Gelatinase B (MMP-9) Is Not Essential in the Normal Kidney and Does Not Influence Progression of Renal Disease in a Mouse Model of Alport Syndrome
2000; Elsevier BV; Volume: 157; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64541-5
ISSN1525-2191
AutoresKaya L. Andrews, Tomoko Betsuyaku, Sharon A. Rogers, J. Michael Shipley, Robert M. Senior, Jeffrey H. Miner,
Tópico(s)Connective tissue disorders research
ResumoMatrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB−/− and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB−/− embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred withCol4a3−/− mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome. Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB−/− and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB−/− embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred withCol4a3−/− mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome. Matrix metalloproteinases (MMPs) are a large family of zinc-requiring enzymes that degrade extracellular matrices. They are expressed in a variety of tissue locations pertinent to development, reproduction, health, and disease. Accordingly, MMPs are thought to be involved in implantation, embryogenesis, normal matrix remodeling, wound healing, tumor metastasis, angiogenesis, inflammation, atherosclerosis, and emphysema.1Matrisian LM Metalloproteinases and their inhibitors in matrix remodeling.Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1535) Google Scholar, 2Birkedal-Hansen H Moore WG Bodden MK Windsor LJ Birkedal-Hansen B DeCarlo A Engler JA Matrix metalloproteinases: a review.Crit Rev Oral Biol Med. 1993; 4: 197-250Crossref PubMed Scopus (2655) Google Scholar, 3Stetler-Stevenson WG Aznavoorian S Liotta LA Tumor cell interactions with the extracellular matrix during invasion and metastasis.Annu Rev Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1529) Google Scholar, 4Shapiro SD Mighty mice: transgenic technology "knocks out" questions of matrix metalloproteinase function.Matrix Biol. 1997; 15: 527-533Crossref PubMed Scopus (43) Google Scholar, 5Vu TH Werb Z Gelatinase B: structure, regulation, and function.in: Parks WC Mecham RP Matrix Metalloproteinases. Academic Press, San Diego1998: 115-148Crossref Google Scholar, 6Parks WC Mecham RP Matrix Metalloproteinases. Academic Press, San Diego1998Google Scholar Gelatinases are MMPs first identified based on their ability to cleave denatured collagen (gelatin). They include gelatinase A (gelA), also known as MMP-2 and 72-kd gelatinase, and gelatinase B (gelB), also known as MMP-9 and 92-kd gelatinase. In addition to their gelatinolytic activities, gelA and gelB possess the ability to cleave type IV collagen and entactin, which are major components of all basement membranes, as well as other extracellular matrix molecules, including elastin and proteoglycans.6Parks WC Mecham RP Matrix Metalloproteinases. Academic Press, San Diego1998Google Scholar Thus, gelA and gelB could be involved in both normal and aberrant degradation or remodeling of basement membranes in vivo. Gelatinase B has garnered special attention for its potential involvement in kidney development and disease. Several groups have documented expression and localization of gelB in the developing, mature, and diseased kidney.7McMillan JI Riordan JW Couser WG Pollock AS Lovett DH Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy.J Clin Invest. 1996; 97: 1094-1101Crossref PubMed Scopus (142) Google Scholar, 8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar, 9Tanney DC Feng L Pollock AS Lovett DH Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.Dev Dyn. 1998; 213: 121-129Crossref PubMed Scopus (42) Google ScholarHowever, the expression patterns reported for gelB during kidney development are conflicting: in the mouse, gelB protein was detected in metanephric mesenchyme,8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar whereas in the rat, gelB was associated with endothelial and mesangial cells.9Tanney DC Feng L Pollock AS Lovett DH Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.Dev Dyn. 1998; 213: 121-129Crossref PubMed Scopus (42) Google Scholar Gelatinase B is also expressed by a variety of kidney-derived cellsin vitro.10Rankin CA Suzuki K Itoh Y Ziemer DM Grantham JJ Calvet JP Nagase H Matrix metalloproteinases and TIMPS in cultured C57BL/6J-cpk kidney tubules.Kidney Int. 1996; 50: 835-844Crossref PubMed Scopus (50) Google Scholar, 11Yokoo T Kitamura M Dual regulation of IL-1 beta-mediated matrix metalloproteinase-9 expression in mesangial cells by NF-kappa B and AP-1.Am J Physiol. 1996; 270: F123-F130PubMed Google Scholar, 12Martin J Steadman R Knowlden J Williams J Davies M Differential regulation of matrix metalloproteinases and their inhibitors in human glomerular epithelial cells in vitro.J Am Soc Nephrol. 