Tubular damage is reduced in double mutant mice deficient of collagen XV and XVIII compared to the WT mice.

<p><i>A–D:</i> Paraffin-embedded sections stained with periodic acid Schiff's (PAS) reagent in sham operated WT(day 1; <i>A</i>), in sham-operated <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (day 1; <i>B</i>) and in WT (<i>C</i>) and <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (<i>D</i>) at day 5 after renal I/R. WT mice showed tubular casts, tubular widening and flattening, and loss of nuclei in tubular cells (black arrows). Such damage was not observed in double collagen mutant mice kidneys. Scale bars 50 µm. <i>E:</i> Percentage of tubular damage was determined at different time points after I/R (see methods) in the <i>Col15a1^−/−</i>, <i>Col18a1^−/−</i> and <i>Col15a1^−/−×Col18a1^−/−</i> double mutant mice compared to the WT controls (*: day 5 p<0.01 double mutant mice compare to WT). Data is presented as mean percentage ± SEM.</p

Increased expression of TNF-α and MCP-1 in double mutant mice, lacking both collagen XV and XVIII compared to WT.

<p><i>A–B:</i> Immunofluorescent staining of MCP-1 (red) in WT (<i>A</i>) and double mutant mice (<i>B</i>) at day 5. Double mutant mice showed an increased expression of MCP-1 in peri-tubular capillaries. Scale bars 20 µm. <i>C:</i> mRNA expression of MCP-1 in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (***: p<0.001 double mutant mice compared to WT). <i>D:</i> mRNA expression of TNF-α in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (**: p<0.01 double mutant mice compared to WT).</p

Reduced neutrophil influx after renal I/R in the mutant mice for collagen XV and XVIII.

<p><i>A:</i> Immunofluorescent staining for collagen IV, XV and XVIII (green) and neutrophils (red) in WT and double mutant mice at day 1 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of neutrophils around these capillaries. Less neutrophil influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signals (in green). Nuclei are shown in blue. Scale bars 20 µm. <i>B:</i> Immunofluorescent staining for neutrophils (red) and nuclei (blue) in WT and double mutant sham operated mice at day 1 after I/R showed the absence of neurophils in renal tissues. Scale bars 20 µm. <i>C:</i> Number of neutrophils per HPF (High Power Field) at different timepoints after I/R. At day 1 after reperfusion double mutant mice showed a decreased number of neutrophils compared to WT mice (p<0.05). At day 5 significantly less neutrophils were observed in kidneys of <i>Col15a1^−/−</i> and <i>Col18a1^−/−</i> mice compared to WT (p<0.05) as well as in those of double mutant mice compared to WT (p<0.01). Data is presented as mean ± SEM. *: p<0.05, **: p<0.01.</p

MCP-1-induced monocyte migration is increased in the presence of immobilized heparin-albumin and glycosylated collagen XVIII.

<p><i>A:</i> MCP-1 dose dependently increased the migration of monocytes over a porous membrane. Immobilization of heparin-albumin, mimicking an artificial BM HSPG, promotes monocyte transmigration. Spontaneous migration over albumin-coated membrane in the absence of MCP-1 was set as 1 and the other values were calculated accordingly. The error bars represent SEM. <i>B:</i> Transmigration of monocytes towards MCP-1 (10 ng/ml) was increased in the presence of heparin-albumin and collagen XVIII with long GAG chains. Heparin-albumin increased the monocyte migration significantly compared to albumin coated membrane (p<0.01). N-terminal fragment of short collagen XVIII with long GAG chains promoted transmigration significantly compared to albumin and N-terminal fragment without GAG chain (both p<0.05). Relative to albumin, also the full-length short collagen XVIII promoted MCP-1-induced monocyte transmigration to some extent (not significant). Data is calculated relative to migration over albumin-coated membrane towards 10 ng/ml MCP-1. The error bars represent SEM. *: p<0.05, **: p<0.01.</p

Production and characterization of recombinant mouse Tsp1-C18 and Short-XVIII by Western blotting with anti-all-18 antibody.

<p><i>A:</i> Gel filtration fractions of recombinant Tsp1-C18 with variable degree of glycosylation and non-glycosylated core protein of ∼55 kDa separated by 10% SDS-PAGE. Fractions 29 and 33 with Long and Intermediate GAG chains, respectively, and 39–40 containing core Tsp-C18 were used for binding and migration experiments. <i>B:</i> Expression of full-length Short-XVIII by two representative stable 293-EBNA clones 1 and 2. Apparently non-glycosylated Short-XVIII of ∼180 kDa was detected in cell lysates (L). Conditioned cell culture media (CM) contained highly glycosylated recombinant Short-XVIII migrating as a smear above 200 kDa in 7% SDS-PAGE. Control 293-EBNA cell lysate and CM did not show reactivity for anti-all-18 antibody with short exposure. <i>C:</i> Heparitinase I treatment of intermediate Tsp1-C18 (fractions 31 and 33, MW ∼130 kDa) removed most GAG chains and revealed a core protein of ∼55 kDa. Heparitinase I treatment did not alter the size of low MW Tsp1-C18 (fractions 39–40) suggesting that this band represents non-glycosylated core protein. <i>D:</i> Heparitinase I and chondroitinase ABC treatments of intermediate Tsp1-C18 (fractions 31 and 33) indicate that most of the GAGs within recombinant Tsp1-C18 produced in HEK-293 cells are HS chains.</p

Reduced macrophage/monocyte influx after renal I/R in double mutant mice for collagen XV and XVIII.

