An intracellular matrix metalloproteinase-2 isoform induces tubular regulated necrosis: implications for acute kidney injury

Abstract

Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with preexisting CKD. Oxidative stress is a common theme in models of AKI induced by ischemia-reperfusion (I-R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an NH₂-terminal truncated MMP-2 (NTT-MMP-2) isoform that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14-mo-old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. Although morphologically normal at the light microscopic level at 4 mo, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition, and mitophagy. To determine whether NTT-MMP-2 expression enhances sensitivity to I-R injury, we performed unilateral I-R to induce mild tubular injury in wild-type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation, and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 "primes" the kidney to enhanced susceptibility to I-R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.

Keywords: acute kidney injury; chronic kidney disease; innate immunity; matrix metalloproteinase-2; mitochondria.

Fig. 1.

The NH₂-terminal truncated matrix metalloproteinase-2 (NTT-MMP-2) isoform, in contrast to the full-length MMP-2 (FL-MMP-2) isoform, is induced by increasing age. I: immunohistochemical (IHC) staining for the FL-MMP-2 isoform of renal cortex from 4- and 14-mo-old wild-type FVB/N mice. There is a low level of FL-MMP-2 cytoplasmic staining of 4-mo-old renal cortex that is not increased at 14 mo (images A and B). II: IHC staining of renal cortex from 4- and 14-mo-old wild-type FVB/N mice, using an antibody directed against the S1′ substrate-binding loop of MMP-2 (α-MMP-2 S1′). There is no detectable IHC signal in the 4-mo renal cortex (image A). There is a strong IHC signal present in the renal cortex of 14-mo-old wild-type FBV/N mice (image B). The staining is concentrated in a filamentous pattern in the basolateral compartment of the tubular epithelial cells (image C, arrow). The filamentous staining pattern, consistent with a mitochondrial association, is particularly prominent in image D, which used pseudocolor enhancement of an image obtained with Nomarski optics (arrow). III: quantitative PCR (qPCR) of FL-MMP-2 and NTT-MMP-2 isoform transcript abundance from renal cortices of 4- and 14-mo-old wild-type FVB/N mice. In contrast to FL-MMP-2, there is a statistically significant increase in NTT-MMP-2 transcript abundance as a function of age. (I: images A and B, ×200; II: images A and B, ×200; image C, ×600; image D, ×1,200; *P < 0.05, n = 6 for each study group). TBM, tubular basement membrane.

Fig. 2.

MMP-2 isoform expression is increased in the HypoE model of human type II cardiorenal syndrome. HypoE mice were placed on a high-fat diet (HFD) for 22 days, followed by HypoE allelic correction and return to normal chow (NC) for 6 wk. I, top: left ventricular sections from controls (image A), after 22 days of HFD (image B), and at 6 wk following allelic correction and return to normal chow (image C). There is evidence for extensive acute myocardial infarction with cardiomyocyte necrosis (arrow) and inflammatory infiltration in image B. Image C demonstrates areas of healed myocardial infarction with fibrosis (arrow) and cardiomyocyte hypertrophy characteristic of ischemic cardiomyopathy. I, bottom: renal cortical sections of controls (image D), at 22 days of HFD (image E), and at 6 wk following allelic correction and return to normal chow (image F). There are foci of tubular epithelial cell necrosis and inflammation at 22 days of HFD (image E, arrows). Image F demonstrates a typical wedge-shaped cortical infarct characteristic of atherosclerotic arteriolar occlusion (outlined by arrows) (hematoxylin-eosin; images A–F, ×220) II: immunohistochemical staining with anti-FL-MMP-2 and α-MMP-2 S1′ antibodies of control renal cortices (images A and C) and at 6 wk following HypoE allelic correction and return to normal chow (images B and D). Immunohistochemical signal for both isoforms is increased in images B and D. The immunohistochemical signal is most prominent within dilated tubules (arrows) (images A–D, ×200). III: qPCR of FL-MMP-2 and NTT-MMP-2 isoform transcript abundance in control renal cortices and at 6 wk following HypoE allelic repair and return to normal diet. There are statistically significant increases in the transcript abundance of both MMP-2 isoforms at 6 wk following HypoE allelic repair and return to normal diet (*P < 001; n = 8 for each study group).