1998; 9: 1629-1637Crossref PubMed Google Scholar Together, these studies suggest that gelB could be involved in the branching morphogenesis fundamental to renal development, in the vascularization of glomeruli, and in the aberrant accumulation or breakdown of extracellular matrix that is associated with diverse renal diseases. The hypothesis that gelB (as well as other MMPs) could play an important role in the kidney is an attractive one. The kidney is a basement membrane-rich organ that relies on its basement membranes, especially the glomerular basement membrane (GBM), for proper function. The mature GBM, a major component of the kidney's ultrafiltration barrier between the vasculature and the urinary space, is the product of complex developmental transitions in expression and deposition of basement membrane proteins.13Abrahamson DR St. John PL Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.J Histochem Cytochem. 1992; 40: 1943-1953Crossref PubMed Scopus (33) Google Scholar, 14Miner JH Sanes JR Collagen IV α3, α4, and α5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches.J Cell Biol. 1994; 127: 879-891Crossref PubMed Scopus (357) Google Scholar, 15Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkins NA Copeland NG Sanes JR The laminin α chains: expression, developmental transitions, and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar, 16Sorokin LM Pausch F Durbeej M Ekblom P Differential expression of five laminin α (1–5) chains in developing and adult mouse kidney.Dev Dyn. 1997; 210: 446-462Crossref PubMed Scopus (134) Google Scholar, 17Miner JH Developmental biology of glomerular basement membrane components.Curr Opin Nephrol Hypertens. 1998; 7: 13-19Crossref PubMed Scopus (107) Google Scholar These transitions are characterized by the deposition of new components and the elimination of old ones from the developing GBM, a remodeling process that could require MMPs. Both gelB blocking antibodies and the tissue inhibitor of metalloproteinase-1 (TIMP-1), a natural inhibitor of gelB, inhibit ureteric bud branching in cultured embryonic mouse kidneys.8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar TIMP-2, a natural inhibitor of gelA, exhibits similar activity.18Barasch J Yang J Qiao J Tempst P Erdjument-Bromage H Leung W Oliver JA Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros.J Clin Invest. 1999; 103: 1299-1307Crossref PubMed Scopus (95) Google Scholar It was concluded that gelB is required for branching morphogenesis of the ureteric bud in vitro.8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar Insofar as metanephric organ culture is a model for normal renal development,19Saxen L Organogenesis of the kidney. Cambridge University Press, Cambridge UK1987Crossref Google Scholar these results suggest a crucial role for gelB in kidney development in vivo. In support of this, ilomastat, an inhibitor of multiple MMPs, also reduces ureteric bud branching in vitro.18Barasch J Yang J Qiao J Tempst P Erdjument-Bromage H Leung W Oliver JA Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros.J Clin Invest. 1999; 103: 1299-1307Crossref PubMed Scopus (95) Google Scholar We have taken advantage of gelB-deficient mice to investigate the necessity of gelB for kidney development, structure, and functionin vivo. These mice harbor a targeted null mutation inMmp9, the gene that encodes gelB, and produce no active gelB. Gelatinase B−/− mice are viable and fertile but exhibit transient aberrant skeletal growth plate vascularization and ossification.20Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar Although the long-term viability of gelB−/− mice suggests that there are likely no severe defects in kidney structure or function, we thought it important to look for both subtle and obvious kidney defects in a systematic fashion. In addition, we considered the possibility that gelB plays an important role in the progression of Alport syndrome, a hereditary disease of collagen IV that affects the GBM and leads to glomerulonephritis and renal failure.21Kashtan CE Michael AF Alport syndrome: from bedside to genome to bedside.Am J Kidney Dis. 1993; 22: 627-640PubMed Scopus (39) Google Scholar, 22Tryggvason K Zhou J Hostikka SL Shows TB Molecular genetics of Alport syndrome.Kidney Int. 1993; 43: 38-44Crossref PubMed Scopus (154) Google Scholar, 23Gubler M-C Antignac C Deschenes G Knebelmann B Hors-Cayla MC Grunfeld J-P Broyer M Habib R Genetic, clinical, and morphologic heterogeneity in Alport's syndrome.Adv Nephrol. 