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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 10, 2004 as doi:10.1096/fj.04-2031fje. |
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,1
* Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy;
DISTBIMO, University of Genoa Medical School, Genoa, Italy;
Department of Clinical Sciences, "La Sapienza" University, Rome, Italy
1Correspondence: Dipartimento di Scienze Cliniche (Endocrinologia), Viale del Policlinico, 155 Rome 00161, Italy. E-mail: giuseppe.pugliese{at}uniroma1.it
SPECIFIC AIMS
In a previous report, we showed that mice lacking galectin-3, a multifunctional lectin recently identified as an AGE receptor (AGE-R3), develop accelerated diabetic glomerulopathy as compared with those expressing galectin-3. This study aimed to further investigate the role of galectin-3 in the pathogenesis of diabetic renal disease by verifying whether accelerated glomerulopathy was attributable to the lack of galectin-3 AGE receptor function.
Adult (4 month-old) galectin-3 knockout (KO) mice, obtained by gene ablation, and coeval wild-type (WT) C57BL6 mice were injected i.p. daily for 3 months with 30 µg N
-carboxymethyllysine (CML) -modified or unmodified mouse serum albumin (MSA). Untreated mice of both genotypes served as normal controls (NC). Changes in renal function and structure were assessed along with cell and extracellular matrix (ECM) turnover, circulating and kidney tissue AGE levels, renal AGE receptor gene expression and AGE signaling.
PRINCIPAL FINDINGS
1. Renal function: more marked CML-induced increases in proteinuria in KO vs. WT mice
Serum creatinine was not affected by either CML or MSA treatment in both WT and KO mice (not shown).
Total proteinuria and albuminuria increased in both genotypes upon injection of CML, though the extent of increases vs. MSA-treated and NC mice was significantly higher in KO than WT mice (3.5–5.0-fold vs. 2.0–2.5-fold increments). Urinary protein and albumin excretion rates were slightly, though not significantly, higher in KO-MSA vs. KO-NC, WT-MSA and WT-NC (Fig. 1
A, B).
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2. Renal structure: more pronounced glomerular lesions in CML-injected KO vs. WT mice
Kidney weights increased insignificantly (less than 10%) in CML-treated vs. MSA-treated and NC mice, with no difference occurring between KO and WT.
Glomerular sclerosis index increased significantly in CML-treated vs. MSA-treated and NC animals of both genotypes, with higher values in KO-CML vs. WT-CML mice (1.35±0.11 vs. 0.77±0.11, P<0.001); mean glomerular area and volume as well as mean mesangial area and mesangial fractional area increased in both KO and WT mice injected with CML vs. those treated with MSA or left untreated. Increases over MSA-treated or NC mice were significantly more pronounced in KO-CML (from +30%–70%) than WT-CML (from +15%–40%) (Fig. 1C-F
).
3. Cell turnover: no difference among experimental groups in proliferation and death rates
Cell proliferation and death rates, as assessed by bromodeoxyuridine incorporation and active caspase-3 staining, did not differ among experimental groups at both glomerular and tubular level.
4. Matrix turnover: more marked CML-induced up-regulation of ECM and TGF-ß and down-regulation of matrix metalloproteinase-2 and 14 kidney cortex mRNA levels in KO vs. WT mice
Transcripts for fibronectin, laminin B1, collagen IV 
1 chain, and TGF-ß1 were up-regulated in CML-treated vs. MSA-treated and NC mice, with significantly higher levels in KO-CML vs. WT-CML animals (from +34% for TGF-ß1 to +49% for laminin B1, P<0.001).
Matrix metalloproteinase (MMP) -2 and MMP-14 (or membrane type 1-MMP, MT1-MMP) mRNA levels were reduced in CML-treated mice, again more markedly in KO (–44% and –54%, respectively, vs. corresponding NC mice) vs. WT (–32% and –34%) mice. MMP-9 and tissue inhibitor of metalloproteinase (TIMP) -2 gene expression was not significantly modified by CML administration, though there was a trend toward reduced MMP-9 and increased TIMP-2 mRNA levels.
5. AGE receptors: altered basal and CML-induced AGE receptor mRNA expression in KO vs. WT mice
KO-NC showed significantly increased mRNA expression (+35%, P<0.001) of 80K-H/AGE-R2 and RAGE and reduced transcript level of OST-48/AGE-R1 (–7%, P=NS) and macrophage scavenger receptor (MSR) -A type II (–13%, P<0.05) vs. WT-NC mice.
MSA injection did not affect AGE receptor gene expression, whereas CML treatment increased significantly (P<0.001) 80K-H/AGE-R2, RAGE and MSR-A type II, but not AGE-R1 mRNA levels. CML-induced up-regulation of 80K-H/AGE-R2 and RAGE was more marked in KO than WT mice (+60% and +70% vs. +46% and +44% over corresponding NC mice) and was at variance with that of MSR-A type II, which was less pronounced in KO than WT mice (+12% vs. +44% over corresponding NC mice). Transcript level for OST-48/AGE-R1 was not influenced by any treatment. Galectin-3 gene expression increased significantly in WT-CML mice (+59% vs. WT-NC, P<0.001).
6. AGE levels: more marked increases in CML-injected KO vs. WT mice
Serum AGEs increased in CML-treated mice from both genotypes. Increments vs. MSA-treated or NC mice were more pronounced in KO vs. WT mice (12.07±1.40 vs. 8.47±1.26 AGE U/mL serum, P<0.001).