Fig. 3.

Characterization of NTT-MMP-2 renal proximal tubular-specific transgenic mice is outlined. I: the NTT-MMP-2 transgene consists of the type I γ-GT promoter driving expression of the NTT-MMP-2/eGFP expression cassette. The relative location of the S1′ substrate-binding loop is depicted. II: Western blot of renal cortical extracts for the NTT-MMP-2/eGFP transgenic fusion protein from transgenic founders and wild-type littermate controls shows expression of the 92-kDa NTT-MMP-2/enhanced green fluorescent protein (eGFP) fusion protein. III: gelatin in situ zymography of control (image A) and NTT-MMP-2/eEGF transgenic renal cortices (image B) shows enzymatic activity of the NTT-MMP-2/eGFP transgene. Gelatinase activity is concentrated in the basolateral aspects of tubular epithelial cells (arrows) (images A and B, ×200).

Fig. 4.

Cellular trafficking of the NTT-MMP-2/eGFP transgenic (Tg) fusion protein; mitochondrial association shows correct cellular processing. I: there is no detectable IHC signal in the renal cortex of wild-type (WT) controls using the α-MMP-2 S1′ and α-eGFP antibodies (images A and C). IHC signal for both antibodies is present in identical basolateral distributions (images B and D, arrows) in the renal cortices of the NTT-MMP-2/eGFP Tg mice. These findings are consistent with a normal cellular trafficking of the NTT-MMP-2/eGFP fusion protein (images A–D, ×300). II: mitochondrial association of the NTT-MMP-2/eGFP fusion protein is demonstrated by digitonin solubilization. Mitochondria were isolated as detailed in materials and methods and incubated with increasing concentrations of digitonin. Supernatants of pelleted mitochondria were examined by Western blot using a rabbit antibody against eGFP. There is a digitonin concentration-dependent release of the 92-kDa NTT-MMP-2/eGFP fusion protein consistent with a mitochondrial localization with the outer membrane/intramembranous space (left). Quantitative densitometry of the NTT-MMP-2/eGFP fusion protein is shown on the right.

Fig. 5.

Proximal tubule transgenic expression of the NTt-MMP-2 isoform induces epithelial cell necrosis. Semithin (0.5 µm) toluidine blue-stained sections of 4-mo-old wild-type littermate control renal cortex (image A) and NTT-MMP-2/eGFP transgenic renal cortex (image B) are shown. In the transgenic kidneys there are foci of tubular epithelial cells with the typical morphological features of necrosis with cytoplasmic and nuclear swelling and loss of organelles (image B, arrows). (images A and B, ×1,200).

Fig. 6.

Prolonged expression of the NTT-MMP-2/eGFP transgene (8 mo) induces extensive tubular epithelial cell necrosis, tubular atrophy, and mononuclear inflammation. I: periodic acid-Schiff (PAS)-stained cortical sections of 8-mo-old wild-type kidney show normal tubular epithelial cell structure (image A). Eight-month-old NTT-MMP-2 transgenic kidneys demonstrate extensive tubular epithelial cell necrosis with shedding into the tubular lumen (image B, arrows) and formation of atrophic tubular structures (image C, arrow). There is tubular basement membrane replication with thickening (image D) and with occasional acellular cyst formation (image E, arrow) associated with intense interstitial mononuclear cell infiltration (image F, arrow). (images A–D, ×200; image E, ×300; image F, ×400). II: Picrosirius Red staining of NTT-MMP-2 transgenic kidneys does not demonstrate an increase in interstitial collagen. Cortical sections from 8-mo-old wild type (image A) and NTT-MMP-2 transgenic mice (image B) stained with Picrosirius Red show minimal amounts of interstitial collagen (arrows, ×200).

