# Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction

Maria K. Tveitarås, Trude Skogstrand, Sabine Leh, Frank Helle, Bjarne M. Iversen, Christos Chatziantoniou, Rolf K. Reed, Michael Hultström

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## To cite this version:

Maria K. Tveitarås, Trude Skogstrand, Sabine Leh, Frank Helle, Bjarne M. Iversen, et al., Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction. PLoS ONE, 2015, 10 (12), pp.e0143390. 10.1371/journal.pone.0143390.t002. [hal-01257793](https://hal.sorbonne-universite.fr/hal-01257793)

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RESEARCH ARTICLE

# Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction

Maria K. Tveitarås<sup>1,2</sup>, Trude Skogstrand<sup>1,2,3</sup>, Sabine Leh<sup>2,4</sup>, Frank Helle<sup>2</sup>, Bjarne M. Iversen<sup>2†</sup>, Christos Chatziantoniou<sup>5</sup>, Rolf K. Reed<sup>1,6</sup>, Michael Hultström<sup>1,2,7,8*</sup>

1 Department of Biomedicine, University of Bergen, Bergen, Norway, 2 Department of Clinical Medicine, University of Bergen, Bergen, Norway, 3 Department of Medicine, Haukeland University Hospital, Bergen, Norway, 4 Department of Pathology, Haukeland University Hospital, Bergen, Norway, 5 Inserm UMR 702, Université Pierre et Marie Curie, Paris VI, Paris, France, 6 Center for Cancer Biomarkers, CCBIO, University of Bergen, Bergen, Norway, 7 Department of Medical Cellbiology, Uppsala University, Uppsala, Sweden, 8 Anaesthesiology and Intensive Care Medicine, Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

† Deceased.

* michael.hultstrom@mcb.uu.se

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## OPEN ACCESS

**Citation:** Tveitarås MK, Skogstrand T, Leh S, Helle F, Iversen BM, Chatziantoniou C, et al. (2015) Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction. PLoS ONE 10(12): e0143390. doi:10.1371/journal.pone.0143390

**Editor:** Nikos K Karamanos, University of Patras, GREECE

**Received:** April 13, 2014

**Accepted:** November 4, 2015

**Published:** December 16, 2015

**Copyright:** © 2015 Tveitarås et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper.

**Funding:** The study was supported by the Western Health Authority of Norway (Helse Vest) (project no: 911685) www.helsevest.no, and the Swedish Society For Medical Research (SSMF) www.ssmf.se. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## Abstract

Matrix Metalloproteinase-2 (Mmp2) is a collagenase known to be important in the development of renal fibrosis. In unilateral ureteral obstruction (UUO) the obstructed kidney (OK) develops fibrosis, while the contralateral (CL) does not. In this study we investigated the effect of UUO on gene expression, fibrosis and pelvic remodeling in the kidneys of Mmp2 deficient mice (Mmp2-/-), heterozygous animals (Mmp2+/-) and wild-type mice (Mmp2+/+) Sham operated animals served as controls (Cntrl). UUO was prepared under isoflurane anaesthesia, and the animals were sacrificed after one week. UUO caused hydronephrosis, dilation of renal tubules, loss of parenchymal thickness, and fibrosis. Damage was most severe in Mmp2+/- mice, while both Mmp2-/- and Mmp2+/- groups showed considerably milder hydronephrosis, no tubular necrosis, and less tubular dilation. Picrosirius red quantification of fibrous collagen showed 1.63±0.25% positivity in OK and 0.29±0.11% in CL (p<0.05) of Mmp2+/-, Mmp2-/- OK and Mmp2-/- CL exhibited only 0.49±0.09% and 0.23 ±0.04% (p<0.05) positivity, respectively. Mmp2+/- OK and Mmp2+/- CL showed 0.43 ±0.09% and 0.22±0.06% (p<0.05) positivity, respectively. Transcriptomic analysis showed that 26 genes (out of 48 examined) were differentially expressed by ANOVA (p<0.05). 25 genes were upregulated in Mmp2+/- OK compared to Mmp2+/- CL: Adamts1, -2, Col1a1, -2, -3a1, -4a1, -5a1, -5a2, Dcn, FbIn1, -5, Fmod, Fn1, Itga2, Loxl1, Mgp, Mmp2, -3, Nid1, Pdgfb, Spp1, Tgfb1, Timp2, Trf, Vim. In Mmp2-/- and Mmp2+/- 18 and 12 genes were expressed differentially between OK and CL, respectively. Only Mmp2 was differentially regulated when comparing Mmp2-/- OK and Mmp2+/- OK. Under stress, it appears that Mmp2+/- OK responds with less Mmp2 upregulation than Mmp2+/- OK, suggesting that there is a threshold level of Mmp2 necessary for damage and fibrosis to occur. In
and UUO (n = 10), Mmp2+/- Control (Cntrl+/-) (n = 10) and UUO (n = 11), and Mmp2-/- Control (Cntrl-/-) (n = 10) and UUO (n = 9) (Table 1). The control groups were sham operated.