1993; 22: 15-35PubMed Google Scholar, 24Antignac C Molecular genetics of basement membranes: the paradigm of Alport syndrome.Kidney Int. 1995; 47: S29-S33Google Scholar, 25Kashtan CE Alport syndrome.Kidney Int. 1997; 51: S69-S71PubMed Google Scholar It has been hypothesized that Alport GBM may be abnormally susceptible to endoproteolysis by proteases normally found in the kidney.26Kalluri R Shield III, CF Todd P Hudson BG Neilson EG Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis.J Clin Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (265) Google Scholar Because gelB is one such protease, we determined the effects of its absence on the progression of renal disease in a mouse model of Alport syndrome. Production of gelB−/− and Col4a3−/− mice has been described.20Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar, 27Miner JH Sanes JR Molecular and functional defects in kidneys of mice lacking collagen α3(IV): implications for Alport syndrome.J Cell Biol. 1996; 135: 1403-1413Crossref PubMed Scopus (256) Google Scholar For timed matings, noon on the day a vaginal plug was found was considered ∼E0.5. To produce gelB−/−;Col4a3−/− and gelB+/−; Col4a3−/− mice, gelB+/− or−/−; Col4a3+/− females were mated with gelB−/− or+/−; Col4a3−/− males, respectively. Mice were genotyped by Southern blot (for gelB) and by PCR (forCol4a3) using DNA from tail biopsies. All procedures were performed in accordance with Institutional Animal Care and Use Committee regulations. For zymography, E12.5 kidney pairs were removed from each embryo and placed into 40 μl of medium.28Rogers SA Ryan G Hammerman MR Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro.J Cell Biol. 1991; 113: 1447-1453Crossref PubMed Scopus (138) Google Scholar Organs were incubated at 37°C for 48 hours. The medium was harvested and 15 μl were subjected to zymography as described.29Brenner CA Adler RR Rappolee DA Pedersen RA Werb Z Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development.Genes Dev. 1989; 3: 848-859Crossref PubMed Scopus (241) Google Scholar DNA was prepared from remaining portions of the embryos for genotyping. For developmental analyses, E12.5 to E13.5 kidneys were cultured on a Nuclepore filter (Whatman-Nuclepore, Tewksbury, MA) for several days as previously described.28Rogers SA Ryan G Hammerman MR Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro.J Cell Biol. 1991; 113: 1447-1453Crossref PubMed Scopus (138) Google Scholar Organs were photographed daily to document their development. RNA was isolated from pooled embryonic and individual adult kidneys as described.30Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63282) Google Scholar Twenty μg of wild-type E15.5 tissue RNA or 50 μg of adult tissue RNA were treated with 1.5 μg or 2.5 μg of RNase-free DNase I (Promega, Madison, WI), respectively, in a buffer containing 40 mmol/L Tris, pH 7.9, 10 mmol/L CaCl2, 6 mmol/L MgCl2, and 10 mmol/L NaCl at 37°C for 20 minutes. The RNA was extracted once with phenol/chloroform (1:1) then with chloroform, and then ethanol precipitated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on 1 μg RNA with the GeneAmp RNA PCR Kit (PE Applied Biosystems, Forest City, CA). Mock reactions were done in the absence of RT. One half of each reaction was amplified with gelA or gelB primers, whereas the other half was amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Gelatinase A primer sequences were 5′-CTTTGCAGGAGACAAGTTCTGG-3′ and 5′-TTAAGGTGGTGCAGGTATCTGG-3′ (701-bp product); gelB primer sequences were 5′-CCATGAGTCCCTGGCAG-3′ and 5′-ATGACAATGTCCGCTTCG-3′ (505-bp product); and the GAPDH primer sequences were 5′-CCATGTTTGTGATGGGTGTGAACC-3′ and 5′-TGTGAGGGAGATGCTCAGTGTTGG-3′ (712-bp product). The gelA cycling conditions were as follows: 94°C for 1 minute; five cycles of 94°C for 20 seconds, 75°C for 1 minute; five cycles of 94°C for 20 seconds, 70°C for 1 minute; and 34 cycles of 94°C for 20 seconds, 62°C for 30 seconds, 72°C for 1 minute. Gelatinase B cycling conditions were as follows: 94°C for 1 minute; five cycles of 94°C for 20 seconds, 65°C for 1 minute; five cycles of 94°C for 20 seconds, 60°C for 1 minute; and 40 cycles of 94°C for 20 seconds, 50°C for 30 seconds, 72°C for 1 minute. GAPDH cycling conditions consisted of an initial denaturation at 94°C for 1 minute, then 30 cycles of 94°C for 20 seconds and 65°C for 1 minute. For conventional histology, tissues were fixed in 4% buffered formaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and periodic-acid Schiff reagents. For electron microscopy, tissues were fixed in 2% glutaraldehyde/2% paraformaldehyde/0.15 mol/L cacodylate buffer and processed, embedded in plastic, sectioned, and stained by standard methods in the Electron Microscopy Facility of the Department of Cell Biology and Physiology, Washington University School of Medicine. For immunofluorescence, tissues were frozen fresh and sectioned at ∼7 μm on a cryostat. Antibodies were diluted in 1% (w/v) bovine serum albumin in phosphate-buffered saline (PBS) and