TIMP-1 levels are related to a pathological remodeling of target organs as heart, vessels, or kidney during the development of diabetic nephropathy, albuminuria, arterial stiffness, or hypertension (16, 19–21), among other pathological situations. For that reason, an adequate, accurate, and direct measurement of the MMP-9–TIMP-1 interactions is completely necessary and has broad preclinical applicability since it would avoid indirect estimations derived from MMP-9/TIMP-1 calculations using classical ELISAs which may lead to unreliable results.…”

“…To directly evaluate this, we used the AlphaLISA ® immunoassay to detect endogenous/native MMP-9–TIMP-1 complexes in human plasma from patients with different pathophysiological conditions in which MMP-9 activity was altered. Representative data from MMP-9–TIMP-1 AlphaLISA ® interactions in human plasma samples are shown in a study recently published by our laboratory (16). Similar to that study, here we analyzed MMP-9–TIMP-1 immunocomplexes in plasma samples from hypertensive patients with or without albuminuria as an indicator of endothelial dysfunction and arterial stiffness (22–25).…”

“…AlphaLISA ® immunoassay (amplified luminescent proximity homogeneous assay, ALPHA) is a bead-based non-radioactive assay that uses luminescent oxygen-channeling chemistry (17, 18). We have recently developed a specific AlphaLISA ® -based assay to directly analyze MMP-9–TIMP-1 complexes in human plasma (16). Here, we describe the step-by-step protocol of this no-wash, rapid, and one-step homogeneous immunoassay for automatically determining specific MMP-9–TIMP-1 complexes using both recombinant human MMP-9 and TIMP-1 proteins and also endogenous/native MMP-9 and TIMP-1 proteins from samples of human plasma.…”