## Ethics Statement

The experiments were conducted in accordance with the guidelines of, and with approval obtained from the Norwegian State Board for Biological Experiments with Living Animals (Approval No: 2009-1899). All surgery was carried out under isoflurane anaesthesia.

## Ureteral obstruction

UUO was prepared under isoflurane anaesthesia. The left ureter was identified through a sub-costal incision, and obstructed using a silk ligature at the level of the lower pole of the kidney. The animals were sacrificed under isoflurane anaesthesia one week after obstruction. The abdominal aorta was dissected and cannulated in order to perfuse the animal with ice-cold PBS before the kidneys were removed. The kidneys were cut in transverse slices that were either stabilized in RNA-later, or fixed in 4% formaldehyde, processed and embedded in paraffin.

## Histology

Morphological damage was investigated by light microscopy using 3 µm sections, stained with Periodic Acid-Schiff (PAS). In order to monitor the amount of collagen in the different experimental groups, 7 µm sections were stained with Picrosirius Red and examined according to our previously published protocol [9]. Briefly, digital images were captured randomly under constant polarized light in a Leica DMLB microscope connected to a CCD ColorView IIIu camera. Image acquisition and analysis was performed using CellD version 2.4. Intensity was separated as a gray-scale image from the HSI colour-space. The detection threshold was the same for all images. Collagen content was expressed as percent positive pixels of total pixels.

## Quantitative RTPCR

RNA was extracted from the kidneys using an RNeasy mini kit (Qiagen, West Sussex, UK) as described in the protocol provided by the manufacturer, and cDNA was synthesised from RNA using Reverse Transcriptase Core Kit obtained from Eurogentec (Seraing, Belgium). A custom-made Low Density Array (LDA) from Applied Biosystems was used to determine the mRNA expression levels of a selection of genes. The method is based on the well-established quantitative RT-PCR (QRT-PCR) technique, however, LDA has the benefit of enabling quantification of several genes simultaneously and at the same time maintaining the sensitivity of QRT-PCR [20]. 18s ribosomal RNA was used as a standard. In addition Gapdh, Tbp, Pgk1 and Ppia were used as housekeeping genes.

## Statistical analysis

Data is presented as means ± standard error of the mean (SEM), except for comparison of the expression of individual genes where the fold-change is used. The probability of chance difference was tested using ANOVA, with Fisher's test and a priori contrasts to test individual comparisons. Comparisons between CL and OK were paired. The Bonferroni correction was used for gene-expression data, separately across samples and across genes for each comparison made. P < 0.05 was accepted as statistically significant.
<table><caption>Table 1. Bodyweight and age of the mice at the start of the experiment.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Mmp2+/+</th><th colspan="2">Mmp2-/-</th><th colspan="2">Mmp2-/-</th></tr><tr><th>Control</th><th>UUO</th><th>Control</th><th>UUO</th><th>Control</th><th>UUO</th></tr></thead><tbody><tr><th>n =</th><td>10</td><td>10</td><td>10</td><td>11</td><td>10</td><td>9</td></tr><tr><th>Bodyweight (g)</th><td>31±0.4</td><td>23±0.3</td><td>35±1</td><td>38±2</td><td>29±2</td><td>29±2</td></tr><tr><th>Age (weeks)</th><td>14±0</td><td>9±0</td><td>19±1</td><td>37±2</td><td>20±2</td><td>41±6</td></tr></tbody></table>