<p><i>A:</i> Immunofluorescent staining for collagen IV, XV and XVIII (green) and macrophages (red) in WT and double mutant mice at day 5 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of macrophage/monocytes around these capillaries. Less macrophage influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signal (in green). The nuclei are stained in blue. Scale bars 20 µm. <i>B:</i> Immunofluorescent staining for macrophages (red) and nuclei (blue) in WT and double mutant sham operated mice at day 5 after I/R showed no macrophage/monocyte in renal tissues. Scale bars 20 µm. <i>C:</i> Number of macrophage/monocytes per HPF (High Power Field) at different timepoints after I/R. At day 5 significantly less macrophage/monocytes were observed in kidneys of <i>Col15a1^−/−×Col18a1^−/−</i> mice compared to WT (p<0.05). Data is presented as mean ± SEM. *: p<0.05.</p

Serum urea levels in WT, collagen XV and/or XVIII deficient mice after renal I/R.

<p>WT mice showed an increased serum urea levels at day 5 after I/R while all collagen mutant mice (<i>Col15a1^−/−</i> and <i>Col18a1^−/−</i> p<0.05, and <i>Col15a1^−/−×Col18a1^−/−</i> p<0.01) had significantly lower serum urea levels compared to WT at this time point. The results are expresses as serum urea mmol/l ± SEM. *: p<0.05, **: p<0.01.</p

Tubular cell activation marker VCAM-1 is reduced in double mutant mice, deficient of collagen XV and XVIII compared to the WT mice.

<p><i>A–D:</i> Cryosections stained for VCAM-1 expression in sham operated (day 1; <i>A</i>), in sham-operated <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (day 1; <i>B</i>) and in WT (<i>C</i>) and <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (<i>D</i>) at day 5. WT mice showed an upregulation of VCAM-1 in tubular compartment while sham and double collagen deficient mice showed a weak VCAM-1 expression in between of tubuli (peritubular capillaries). Scale bars 50 µm. <i>E:</i> Tubular cell activation was quantified as percentage of VCAM-1 expression in tubular cells at different timepoints after I/R in the <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice (**: at day 5 p<0.01 double mutant mice compared to WT) (see methods). Data is presented as mean percentage ± SEM. <i>F:</i> Quantitative RT-PCR on RNA isolated from renal tissue, day 5 after I/R. VCAM-1 mRNA expression is reduced in all mutant mice, and reached statistical significance in the double KO mice (***: p<0.001).</p

Arresten inhibits migration of HSC-3 cells.

<p><b>A.</b> 30 000 Ctrl-HSC and Arr-HSC cells were allowed to migrate through Transwell inserts and the number of migrated cells was counted under a microscope at 50×magnification. Mann-Whitney U-test, ***p<0.001, (n = total number of fields analyzed, 2–4 fields per Transwell insert). <b>B.</b> 30 000 HSC-3 cells were allowed to migrate through Transwell inserts in the presence of human recombinant purified arresten (5 and 20 µg/ml) and the number of migrated cells was counted as described above. Mann-Whitney U-test, **p<0.01, (n = total number of fields analyzed, 3–5 fields per Transwell insert). <b>C.</b> Scratch wound healing assay with Ctr-HSC and Arr-HSC clones in which the closure of the wound was measured at 0, 16 and 48 h. Scale bar 50 µm<b>. </b><b>E.</b> Quantification of scratch wound healing in the Ctrl-HSC and Arr-HSC clones. Mann-Whitney U-test, ***p<0.001, (n = 70 fields at 0, 16 and 48 h per clone).</p

Arr-HSC cell spreading is impaired in the presence of a function-blocking antibody to α1 integrin. A.

<p>The impedance, reflecting cell adhesion and spreading, was measured for Ctrl-HSC and Arr-HSC cells using electric cell-substrate impedance sensing (ECIS) (mean of duplicate wells of representative ECIS plates). The Arr-HSC cells showed markedly higher impedance at a low frequency than the control cells. <b>B</b>. Treatment of Arr-HSC cells with a specific function-blocking α1 antibody reduced the impedance when compared to the untreated Arr-HSC cells. Treatment of Arr-HSC cells with integrin α2 antibody almost completely abolished the cell spreading. <b>C</b>. Ctrl-HSC cells showed reduced spreading in the presence of integrin α2 antibody while α1 antibody had no effect on impedance.</p