Fig. 7.

TUNEL staining of NTT-MMP-2/eGFP transgenic kidneys demonstrates nuclear DNA fragmentation in tubular epithelial cells. Renal cortical sections from 8-mo-old mice were stained using the TUNEL method to detect fragmented DNA. There was very rare TUNEL staining in wild-type kidneys (image A), whereas there was extensive TUNEL staining of tubular epithelial cells in the NTT-MMP-2eGFP transgenic kidneys (image B). This frequently involved entire tubular structures (image C, arrows) (images A and B, ×200; image C, ×400).

Fig. 8.

NTT-MMP-2 induces the mitochondrial permeability transition (MPT). I: transmission electron microscopy of wild-type renal proximal tubule cells reveals normal mitochondrial ultrastructure with intact mitochondrial membranes and highly organized cristae (image A). In contrast, the mitochondria in proximal tubule cells of NTT-MMP transgenic mice are grossly distorted in shape, visibly swollen with disorganized cristae, and display evidence of rupture (image B, arrows) (images A and B, ×1,500). II: direct demonstration that NTT-MMP-2 induces the MPT in vitro; human proximal tubular epithelial HK2 cells preloaded with tetramethylrhodamine ethyl ester (TMRE) were transiently transfected with the cDNA encoding NTT-MMP-2. NTT-MMP-2 induced a concentration-dependent reduction in TMRE signal, consistent with mitochondrial depolarization and the MPT.

Fig. 9.

Ultrastructural analysis of 4-mo-old NTT-MMP-2 transgenic mice reveals regulated necrosis, inflammation, and autophagy (mitophagy). Image A: wild-type kidney with normal proximal tubular epithelial cell ultrastructure. There are abundant basolateral mitochondria arranged in linear arrays. There is a compact nuclear structure with prominent heterochromatin. Image B: proximal tubular epithelial cells undergoing regulated necrosis in NTT-MMP-2 transgenic mice. Features include loss of plasma membrane integrity, nuclear expansion, loss of cytosolic organization, and mitochondrial disruption (arrow). Image C: mononuclear cell (arrow) adherent to a tubular epithelial cell in an early phase of regulated necrosis. Image D: tubular lumen filled with necrotic tubular epithelial cell debris (white arrow). Autophagic vesicles present in tubular epithelial cell (black arrow). Image E: higher power image of typical autophagic vesicles in tubular epithelial cells (black arrow). Image F: autophagic vesicles containing lamellar inclusions (black arrows) characteristic of mitochondria (mitophagy). (Images A and B, ×1,400; image C, ×2,500; image D, ×900; image E, ×4,600; image F, ×6,000).

Fig. 10.

NTT-MMP-2 induces renal tubular epithelial reactive oxygen species (ROS). Renal cortical frozen sections from 4- (image A: wild type; image B: NTT-MMP-2 transgenic) and 8-mo-old (image C: wild type; image D: NTT-MMP-2 transgenic) mice were stained with the ROS detection agent 2′-7′-DCF-diacetate (DCF), as detailed in materials and methods. No ROS signal was detected in the wild-type kidneys at either 4 or 8 mo of age (images A and C). Foci of DCF signal were present in the cortices of the 4-mo-old NTT-MMP-2 transgenic mice (image B). Diffuse DCF signal in a characteristic punctuate patterns characteristic of a mitochondrial localization was present in the renal cortices of 8-mo-old NTT-MMP-2 transgenic mice (×200).

Fig. 11.