doi:10.1371/journal.pone.0143390.t001

# Results

## Kidney damage in UUO

A total of 60 mice were used in the UUO experiment (Table 1). UUO caused severe hydronephrosis in Mmp2+/+, which was accompanied by tubular dilation, necrosis and atrophy, while Mmp2-/- and Mmp2+/+ showed considerably milder hydronephrosis, no tubular necrosis, and less tubular dilation (Figs 1 and 2). Automatic image analysis of collagen content in Picrosirius Red stained sections showed 1.63±0.25% positivity in OK and 0.29±0.11% in CL (p<<0.05) of Mmp2+/+, whereas Mmp2-/- OK and Mmp2-/- CL only showed 0.49±0.09% and 0.23±0.04% (p<0.05) positivity, respectively, and Mmp2+/+ OK and Mmp2+/+ CL exhibited 0.43±0.09% and 0.22±0.06% (p<0.05) positivity, respectively (Figs 3 and 4). There were no significant discrepancies between the levels of fibrosis in the control kidneys and the CL kidneys in any of the groups.

## Gene expression

Using RTPCR, 26 out of 48 genes examined showed differential expression when ANOVA was applied across groups (Table 2). None of the housekeeping genes showed significant changes, and the results were comparable when using the different genes for normalisation. Data are presented using 18S as standard. There was no difference in gene expression between CL and control kidneys in any of the groups.

The greatest difference in gene expressions was seen when comparing the OK and CL in the Mmp2+/+ mice, where 25 genes were significantly altered (Table 2). A comparison of Mmp2-/- OK vs Mmp2-/- CL identified 18 significantly altered genes. Mmp2+/+ OK displayed 12 significantly altered genes when compared to Mmp2+/+ CL. Cntrl+/- did not differ from Cntrl+/- in any of the 48 tested genes. After UUO, Mmp2 expression was 7-fold and 5.5-fold induced in Mmp2+/+ OK and Mmp2+/+ OK, respectively, when compared to Mmp2-/- OK. Comparisons between Mmp2-/- OK vs Mmp2-/- CL, and Mmp2+/+ OK vs Mmp2+/+ CL showed that five genes in common did not respond in the same manner as in the Mmp2+/+ OK vs Mmp2+/+ CL comparison. These genes are Lox11, Fbln5, Fmod, Fbln1 and Col5a2. None of these genes were significantly altered after UUO in Mmp2-/- and Mmp2+/+ mice. In contrast, all five genes were upregulated in Mmp2+/+ mice. Fmod is downregulated in both Mmp2-/- and Mmp2+/+ OK groups compared to Mmp2+/+ OK. Mmp9 showed a 2.2-fold upregulation in Mmp2-/- OK when compared to Mmp2-/- CL mice. Only Mmp2 was differentially regulated when comparing Mmp2-/- OK and Mmp2+/+ OK. In addition to the housekeeping genes, 17 genes were not differentially expressed between any of the groups.