incubated on sections for 1 to 2 hours. After rinsing off unbound primary antibody with PBS, secondary antibodies were applied for 1 to 2 hours. Sections were rinsed again and then mounted in glycerol-para-phenylenediamine and observed with epifluorescent illumination. Primary antibodies were as follows: rabbit anti-mouse collagen α1(IV)/α2(IV) was purchased from Collaborative Biomedical Products/Becton Dickinson Labware (Franklin Lakes, NJ). Rabbit anti-mouse collagen α4(IV) and laminin α5 have been described.14Miner JH Sanes JR Collagen IV α3, α4, and α5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches.J Cell Biol. 1994; 127: 879-891Crossref PubMed Scopus (357) Google Scholar, 15Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkins NA Copeland NG Sanes JR The laminin α chains: expression, developmental transitions, and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar Monoclonal rat anti-mouse laminin-1 antibodies 5A2 and 8B331Abrahamson DR Irwin MH St. John PL Perry EW Accavitti MA Heck LW Couchman JR Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains.J Cell Biol. 1989; 109: 3477-3491Crossref PubMed Scopus (82) Google Scholar were produced and generously provided by Dale Abrahamson, University of Kansas Medical Center, Kansas City, KS. These antibodies recognize laminin β1 and α1, respectively.32Martin PT Ettinger AJ Sanes JR A synaptic localization domain in the synaptic cleft protein, s-laminin/laminin β2.Science. 1995; 269: 413-416Crossref PubMed Scopus (82) Google Scholar, 33Miner JH Cunningham J Sanes JR Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin α5 chain.J Cell Biol. 1998; 143: 1713-1723Crossref PubMed Scopus (392) Google Scholar Fluorescein isothiocyanate and Cy3-labeled secondary antibodies were purchased from ICN/Cappel (Costa Mesa, CA) and Jackson Immuno Research Laboratories (West Grove, PA). To obtain urine and blood at the time of sacrifice, mice were first mildly anesthetized by inhalation of Metofane (Mallinckrodt Veterinary, Mundelein, IL) and then deeply anesthetized by an intraperitoneal injection of ketamine/xylazine. Urine was obtained directly from the bladder by aspiration and blood was obtained by cardiac puncture from the right ventricle with a heparin-treated syringe and 27-gauge needle. Serum was obtained by microcentrifugation of the blood at 7,000 rpm for 1 minute. Blood creatinine and urea nitrogen concentrations and urinary creatinine and protein concentrations were measured with a Cobas Mira Plus analyzer (Roche, Somerville, NJ). Before proceeding with a detailed study of the effects of gelB deficiency on kidney development and function, we wished to confirm that in the mouse background we were using, embryonic kidneys produced gelB. We also wanted to ensure that gelB activity was absent in kidneys from embryos that were genotypically gelB−/− (as determined by Southern analysis). To address both these issues, we arranged timed matings of gelB−/− females with gelB+/− males and isolated embryos at E12.5. Both kidneys of each embryo were removed and cultured together in 40 μl of medium for 48 hours. The medium was harvested, and a 15-μl aliquot was subjected to gelatin zymography to detect gelatinolytic activity. Neutrophil extract, which is rich in gelB proenzyme, was used as a positive control. Medium conditioned by all gelB+/− kidneys produced lysis bands corresponding to gelB (∼102 kd) and gelA (∼60 to 64 kd triplet), but medium conditioned by gelB−/− kidneys produced only the gelA triplet (Figure 1A). The intensity of the gelA bands did not vary significantly between gelB+/− and −/− lanes, suggesting that there is no compensatory up-regulation of gelA in the absence of gelB. The identification of the triplet as gelA is based on the fact that the bands are apparent in the gelB−/− lanes and thus cannot result from gelB activity. Although we could detect ample gelB activity in media conditioned by normal embryonic kidneys (in agreement with Lelongt et al8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar), we could not detect activity in lysates from freshly isolated E12.5 to E15.5 kidneys (data not shown). To further assess expression of gelB, we pooled kidneys from litters of normal E13.5 and E15.5 embryos and prepared total RNA. The RNA was treated with DNase and subjected to RT-PCR with gelB-specific primers, and we detected a band of the appropriate size (505 bp) at both ages (Figure 1B). Thus, gelB is expressed by the embryonic kidney in vivo, and, taken together with the protein data presented here and previously,8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar, 9Tanney DC Feng L Pollock AS Lovett DH Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.Dev Dyn. 1998; 213: 121-129Crossref PubMed Scopus (42) Google Scholar it is plausible