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1 Supplementary Materials: Supplemental Figure 1: Profibrotic markers in wt/wt or ex/ex mouse kidneys 42 days post IRI. The levels of fibronectin and αsma were examined by immunostaining in ADAM17 wt/wt or ADAM17 ex/ex mouse kidneys 42 days post ischemia (left: representative images, right: quantification, n=4, scale bar: 100 µm). *: P<0.05; **: P<0.01 as determined by an unpaired 2- tailed Student s t test. ADAM17: a-disintegrin-and-metalloprotease-17, IRI: ischemia-reperfusion-injury, αsma: alpha smooth muscle actin 1
2 Supplemental Figure 2: Kidney stroma-specific ADAM17 knockout does not protect against fibrosis after UUO. A. Stroma-specific knock out (FoxD1-Cre) of ADAM17 was examined by immunostaining in the UUO model 7 days post obstruction. αsma (a myofibroblast marker) is strongly co-localized with ADAM17 in kidney stroma in wt but not in 2
3 ADAM17fl/fl - FoxD1 Cre+/- mice. B. Induction of fibronectin and αsma was examined by Immunostaining (left: representative images, right: quantification, n=6-7, scale bar: 100 µm). ADAM17: a-disintegrin-and-metalloprotease-17, αsma: alpha-smooth-muscle-actin, UUO: unilateral-ureteral-obstruction 3
4 Supplemental Figure 3: ADAM17 pathway induction by UUO in wt/wt or ex/ex mouse kidneys. A, B. qpcr analysis of mrna expression levels of ADAM17 pathway components in ex/ex or wt/wt mice at 7 days of UUO, expressed as fold over respective sham-injured mice (n=5). C. UUO (7 days) -induced EGFR phosphorylation (Y1068) was examined by western blot in wt/wt or ex/ex mice (left: quantification, n=3, right: representative blot). GAPDH was used as loading control. *: P<0.05; **: P<0.01;***:P<0.001 as determined by an unpaired 2-tailed Student s t test. ADAM17: a-disintegrin-and-metalloprotease-17, AREG: amphiregulin, EGF: epidermal-growthfactor, EGFR: epidermal-growth-factor-receptor, EREG: epiregulin, HB-EGF: heparin-binding- 4
5 epidermal-growth-factor-like growth factor, TGFα: transforming-growth-factor-alpha, TNFα: tumor-necrosis-factor-alpha, TNFR: tumor-necrosis-factor-receptor, UUO: unilateral-ureteralobstruction 5
6 Supplemental Figure 4: ADAM17 pathway and proinflammatory markers induction by different EGFR ligands in HPTC. qpcr analysis of ADAM17 pathway components and proinflammatory marker mrna expression after addition of equimolar amounts of different EGFR ligands (HB-EGF, EREG, TGFα, EGF, AREG) or of TNFα for 24h. Average results of 3 independent experiments are shown as fold over control after hierarchical clustering of the added factors. ADAM10: a-disintegrin-and-metalloprotease-10, ADAM17: a-disintegrin-and-metalloprotease- 17, AREG: amphiregulin, EGF: epidermal-growth-factor, EGFR: epidermal-growth-factorreceptor, EREG: epiregulin, HB-EGF: heparin-binding-epidermal-growth-factor-like growth 6
7 factor, MCP1: monocyte-chemoattractant-protein-1, MIP1A: macrophage-inflammatory-protein- 1-alpha, MIP1B: macrophage-inflammatory-protein-1-beta, RANTES: regulated-on-activationnormal-t-cell-expressed-and-secreted, TGFα: transforming-growth-factor-alpha, TGFβ: transforming-growth-factor-beta, TNFα: tumor-necrosis-factor-alpha, TNFR: tumor-necrosisfactor-receptor. 7
8 Supplemental Table 1: Patient characteristics of AKI, CKD patients and healthy volunteers included in urinary ELISA measurements. Healthy controls Gender Race Age F White 49 M White 50 M White 50 F Asian 41 F Black 28 M White 27 F Asian 28 F White 37 M White 28 F Asian 28 CKD patients Gender Race Age egfr Protein/Cr ACE/ARB Diuretic DM Cause SBP DBP Male Black FSGS Female Hispanic DM