## Discussion

The present study demonstrates that Mmp2-/- and Mmp2+/+ mice display less pelvic-remodeling and fibrosis compared to Mmp2+/+ mice after UUO. Both Mmp2-/- and Mmp2+/+ mice
<table><caption>Table 2. Differential gene expression between strains (Mmp2 +/+, +/-, and -/-), and as paired comparisons between the obstructed (OK) and contra-lateral kidneys (CL) of the same animal.</caption><thead><tr><th>Gene</th><th>Average<br>CT-value<br>± SEM</th><th>OK-/-<br>vs<br>OK+/+</th><th>OK-/-<br>vs<br>OK+/-</th><th>OK+/-<br>vs<br>OK+/+</th><th>OK-/-<br>vs<br>CL-/-</th><th>CL-/-<br>vs<br>Cntrl-/-</th><th>OK+/-<br>vs<br>CL+/-</th><th>CL+/-<br>vs<br>Cntrl+/-</th><th>OK+/-<br>vs<br>CL+/+</th><th>CL+/-<br>vs<br>Cntrl+/+</th><th>Cntrl-/-<br>vs<br>Cntrl+/+</th><th>Cntrl-/-<br>vs<br>Cntrl-/-</th><th>Cntrl+/-<br>vs<br>Cntrl+/+</th></tr></thead><tbody><tr><td>Mmp2 †</td><td>33.05 ± 0.28</td><td>-7.060 *</td><td>-5.774 *</td><td>-1.287 *</td><td>-0.907</td><td>0.704</td><td>3.973 *</td><td>-0.345</td><td>4.079 *</td><td>0.455</td><td>-2.323 *</td><td>-1.942 *</td><td>-0.381</td></tr><tr><td>Adamts2 †</td><td>31.06 ± 0.22</td><td>-0.666</td><td>0.479</td><td>-1.146 *</td><td>2.748 *</td><td>0.532</td><td>3.122 *</td><td>0.247</td><td>3.867 *</td><td>0.033</td><td>-0.046</td><td>0.569</td><td>-0.615</td></tr><tr><td>Dcn †</td><td>28.22 ± 0.18</td><td>-0.533</td><td>0.534</td><td>-1.067 *</td><td>1.774 *</td><td>0.914</td><td>1.269</td><td>0.801</td><td>3.078 *</td><td>-0.567</td><td>-0.710</td><td>-0.084</td><td>-0.626</td></tr><tr><td>Mgp †</td><td>24.65 ± 0.32</td><td>0.829</td><td>0.421</td><td>0.408</td><td>2.853 *</td><td>1.599</td><td>3.673 *</td><td>0.420</td><td>3.555 *</td><td>-0.152</td><td>-0.219</td><td>0.062</td><td>-0.280</td></tr><tr><td>Adamts1 †</td><td>29.84 ± 0.24</td><td>0.726</td><td>0.435</td><td>0.291</td><td>3.840 *</td><td>-0.241</td><td>3.591 *</td><td>-0.626</td><td>2.463 *</td><td>-0.012</td><td>-0.423</td><td>-0.199</td><td>-0.224</td></tr><tr><td>Spp1 †</td><td>23.72 ± 0.28</td><td>0.870</td><td>0.483</td><td>0.387</td><td>3.409 *</td><td>1.432</td><td>4.996 *</td><td>-0.538</td><td>3.337 *</td><td>0.887</td><td>0.255</td><td>0.100</td><td>0.154</td></tr><tr><td>Col4a1 †</td><td>25.82 ± 0.16</td><td>-0.187</td><td>0.340</td><td>-0.527</td><td>2.463 *</td><td>0.071</td><td>0.977</td><td>1.208</td><td>2.160 *</td><td>0.349</td><td>-0.212</td><td>-0.009</td><td>-0.203</td></tr><tr><td>Fn1 †</td><td>29.61 ± 0.25</td><td>-0.782</td><td>0.149</td><td>-0.930</td><td>1.713</td><td>1.742</td><td>4.074 *</td><td>-0.643</td><td>4.371 *</td><td>0.295</td><td>0.429</td><td>0.125</td><td>0.304</td></tr><tr><td>Col1a2 †</td><td>26.48 ± 0.23</td><td>-0.845</td><td>0.475</td><td>-1.320</td><td>3.921 *</td><td>-0.011</td><td>3.598 *</td><td>-0.133</td><td>4.415 *</td><td>0.123</td><td>-0.217</td><td>0.029</td><td>-0.246</td></tr><tr><td>Vim †</td><td>27.38 ± 0.17</td><td>-0.369</td><td>0.403</td><td>-0.772</td><td>2.109 *</td><td>0.633</td><td>1.032</td><td>1.342</td><td>3.098 *</td><td>0.065</td><td>0.051</td><td>0.034</td><td>0.016</td></tr><tr><td>Nid1 †</td><td>29.28 ± 0.15</td><td>-0.358</td><td>0.132</td><td>-0.490</td><td>1.578 *</td><td>0.620</td><td>1.296 *</td><td>0.774</td><td>1.898 *</td><td>0.764</td><td>0.106</td><td>0.004</td><td>0.103</td></tr><tr><td>Mmp3 †</td><td>32.99 ± 0.29</td><td>0.052</td><td>0.600</td><td>-0.548</td><td>4.706 *</td><td>0.870</td><td>4.741 *</td><td>-0.138</td><td>4.228 *</td><td>0.029</td><td>-1.267</td><td>-0.374</td><td>-0.893</td></tr><tr><td>Trf †</td><td>30.29 ± 