| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* The Department of Medicine, SFVAMC/University of California, San Francisco; and
the Department of Pathology, University of California, San Francisco, USA
1Correspondence: 111J Medical Service, SFVAMC, 4150 Clement St., San Francisco, CA 94121, USA. E-mail: david.lovett{at}med.va.gov
SPECIFIC AIMS
The increasing incidence of obesity, with attendant hypertension and diabetes mellitus, has led to an epidemic of chronic kidney disease (CKD) in the industrialized world. CKD, regardless of the primary etiology, is characterized by glomerulosclerosis, loss of functional nephron units via tubular atrophy, and development of interstitial fibrosis. Recent studies have emphasized the potential role of epithelial mesenchymal transition (EMT) in the development of CKD. We recently demonstrated that a specific member of the matrix metalloproteinase (MMP) gene family was necessary and sufficient to induce EMT in vitro. The current study addressed the hypothesis that transgenic expression of active MMP-2 in the renal proximal tubule would be sufficient to trigger EMT and renal failure in vivo. In addition, we hypothesized that the initial effect of MMP-2 would be the disruption of tubular basement membrane (TBM) integrity, a process sufficient to trigger EMT in vitro.
PRINCIPAL FINDINGS
1. Generation and validation of renal proximal tubule-specific MMP-2 transgenics
The transgenic construct consisted of an expression cassette for constitutively active MMP-2, achieved through mutation of the prodomain, coupled to a c-myc epitope tag to distinguish transgenic from native MMP-2 protein. Expression was driven by the renal proximal tubule-specific type I 
GT promoter.
2. Transgenic MMP-2 induces renal tubular epithelial mesenchymal transition
Transmission electron microscopy (TEM) was consistent with EMT of the proximal tubular epithelial cells. Many of the proximal tubular epithelial cells examined contained filamentous actin bundles (Fig. 1
A), and there was widespread evidence for MMP-2-mediated basement membrane proteolytic alteration, as evidenced by patchy lucencies and lytic areas (Fig. 1A
). The foci of EMT at sites of overt TBM disruption and interstitial invasion revealed dense bundles of interstitial collagen in close apposition to cells with a mixed fibroblastic and mesenchymal phenotype, with abundant rough endoplasmic reticulum (RER) and secretory vesicles (Fig. 1B
). The intrinsic interstitial fibroblasts also demonstrated an activated morphological phenotype (Fig. 1C
), with conspicuous RER and organized pericellular bundles of interstitial collagen. The TBM were also remarkable for frequent areas of reduplication with inclusions, consistent with persistent dysfunctional turnover (Fig. 1D
).
|
While the transgenic kidneys appeared normal when examined with hematoxylin/eosin staining at 4 months of age (Fig. 2
A, cf. a, b), there was extensive loss of tubular epithelial cell staining for the epithelial marker cytokeratin (cf. Fig. 2c, d
) and loss of ZO-1 in the adherens junctions of the tubules (cf. Fig. 2e, f
). This loss of epithelial marker expression was associated with variable increases in mesenchymal marker expression (Fig. 2B
). There was widespread expression of fibroblast-specific protein-1 (FSP-1) in the epithelial cells of the MMP-2 transgenics and intense staining of interstitial fibroblasts (Fig. 2B
, cf. a, b). Heat shock protein-47 (HSP-47) is a molecular chaperone involved in the processing and secretion of interstitial collagens and there was extensive tubular epithelial cell expression of this mesenchymal marker in the MMP-2 transgenics (cf. Fig. 2c, d
). Expression of 
-smooth muscle actin (-SMA, cf. Fig. 2e, f
) and vimentin (cf. Fig. 2g, h
) were restricted to a more limited number of tubular epithelial and interstitial cells. Thus, there exists a range of cellular phenotypes in the MMP-2 transgenics at 4 months, suggesting that FSP-1/S100A4 and HSP-47 represent early and widely expressed markers of epithelial mesenchymal transition, while -SMA and vimentin reflect acquisition of a fully developed mesenchymal phenotype by a considerably more limited subset of tubular epithelial cells. These IHC studies define a second, or intratubular, pattern of EMT in which tubular epithelial cells assume various degrees of a mesenchymal phenotype without overt invasion of the interstitial space.
|
Quantitative polymerase chain reaction (PCR) analysis for FSP-1/S100A4, HSP-47, vimentin and the 1 chain of type I collagen (COL1A1) confirmed the IHC analysis (Fig. 2C
). Transcript abundance was significantly increased at 4 months for FSP-1/S100A4, HSP-47 and the 1 type I collagen chain. At this time there was no significant increase in vimentin transcript abundance, consistent with the limited expression observed with IHC. Transcript abundance for FSP-1/S100A4, HSP-47, and COL1A1 remained elevated at 8 months, although to a lesser degree than that observed at 4 months. Vimentin transcript abundance was increased nearly 3-fold compared to age-matched controls at 8 months consistent with a progressive evolution to a more fully mesenchymal phenotype.