Upon CML-treatment, AGEs accumulated in the kidneys of both genotypes, with more pronounced increases vs. MSA-treated or NC mice detected in KO vs. WT mice (33.49±6.21 vs. 18.82±2.47 AGE U/mg tissue, P<0.001). Differences between KO and WT mice were even more marked than those detected for circulating levels (+78% vs. +42%). Immunostaining for CML increased in tubuli and became detectable in glomeruli from CML-treated vs. control animals, with more intense and diffuse positivity in KO vs. WT mice.
7. AGE signaling: more pronounced increases in renal 4-hydroxy-2-nonenal content and nuclear factor 
B activity in CML-treated KO vs. WT mice
Renal content of 4-hydroxy-2-nonenal (HNE), a product of oxidative stress, was increased in CML-treated vs. MSA-treated and NC mice, with a much more evident positivity of immunostaining in KO than WT mice at both glomerular (33.43±3.18 vs. 16.38±2.03% of glomerular area, P<0.001) and tubular level.
Kidney cortex nuclear factor (NF) B/p65 activity increased in both genotypes in response to CML. Increases were more marked in KO-CML than WT-CML mice (0.97±0.21 vs. 0.54±0.12 OD/sample, P<0.001).
CONCLUSIONS AND SIGNIFICANCE
Mice lacking galectin-3 developed accelerated glomerular disease when injected with CML-modified proteins, as compared with mice expressing it. This differential renal outcome was associated with: 1) higher circulating AGE levels, renal tissue AGE and HNE content, and kidney NFB activity, despite injection of equal doses of CML; and 2) an altered basal and CML-induced expression pattern of the other AGE receptors, with higher AGE-R2 and RAGE and lower MSR-A type II mRNA levels, in KO than WT mice.
In this study, we used CML-modified albumin, the major AGE found in vivo and a known RAGE ligand, rather than nonspecific AGE structures. We also injected lower doses for longer duration than in previous reports, thus mimicking more closely the diabetic condition. Both KO and WT mice injected with CML showed changes in renal function and structure mimicking those of diabetic animals and similar to those of rats given AGEs. Changes observed in the kidney cortex gene expression of proteins involved in ECM turnover and AGE receptors also mimic those observed in experimental diabetes and confirm that matrix accumulation is due to an imbalance between increased synthesis and impaired degradation and that TGF-ß up-regulation may sustain this imbalance. Reduced gene expression for MMP-2 and MMP-14/ MT1-MMP (that regulates proenzyme processing of pro-MMP-2/TIMP-2 complex) indicates a decreased expression/activation of MMP-2, with consequent impaired degradation of its substrates fibronectin, laminin, and collagen IV. Lack of significant changes in cell proliferation and death rate indicates that altered cell turnover does not play a major role in the occurrence of renal lesions in this model.
An intriguing finding in this study was that proteinuria, mesangial expansion and up-regulation of ECM gene expression increased more markedly in CML-injected KO vs. WT mice. These differences in the severity of renal injury between the two genotypes occurred despite injection of equal doses of CML, and in association with higher circulating and renal tissue AGE levels and more pronounced increases in renal HNE content and NFB activation in KO vs. WT mice. These findings support the concept that accelerated glomerulopathy in KO mice is due to the lack of galectin-3 AGE receptor function. In the absence of galectin-3, removal of AGEs from extrarenal and renal tissues would be impaired, with consequent more pronounced accumulation of AGEs within renal tissues of KO mice. This in turn would cause enhanced AGE binding to RAGE and the other AGE receptors, increased AGE signaling through redox-sensitive pathways, as evidenced by increased renal HNE content and NFB activation, and ultimately AGE-induced cell activation with altered expression of genes involved in matrix turnover. This sequence of events would be favored by increased content of AGE-R2 and RAGE (and reduced levels of AGE-R1 and MSR-A) and a more marked up-regulation of these receptors in response to CML treatment (with reduced increase in MSR-A) detected in KO vs. WT mice, which facilitates AGE-induced cell activation and impairs AGE removal.
These observations suggest that galectin-3 acts as an AGE receptor to protect from AGE-induced tissue injury. This protection may be provided either directly or through a modulation of the expression of other AGE receptors to favor AGE degradation vs. cell activation (Fig. 2
). Modulation of AGE receptor expression could be mediated, at least partly, via reduced AGE levels. However, the altered AGE receptor pattern observed under normal conditions indicates that galectin-3 influences expression of other AGE receptors independent of circulating and tissue AGE levels. Direct effects of galectin-3 could consist of an interference with RAGE signaling and downstream events and/or transduction of AGE signals through intracellular pathways mediating AGE internalization and degradation. Finally, like soluble RAGE, it may function as an extracellular ligand for AGEs. The finding that galectin-3 behaves differently from RAGE as an AGE receptor supports the concept that AGE receptors are functionally distinct. In fact, AGE binding to RAGE is associated with ROS generation triggering redox-sensitive signaling pathways, with consequent induction of tissue injury.
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These data show that galectin-3 ablation is associated with enhanced susceptibility to AGE-induced renal disease, increased AGE levels and signaling, and altered AGE receptor expression and indicate that galectin-3 is operating in vivo as an AGE receptor to afford protection toward AGE-dependent tissue injury at variance with RAGE.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2031fje;
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