0.23</td><td>0.461</td><td>0.530</td><td>-0.069</td><td>1.550 *</td><td>0.221</td><td>1.201</td><td>0.656</td><td>2.591 *</td><td>0.025</td><td>1.305</td><td>0.616</td><td>0.689</td></tr><tr><td>Col3a1 †</td><td>28.04 ± 0.31</td><td>-0.610</td><td>0.958</td><td>-1.568</td><td>5.332 *</td><td>0.239</td><td>4.743 *</td><td>-0.003</td><td>5.177 *</td><td>0.561</td><td>-0.444</td><td>0.126</td><td>-0.570</td></tr><tr><td>Col1a1 †</td><td>29.56 ± 0.21</td><td>-0.953</td><td>0.671</td><td>-1.624</td><td>4.894 *</td><td>0.349</td><td>4.655 *</td><td>-0.306</td><td>4.914 *</td><td>0.534</td><td>-0.747</td><td>-0.223</td><td>-0.524</td></tr><tr><td>Loxl1 †</td><td>29.77 ± 0.18</td><td>-0.088</td><td>0.172</td><td>-0.261</td><td>1.518</td><td>1.255</td><td>1.147</td><td>1.518</td><td>2.978 *</td><td>-0.067</td><td>0.049</td><td>0.064</td><td>-0.014</td></tr><tr><td>Tgfb1 †</td><td>28.97 ± 0.16</td><td>0.316</td><td>0.329</td><td>-0.014</td><td>2.336 *</td><td>0.309</td><td>0.983</td><td>1.514</td><td>2.450 *</td><td>-0.003</td><td>0.118</td><td>0.181</td><td>-0.063</td></tr><tr><td>Col5a1 †</td><td>32.47 ± 0.23</td><td>-1.296</td><td>0.050</td><td>-1.346</td><td>1.573</td><td>1.698</td><td>2.074 *</td><td>1.065</td><td>3.823 *</td><td>1.038</td><td>0.293</td><td>-0.082</td><td>0.376</td></tr><tr><td>Pdgfb †</td><td>29.29 ± 0.22</td><td>0.640</td><td>0.237</td><td>0.404</td><td>1.701 *</td><td>0.897</td><td>1.189</td><td>1.077</td><td>1.682 *</td><td>0.191</td><td>-0.085</td><td>-0.094</td><td>0.010</td></tr><tr><td>Fbln5 †</td><td>28.53 ± 0.14</td><td>-0.462</td><td>-0.029</td><td>-0.433</td><td>0.824</td><td>0.238</td><td>0.905</td><td>-0.134</td><td>1.575 *</td><td>0.136</td><td>0.186</td><td>-0.321</td><td>0.507</td></tr><tr><td>Fmod †</td><td>31.42 ± 0.10</td><td>-1.316 *</td><td>-0.260</td><td>-1.057 *</td><td>0.309</td><td>0.057</td><td>0.768</td><td>-0.384</td><td>1.217 *</td><td>-0.284</td><td>-0.750</td><td>-0.242</td><td>-0.508</td></tr><tr><td>Mmp9 †</td><td>34.56 ± 0.24</td><td>0.568</td><td>0.864</td><td>-0.296</td><td>2.160 *</td><td>1.186</td><td>0.967</td><td>2.494</td><td>5.068</td><td>-2.000</td><td>0.289</td><td>0.980</td><td>-0.690</td></tr><tr><td>Fbln1 †</td><td>30.71 ± 0.17</td><td>0.374</td><td>0.763</td><td>-0.389</td><td>1.666</td><td>1.588</td><td>1.223</td><td>1.119</td><td>2.411 *</td><td>-0.161</td><td>-0.629</td><td>-0.148</td><td>-0.481</td></tr><tr><td>Timp2 †</td><td>29.50 ± 0.13</td><td>0.319</td><td>0.488</td><td>-0.169</td><td>1.455 *</td><td>0.797</td><td>0.952</td><td>0.885</td><td>1.424 *</td><td>0.308</td><td>-0.201</td><td>0.072</td><td>-0.274</td></tr><tr><td>Col5a2 †</td><td>30.88 ± 0.18</td><td>-0.571</td><td>0.346</td><td>-0.917</td><td>1.280</td><td>1.092</td><td>0.856</td><td>1.236</td><td>2.922 *</td><td>-0.026</td><td>-0.048</td><td>0.065</td><td>-0.114</td></tr><tr><td>Itga2 †</td><td>34.10 ± 0.25</td><td>-0.603</td><td>0.568</td><td>-1.171</td><td>3.203 *</td><td>0.285</td><td>2.148</td><td>-0.567</td><td>3.516 *</td><td>-0.231</td><td>-0.807</td><td>-1.340</td><td>0.532</td></tr><tr><td>Lgals3</td><td>27.31 ± 0.20</td><td>-0.079</td><td>0.289</td><td>-0.368</td><td>1.423</td><td>0.684</td><td>0.699</td><td>1.264</td><td>2.212</td><td>-0.271</td><td>-0.245</td><td>0.144</td><td>-0.389</td></tr><tr><td>Timp1</td><td>31.18 ± 0.42</td><td>-0.642</td><td>0.328</td><td>-0.970</td><td>3.155</td><td>4.151</td><td>3.867</td><td>3.489</td><td>7.435</td><td>-0.743</td><td>-1.256</td><td>0.378</td><td>-1.634</td></tr><tr><td>Itgb3</td><td>33.51 ± 