that gelB could be involved in kidney development in vivo. This would be consistent with the notion that MMPs play roles in branching morphogenesis. The production of gelB−/− mice by targeted mutagenesis presented the perfect opportunity to directly assess the function of gelB in the kidney. Mice lacking gelB exhibit transient delays in ossification of long bones,20Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1513) Google Scholar but they exhibit no other obvious abnormalities. Because gelB is expressed in developing kidney and has been implicated as being involved in kidney development in vitro (Figure 1 and 8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar, 9Tanney DC Feng L Pollock AS Lovett DH Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.Dev Dyn. 1998; 213: 121-129Crossref PubMed Scopus (42) Google Scholar), we looked for structural defects in adult kidneys of mice lacking gelB. The gross appearance of whole kidneys from gelB−/− mice did not differ significantly from those of their gelB+/− littermates (data not shown), and histological analysis of renal architecture using H&E-stained paraffin sections failed to reveal any differences between mutant and control kidneys (Figure 2 and data not shown). Furthermore, ultrastructural analysis of the GBM at several ages did not reveal any defects in the gelB mutants (Figure 2 and data not shown), suggesting that gelB is not involved either in forming or maintaining a proper GBM. Because gelB is thought to be involved in matrix degradation and remodeling, we examined the distribution of several extracellular matrix proteins in the basement membranes of control and mutant adult kidneys. Basement membranes in the mature kidney are heterogeneous, especially in terms of the laminin and collagen IV isoforms that are found associated with the various functionally and anatomically distinct nephron segments.34Miner JH Renal basement membrane components.Kidney Int. 1999; 56: 2016-2024Crossref PubMed Scopus (293) Google Scholar For example, the laminin α1 chain is found primarily in tubular basement membranes associated with proximal tubules and loops of Henle, but it is absent from the GBM and other tubular segments. In contrast, laminin α5 is essentially ubiquitous in renal basement membranes.15Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkins NA Copeland NG Sanes JR The laminin α chains: expression, developmental transitions, and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar, 16Sorokin LM Pausch F Durbeej M Ekblom P Differential expression of five laminin α (1–5) chains in developing and adult mouse kidney.Dev Dyn. 1997; 210: 446-462Crossref PubMed Scopus (134) Google Scholar Likewise, the collagen α3-α5(IV) chains are found in the GBM and in a subset of tubular basement membranes, whereas the collagen α1 and α2(IV) chains are found in all tubular basement membranes, but are absent from the GBM in mice.14Miner JH Sanes JR Collagen IV α3, α4, and α5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches.J Cell Biol. 1994; 127: 879-891Crossref PubMed Scopus (357) Google Scholar In mice lacking gelB, we could detect no alterations from the norm in the distribution of collagen α1 and α2(IV), collagen α4(IV), laminin α1, or laminin α5 (Figure 3 and data not shown). Despite the fact that we could detect no gross structural or molecular differences between gelB+/− and −/− kidneys, the possibility existed that there were functional defects in the gelB−/− kidneys. To look for defects in kidney function, we collected blood and urine from gelB+/− and −/− mice at various ages. Serum creatinine and urea nitrogen levels were not elevated in the gelB−/− mice (data not shown), indicating that there was no reduction in the glomerular filtration rate. Another possibility was that a defect existed in the perm-selectivity of the GBM. Accordingly, we tested urine from mutants for protein and found that concentrations of total protein with respect to creatinine did not vary from the normal range (data not shown). Thus, we could find no defects in renal structure or function in the absence of gelB. Gelatinase B blocking antibodies and TIMP-1 inhibit ureteric bud branching and kidney growth in organ culture at an early stage of kidney development.8Lelongt B Trugnan G Murphy G Ronco PM Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro.J Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (151) Google Scholar We considered the possibility that the lack of gelB in our mutant mice might cause a similar early defect, but that a compensatory mechanism rescues the defect at a later stage, thus resulting in an essentially normal adult kidney. (Indeed, the ossification defect described for these mice is only apparent during a restricted developmental period
Referência(s)