Kidney redox capacity measured by hyperpolarized ¹³C-dehydroascorbate (DHA) MR spectroscopy in vivo shows ongoing oxidative stress in NTT-MMP-2 transgenic kidneys. A: coronal T2-weighted image of a mouse showing both kidneys and typical voxel placement for 3-dimensional chemical shift acquisitions. B: spectrum from a voxel placed in a kidney, showing conversion of DHA into vitamin C (VitC; red voxel). C: in blue, voxel predominantly containing signal from a blood vessel, showing no metabolism of DHA. D: average results obtained in wild-type (WT; n = 4) and NTT-MMP2 transgenic kidneys (NTT-MMP-2; n = 7). Renal VitC-to-DHA + VitC ratios showed a significantly lower reduction of DHA to VitC in NTT-MMP2 mice (P = 0.002). This measure is an index of renal redox capacity and reflects decreased glutathione concentration.

Fig. 12.

Renal proximal tubule expression of NTT-MMP-2 sensitizes kidneys to enhanced ischemia-reperfusion (I-R) injury. I: PAS-stained images at 96 h following I-R injury of contralateral (CL) and kidneys subjected to I-R injury from wild-type (WT) and NTT-MMP-2 transgenic (Tg) mice. Image A: contralateral wild-type kidney after unilateral I-R injury with normal histology. Image B: wild-type kidney after I-R injury with mild to moderate tubular dilation, occasional cast formation, and minimal mononuclear cell infiltration. Image C: contralateral NTT-MMP-2 transgenic kidney after I-R injury with occasional tubular dilation and cast formation (arrows). Image D: NTT-MMP-2 transgenic kidney subjected to I-R injury; there is extensive tubular dilation, cast formation, and cellular infiltration. II: PAS-stained images at 3 wk following I-R injury: Image A: there is mild to moderate tubular dilatation with rare cast formation in the contralateral kidney of the wild-type mice. Image B: there is moderate tubular dilatation, cast formation, and cellular infiltration in the wild-type kidney subjected to I-R injury. Image C: contralateral kidney of the NTT-MMP-2 Tg mouse has moderate to severe tubular dilation and extensive cellular infiltration. Image D: the renal cortex of the NTT-MMP-2 Tg mouse subjected to I-R injury shows extensive loss of tubular structures with cast formation and intense cellular infiltration. III: Picrosirius Red-stained images at 3 wk following I-R injury. Image A: the contralateral wild-type renal cortex has no significant increase in interstitial collagen deposition. Image B: there are occasional patchy foci of interstitial collagen deposition (arrows) in the renal cortices of wild-type kidneys subjected to I-R injury. Image C: the contralateral renal cortices of the NTT-MMP-2 transgenic mice have dense foci of interstitial collagen deposition along with evident tubular basement membrane thickening. Image D: there is extensive collagen deposition in the renal cortices of NTT-MMP-2 kidneys subjected to I-R injury. (I–III, ×200).

Fig. 13.

NTT-MMP-2 enhances the innate immune response to ischemia-reperfusion injury. Transcript levels of 5 innate immunity genes (IL6, OAS-1A, IFIT-1, IRF-7, and CXCL-10) were measured by qPCR in the renal cortex of wild-type (WT) and NTT-MMP-2 transgenic mice (Tg) at 96 h following ischemia-reperfusion injury (A) and the contralateral kidneys (B). Innate immunity transcripts were quantified as detailed in materials and methods at 3 wk in WT and Tg mice following ischemia-reperfusion-treated kidneys (C) and the contralateral kidneys (D) (n = 6/treatment group; *P < 0.05 and **P < 0.01).

Fig. 14.

NTT-MMP-2 induces a prolonged release of mitochondrial DNA danger-associated molecular patterns following ischemia-reperfusion injury. Plasma levels of mitochondrial COX III and CytB DNA were quantified by qPCR as detailed in materials and methods and compared with plasma from sham-operated controls. A: COX III and CytB DNA levels were similarly elevated at 96 h following I-R injury of both wild-type and NTT-MMP-2 Tg mice. B: COX III and Cyt B DNA levels returned to basal levels at 3 wk following I-R injury in the wild-type mice, whereas mitochondrial DNA levels remained significantly elevated in the NTT-MMP-2 transgenic mice (n = 6/treatment group; *P < 0.05).