0.22</td><td>-0.273</td><td>0.262</td><td>-0.534</td><td>1.860</td><td>0.322</td><td>0.406</td><td>1.516</td><td>2.377</td><td>0.911</td><td>0.833</td><td>0.002</td><td>0.831</td></tr><tr><td>Pdgfa</td><td>31.10 ± 0.14</td><td>0.383</td><td>0.366</td><td>0.017</td><td>0.975</td><td>0.782</td><td>0.815</td><td>0.511</td><td>1.418</td><td>-0.204</td><td>-0.159</td><td>-0.064</td><td>-0.095</td></tr><tr><td>Il1b</td><td>32.40 ± 0.19</td><td>-0.147</td><td>0.259</td><td>-0.406</td><td>1.526</td><td>1.701</td><td>1.332</td><td>1.269</td><td>3.335</td><td>-0.494</td><td>-0.533</td><td>-0.368</td><td>-0.166</td></tr><tr><td>Pgk1</td><td>26.20 ± 0.12</td><td>0.040</td><td>-0.164</td><td>0.204</td><td>-0.438</td><td>-0.175</td><td>0.007</td><td>-0.611</td><td>-0.886</td><td>-0.002</td><td>-0.235</td><td>-0.155</td><td>-0.080</td></tr><tr><td>Itgav</td><td>27.80 ± 0.19</td><td>0.081</td><td>-0.048</td><td>0.129</td><td>0.953</td><td>0.236</td><td>0.942</td><td>0.410</td><td>1.513</td><td>-0.128</td><td>0.277</td><td>0.115</td><td>0.162</td></tr><tr><td>Vegfa</td><td>27.77 ± 0.08</td><td>-0.238</td><td>-0.073</td><td>-0.165</td><td>-0.459</td><td>-0.148</td><td>-0.196</td><td>-0.469</td><td>-0.769</td><td>0.295</td><td>-0.104</td><td>-0.131</td><td>0.027</td></tr><tr><td>Gapdh</td><td>22.53 ± 0.21</td><td>-0.034</td><td>-0.031</td><td>-0.003</td><td>-0.162</td><td>-0.205</td><td>0.033</td><td>-0.434</td><td>-0.439</td><td>-0.202</td><td>-0.308</td><td>-0.065</td><td>-0.243</td></tr><tr><td>Vegfc</td><td>32.97 ± 0.11</td><td>0.159</td><td>0.458</td><td>-0.299</td><td>0.649</td><td>0.427</td><td>0.557</td><td>-0.275</td><td>0.766</td><td>-0.072</td><td>-0.224</td><td>-0.337</td><td>0.113</td></tr><tr><td>Nkap</td><td>30.48 ± 0.09</td><td>0.329</td><td>0.132</td><td>0.197</td><td>0.557</td><td>0.061</td><td>0.628</td><td>-0.052</td><td>0.247</td><td>-0.090</td><td>-0.132</td><td>0.089</td><td>-0.221</td></tr><tr><td>Itga10</td><td>34.71 ± 0.12</td><td>0.009</td><td>0.341</td><td>-0.331</td><td>0.506</td><td>-0.373</td><td>0.308</td><td>-0.190</td><td>1.233</td><td>-0.230</td><td>0.880</td><td>0.326</td><td>0.554</td></tr><tr><td>Vegfb</td><td>29.01 ± 0.12</td><td>0.489</td><td>0.056</td><td>0.434</td><td>-0.465</td><td>0.116</td><td>-0.082</td><td>-0.249</td><td>-0.814</td><td>0.074</td><td>0.098</td><td>0.074</td><td>0.024</td></tr><tr><td>Itga11</td><td>34.60 ± 0.16</td><td>-0.295</td><td>0.767</td><td>-1.062</td><td>1.445</td><td>0.832</td><td>1.340</td><td>0.034</td><td>2.641</td><td>-0.138</td><td>-0.069</td><td>-0.136</td><td>0.067</td></tr><tr><td>Pdgfc</td><td>30.78 ± 0.11</td><td>0.203</td><td>0.140</td><td>0.063</td><td>-0.036</td><td>-0.123</td><td>0.309</td><td>-0.418</td><td>-0.412</td><td>0.191</td><td>0.140</td><td>0.190</td><td>-0.049</td></tr><tr><td>Tbp</td><td>30.20 ± 0.70</td><td>0.161</td><td>0.001</td><td>0.160</td><td>0.322</td><td>0.089</td><td>0.375</td><td>-0.218</td><td>0.144</td><td>-0.070</td><td>-0.175</td><td>-0.252</td><td>0.077</td></tr><tr><td>Ppia</td><td>23.73 ± 0.10</td><td>0.214</td><td>0.221</td><td>-0.007</td><td>0.294</td><td>0.060</td><td>0.217</td><td>-0.175</td><td>0.014</td><td>-0.017</td><td>-0.142</td><td>-0.090</td><td>-0.052</td></tr><tr><td>Itgb1bp1</td><td>29.15 ± 0.06</td><td>0.272</td><td>0.086</td><td>0.186</td><td>0.322</td><td>-0.196</td><td>0.161</td><td>-0.029</td><td>-0.197</td><td>0.001</td><td>-0.051</td><td>0.092</td><td>-0.142</td></tr><tr><td>Tff3</td><td>35.70 ± 0.14</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>0.253</td><td>0.679</td><td>NA</td><td>NA</td><td>1.029</td></tr></tbody></table>