3. Quantitative assessment of renal injury in the MMP-2 transgenics:
Renal cortical sections were stained with Picrosirius red and examined by polarizing microscopy. Interstitial collagens were detected by yellow birefringent staining and the collagen volume fraction (CVF) was determined by digital image analysis. At 4 months, MMP-2 transgenics displayed substantial peritubular staining for interstitial collagen in a pattern consistent with tubular epithelial cell synthesis. By 8 months there was extensive deposition of collagen in the interstitium associated with a loss of tubules. At 4 months, the CVF of the MMP-2 transgenics was significantly elevated (transgenics: 5.6±0.5%; controls: 1.5±0.3%, P<0.05). By 8 months of age, there was a modest increase in the control CVF (2.4±0.5%); however, the CVF of the MMP-2 transgenics was greatly increased (28±3%; P<0.01).
Tubular atrophy is a common morphological feature of progressive renal disease and is characterized by apoptotic loss of tubular epithelial cells in response to injury. There was no significant tubular atrophy in the MMP-2 transgenics at 4 months, but by 8 months tubular atrophy was readily evident. At 8 months the semiquantitative tubular atrophy score for the wild-types was 0.5 ± .15, while the MMP-2 transgenics had a tubular atrophy score of 1.9 ± 0.2 (P<0.05). Mononuclear cell infiltration is an additional measure of progressive renal injury and the 8-month-old MMP-2 transgenics had significant degrees of interstitial cellular infiltration Finally, there was a significant increase in the glomerular injury score characterized by mesangial matrix expansion and hypercellularity in the 8-month-old MMP-2 transgenics compared with controls.
The morphological markers of renal injury correlated with a decrease in renal function measured by plasma creatinine. At 8 months, when established structural injury was present, MMP-2 transgenics demonstrated increases in creatinine levels consistent with a loss of 50% of renal function (controls: 0.3±0.06 mg/dl; transgenics: 0.7±0.4 mg/dl; n=6, P<0.01).
CONCLUSIONS AND SIGNIFICANCE
In this report we demonstrate that targeted expression of active MMP-2 in the renal proximal tubule is sufficient to generate the entire spectrum of EMT in the absence of superimposed injury. The most evident initial changes in the transgenic kidneys were widespread lucencies within the tubular basement membrane, consistent with proteolysis by MMP-2.
The current report provides proof-of-principle for a central and initiating role for MMP-2 in the pathogenesis of progressive renal injury. MMP-2 induction and targeted proteolysis of the TBM provides a unifying mechanism integrating the multiple pathophysiologic processes that induce epithelial cell injury. Strategies aimed at the inhibition of MMP-2, especially in the early prefibrotic stages of disease, could have a major impact on the reduction of CKD and its attendant costs and morbidities.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5898fje
This article has been cited by other articles:
|
|
 |
|
  C. C. Berthier, P. R. Wahl, M. L. Hir, H.-P. Marti, U. Wagner, H. Rehrauer, R. P. Wuthrich, and A. L. Serra Sirolimus ameliorates the enhanced expression of metalloproteinases in a rat model of autosomal dominant polycystic kidney disease Nephrol. Dial. Transplant., March 1, 2008; 23(3): 880 - 889. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cohen-Mazor, S. Sela, R. Mazor, N. Ilan, I. Vlodavsky, A. L. Rops, J. van der Vlag, H. I. Cohen, and B. Kristal Are primed polymorphonuclear leukocytes contributors to the high heparanase levels in hemodialysis patients? Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H651 - H658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Boor, K. Sebekova, T. Ostendorf, and J. Floege Treatment targets in renal fibrosis Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3391 - 3407. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Kumpers, F. Gueler, S. Rong, M. Mengel, I. Tossidou, I. Peters, H. Haller, and M. Schiffer Leptin is a coactivator of TGF-beta in unilateral ureteral obstructive kidney disease Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1355 - F1362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Krag, J. R. Nyengaard, and L. Wogensen Combined effects of moderately elevated blood glucose and locally produced TGF-{beta}1 on glomerular morphology and renal collagen production Nephrol. Dial. Transplant., September 1, 2007; 22(9): 2485 - 2496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Daniel, K. Amann, B. Hohenstein, P. Bornstein, and C. Hugo Thrombospondin 2 Functions as an Endogenous Regulator of Angiogenesis and Inflammation in Experimental Glomerulonephritis in Mice J. Am. Soc. Nephrol., March 1, 2007; 18(3): 788 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Catania, G. Chen, and A. R. Parrish Role of matrix metalloproteinases in renal pathophysiologies Am J Physiol Renal Physiol, March 1, 2007; 292(3): F905 - F911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Eddy and A. B. Fogo Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease: Evidence and Mechanisms of Action J. Am. Soc. Nephrol., November 1, 2006; 17(11): 2999 - 3012. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