9 Female Asian Chronic nephritis Female Black DM/HTN Female White CAKUT Male Hispanic Chronic nephritis Female White LITHIUM Female Black DM/HTN Male Black DM Female White DM AKI patients Admission Baseline Peak Creatinine scr scr Reason for AKI Hypotension/hypoperfusion, likely ATN ATN ATN, hemodynamic changes intra-op ATN ATN (post-op) 9
10 ATN (sepsis, hypotension) ATN (post-operative, hemodynamic, vascular) ATN, i.v. contrast Cardiogenic shock, ATN Post-operative AKI 10
11 Supplemental Table 2: Primers used for qpcr. Target gene Synonym Species Direction Sequence ADAM10 human Forward GCTGTGATTGCCCAGATATC CA ADAM10 human Reverse CACCATGAAACTGATGTTAC GGA ADAM17 TACE human Forward AGTGCAGTGACAGGAACAGT C ADAM17 TACE human Reverse GGACACGCCTTTGCAAGTAG AREG human Forward TCGGCTCAGGCCATTATGC AREG human Reverse AATCCATCAGCACTGTGGTC C CCL2 MCP1 human Forward AGCATGAAAGTCTCTGCCGC CCL2 MCP1 human Reverse GGCATTGATTGCATCTGGCT G CCL3 MIP1A human Forward TTCCGTCACCTGCTCAGAAT CCL3 MIP1A human Reverse GCAGCAAGTGATGCAGAGAA C CCL4 MIP1B human Forward CCCAGCCAGCTGTGGTATTC CCL4 MIP1B human Reverse AGCAGCTCAGTTCAGTTCCA 11
12 CCL5 RANTES human Forward CTGCTTTGCCTACATTGCCC CCL5 RANTES human Reverse TCGGGTGACAAAGACGACTG EGF human Forward CTTGGGAGCCTGAGCAGAAA EGF human Reverse TGCACAAGTGTGACTGGAGG EGFR human Forward CGAATGGGCCTAAGATCCCG EGFR human Reverse CTTCGCATGAAGAGGCCGAT EREG human Forward ACGTGTGGCTCAAGTGTCAA EREG human Reverse CACTTCACACCTGCAGTAGT TT GAPDH human/mouse Forward ACCACAGTCCATGCCATCAC GAPDH human/mouse Reverse TCCACCACCCTGTTGCTGTA HBEGF human Forward TTGTGCTCAAGGAATCGGCT HBEGF human Reverse CAACTGGGGACGAAGGAGTC PRLP0 human Forward CGTCCTCGTGGAAGTGACAT PRLP0 human Reverse TAGTTGGACTTCCAGGTCGC TGFA human Forward GTAAAATGGTCCCCTCGGCT TGFA human Reverse GGGTCTGCACTCAGCGG TGFB1 human Forward GGAAATTGAGGGCTTTCGCC TGFB2 human Reverse CCGGTAGTGAACCCGTTGAT TNF TNFA human Forward GCCCATGTTGTAGCAAACCC 12
13 TNF TNFA human Reverse TATCTCTCAGCTCCACGCCA TNFRSF1A TNFR1 human Forward ACGAGTGTGTCTCCTGTAGT TNFRSF1A TNFR1 human Reverse CACTGTGGTGCCTGAGTCC TNFRSF1B TNFR2 human Forward CCCAGGTGAAAAAGAAGCCC TNFRSF1B TNFR2 human Reverse CAGGTGCTGCTGCTCGG Acta2 αsma mouse Forward AGCCATCTTTCATTGGGATG GA Acta2 αsma mouse Reverse TACCCCCTGACAGGACGTTG Adam10 mouse Forward TGCCTGGCCTATGTCTTCAC Adam10 mouse Reverse TATTCCCCCAGAGCTTCCTGA Adam17 Tace mouse Forward TCTGAAGAGTTTGTTCGTCG AGT Adam17 Tace mouse Reverse CTTCTCCACGGCCCATGTAT Areg mouse Forward GCTGAGGACAATGCAGGGTA A Areg mouse Reverse GTGACAACTGGGCATCTGGA Ccl2 Mcp1 mouse Forward CACTCACCTGCTGCTACTCA Ccl2 Mcp1 mouse Reverse GCTTGGTGACAAAAACTACA GC Ccl3 Mip1A mouse Forward CAGCCAGGTGTCATTTTCCTG A 13
14 Ccl3 Mip1A mouse Reverse TCTCAGGCATTCAGTTCCAG G Ccl4 Mip1B mouse Forward GCCAGCTGTGGTATTCCTGA Ccl4 Mip1B mouse Reverse AGCTGCTCAGTTCAACTCCA Ccl5 Rantes mouse Forward CTCACCATATGGCTCGGACA Ccl5 Rantes mouse Reverse CGACTGCAAGATTGGAGCAC Egf mouse Forward AGCATACTCAGCGTCACAGC Egf mouse Reverse GCAGGACCGGCACAAGTC Egfr mouse Forward ACCTCTCCCGGTCAGAGATG Egfr mouse Reverse CTTGTGCCTTGGCAGACTTTC Ereg mouse Forward TGCTTTGTCTAGGTTCCCACC Ereg mouse Reverse GGCGGTACAGTTATCCTCGG Hbegf mouse Forward TCTGGCCGCAGTGTTGTCC Hbegf mouse Reverse GGTTTGTGGATCCAGTGGGA G Pdgfrb Pdgfr1 mouse Forward GTCAATGTCCCCGTCCGTG Pdgfrb Pdgfr2 mouse Reverse CTGCAGGTAGACCAGGTGAC Prpl0 mouse Forward CTCTCGCTTTCTGGAGGGTG Prpl0 mouse Reverse ACGCGCTTGTACCCATTGAT Tgfa mouse Forward CTCTGCTAGCGCTGGGTATC Tgfa mouse Reverse TGGGCACTTGTTGAAGTGAG 14
15 A Tgfb1 Tgfβ mouse Forward CTGCTGACCCCCACTGATAC Tgfb1 Tgfβ mouse Reverse AGCCCTGTATTCCGTCTCCT Tnf Tnfa mouse Forward ATGGCCTCCCTCTCATCAGT Tnf Tnfa mouse Reverse CTTGGTGGTTTGCTACGACG Tnfrsf1a Tnfr1 mouse Forward ACGAATCACTCTGCTCCGTG Tnfrsf1a Tnfr1 mouse Reverse TCGCAAGGTCTGCATTGTCA Tnfrsf1b Tnfr2 mouse Forward GAGATGCCAAGGTGCCTCAT Tnfrsf1b Tnfr2 mouse Reverse AACTGGGTGCTGTGGTCAAC 15
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