(Continued)
<table><caption>**Table 2.** (Continued)</caption><thead><tr><th>Gene</th><th>Average<br>CT-value<br>± SEM</th><th>OK-/-<br>vs<br>OK+/-</th><th>OK-/-<br>vs<br>OK+/-</th><th>OK+/-<br>vs<br>OK+/-</th><th>OK-/-<br>vs<br>CL-/-</th><th>CL-/-<br>vs<br>Cntrl-/-</th><th>OK+/-<br>vs<br>CL+/-</th><th>CL+/-<br>vs<br>Cntrl+/-</th><th>OK+/-<br>vs<br>CL+/-</th><th>CL+/-<br>vs<br>Cntrl+/-</th><th>Cntrl-/-<br>vs<br>Cntrl+/-</th><th>Cntrl-/-<br>vs<br>Cntrl+/-</th><th>Cntrl+/-<br>vs<br>Cntrl+/-</th></tr></thead><tbody><tr><td>Il1a</td><td>35.93 ± 0.31</td><td>-0.429</td><td>1.094</td><td>-1.523</td><td>1.657</td><td>-1.016</td><td>-0.533</td><td>0.624</td><td>1.242</td><td>-0.540</td><td>-0.368</td><td>0.544</td><td>-0.912</td></tr><tr><td>Matn1</td><td>33.92 ± 1.25</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr></tbody></table>

Cntrl denotes kidneys from control animals without obstruction.

† denotes significant variations between all groups, P<0.05 by ANOVA.

* denotes P<0.05 for the individual comparison.

doi:10.1371/journal.pone.0143390.t002

even though Timp2 expression in OK and CL in Mmp2-/- and Mmp2+/Mmp2+/- mice differ, the limited possibility of an increase in Mmp2 at the level of expression would probably reduce this effect. This may be the reason why they are also protected. In our study, Timp1 was not affected by UUO, and this is supported by evidence that elimination of Timp1 alone was not found to be sufficient in order to alter the severity of fibrosis during UUO [29].

The present results did not show consistent findings in the Pdgf system. This is not entirely surprising since the Pdgf system is important in kidney fibrosis [30] and in UUO [31,32], but it is not necessary for the development of fibrosis in the kidney [33]. There was no differential expression of Pdgfa or Pdgfc. On the other hand Pdgfb was differentially expressed after UUO in Mmp2-/- and Mmp2+/Mmp2+/- mice but not in Mmp2+/- animals. This is difficult to explain, especially since antagonists have a positive effect in several models of renal disease. Pdgfb, however, also plays a role in the repair of cellular damage [34].

Tff3, Vgfa, Vgfb, Vgfc, Il1b and Lgals3 were not differentially expressed in the present study even though they have been implicated in kidney disease. Specifically, Tff3 has been suggested as a diagnostic marker in CKD since it increases in both serum and urine during CKD progression [35]. Vegfs have been demonstrated to slow down the progression of renal injury in experimental models and can be administered in renoprotective therapy [36], but overstimulation can induce glomerular pathology. Il1b is known to stimulate proliferation of human renal fibroblasts as well as production of matrix proteins, while Lhals3 has been shown to protect against fibrosis [37,38].

The clinical treatment of ureteral obstruction is primarily surgical, but as a strategy for limiting the destruction of kidney tissue in progressive or recurring disease, some intervention against Mmp2 may be useful. It is further unlikely that reduced Mmp2 expression provides complete protection from the damage in UUO. There is no doubt that a longer period of UUO would lead to more severe damage, even in mice with decreased Mmp2 expression.

In conclusion, both homozygous and heterozygous genetic inactivation of Mmp2 protect mice against hydronephrosis and kidney fibrosis after UUO, as indicated by both histology and gene expression. The genetic mechanism seems to be a reduced ability to respond with Mmp2 upregulation under stress, and this may suggest that there is a threshold level of Mmp2 necessary for pelvic remodelling and genetic events leading to activation of fibrosis.

## Acknowledgments

The study was supported by the Western Health Authority of Norway (Helse Vest), and the Swedish Society for Medical Research (SSMF).

Parts of this study were presented at Experimental Biology 2012 and published as a meeting abstract [39].
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