SUPPLEMENTAL INFORMATION

Supplemental Figure 1

Supplemental Figure 1. Quantitative PCR (qPCR) showing transcripts of Eph/ family in whole kidney samples following UUO kidney injury. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (A) EphA/ephrinA in whole kidneys. Epha2 and efna4 are highly expressed and regulated in d2 and d4 UUO kidneys. (B) EphB/ephrinB in whole kidneys. Ephb4, efnb1 and efnb2 are highly expressed and regulated in d2 and d4 UUO kidneys. N=3/time point.

Supplemental Figure 2

Supplemental Figure 2. Quantitative PCR showing transcripts of Eph/ephrin family gene in primary culture of proximal tubular epithelial cells with or without transforming growth factor (TGF)-β stimulation (10 ng/mL) for 48h. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). TGF-β stimulation significantly increases transcripts of Ephb4, efnb1 and efnb2 in tubular epithelial cells. As we confirmed, these transcripts are increased in whole kidneys following injury, which most likely reflects elevation of these transcripts in tubular epithelial cells. N=3. *P<0.05 vs control.

Supplemental Figure 3

Supplemental Figure 3. Expression of phosphorylated ephrinB (p-ephrinB) in control and d4 UUO kidneys of WT, ephrinB2 5Y and ephrinB2 ΔV mice. (A) Representative images show that p-ephrinB is detected only after kidney injury of WT and ephrinB2 ΔV mice, suggesting that ephrinB2, but not ephrinB1, is activated in d4 UUO kidneys. Moreover, this result means phosphotyrosine-dependent signaling of ephrinB2 is not coupled with PDZ-dependent signaling of ephrinB2. Arrowheads: positive cells, Bar: 10µm. (B) Quantification of p-ephrinB+ cells in d4 UUO kidneys. N=3. *P<0.05, NS: no significant difference. Supplemental Figure 4

Supplemental Figure 4. Apoptosis of PTC MVECs contributes to PTC rarefaction in ephrinB2 ΔV mice, whereas CD31+ leukocytes and homing of endothelial progenitor cells do not play a role in impaired angiogenesis in diseased kidneys of ephrinB2 ΔV mice. (A) Quantification of CD31+ active caspase-3+ cells in control and d10 UUO kidneys (200× HPF). Note increased apoptosis of MVECs in ephrinB2 ΔV (dV) mice after UUO. (B) Percentage of CD31+ CD45+ cells among total CD31+ cells in control and UUO kidneys of WT, ephrinB2 5Y and ephrinB2 ΔV (dV) mice. Note that less than 0.5% of CD31+ cell population expresses CD45. (C) qPCR of SDF-1 (Cxcl12) and its receptor, Cxcr4 transcripts in control and UUO kidneys of WT, ephrinB2 5Y and ephrinB2 ΔV (dV) mice. N=3/group/time point. *P<0.05, NS: no significant difference.

Supplemental Figure 5

Supplemental Figure 5. EphrinB2 ΔV mice demonstrate more PTC rarefaction and more fibrosis than WT mice after unilateral ischemic reperfusion injury (IRI). (A) Immunofluorescence images of CD31+ MVECs in outer medulla of control, d5 and d14 IRI kidneys of WT and ephrinB2 ΔV mice. Bar: 50µm. (B) Peritubular capillary (PTC) density of WT and ephrinB2 ΔV (dV) mice after unilateral IRI. Note prominent PTC rarefaction in ephrinB2 ΔV (dV) mice compared with WT mice following IRI. (C) Images of picro-sirius red stained control and d14 IRI kidney sections of WT and ephrinB2 ΔV mice. Bar: 50µm. (D) Morphometric quantification of fibrillar collagen in control and d14 IRI kidneys of WT and ephrinB2 ΔV (dV) mice. N=3/group/time point. *P<0.05, NS: no significant difference.

Supplemental Figure 6

Supplemental Figure 6. Infiltrating macrophages do not have a major role in enhanced capillary rarefaction and fibrosis in the kidney of ephrinB2 ΔV mice. (A) Immunofluorescence images of F4/80+ macrophages in control, d4 and d10 UUO kidneys of WT, ephrinB2 5Y and ephrinB2 ΔV mice. g: glomerulus. Bar: 20µm. (B) Number of F4/80+ macrophages in control and UUO kidneys. There is no significant difference between groups at all time points. N=3-4/group/time point. NS: no significant difference. (C) qPCR for Trem1 (M1 marker) and Il-10 (IL-10, M2 marker) transcripts in control and UUO kidneys of WT, ephrinB2 5Y and ephrinB2 ΔV (dV) mice. N=3/group/time point. NS: no significant difference. (D) Quantification of CD206+ cells in control and UUO kidneys. Note that there is no difference in number of CD206+ macrophages (M2 macrophages) between groups. N=3/group/time point. NS: no significant difference. Supplemental Figure 7

Supplemental Figure 7. EphA2-receptor does not contribute to capillary rarefaction and fibrosis after kidney injury. (A) Immunofluorescence images of CD31+ MVECs in control and d4 UUO kidneys of WT and EphA2-/- mice. (B) Peritubular capillary density of WT and EphA2 knockout (KO) mice. (C) Immunofluorescence images of αSMA+ myofibroblasts in control and d4 UUO kidneys of WT and EphA2-/- mice. (D) Number of αSMA+ myofibroblasts in WT and EphA2 KO mice. g: glomerulus. Bar: 20µm. N=3/group/time point. NS: no significant difference.

Supplemental Figure 8

Supplemental Figure 8. Primary kidney pericytes isolated from each genotype of mice. (A) Phase contrast images. Morphologically, there is no remarkable difference between three groups of pericytes. Bar: 20µm. (B) Graph showing proliferation of WT and ephrinB2 ΔV (dV) pericytes assessed by BrdU incorporation for short term (6h). N=3/group. NS: no significant difference.

Supplemental Figure 9

Supplemental Figure 9. Gating strategy to obtain MVECs from kidney single cell suspension. (A) Live cells are gated for kidney cells. (B-C) Doublets are excluded using forward (FSC) and side scatter (SSC) gates. (D) CD31+CD11b- cells are identified and sorted for culture. The number shown inside of each gate is the ratio of gated cell number to its parent cell number. (E) qPCR for pericyte transcript (Pdgfrb) or endothelial transcript (CD31, Pecam) in purified kidney pericytes (left) or purified MVECs (right) sorted from normal kidney. Note that CD31 transcript is much more abundantly expressed in MVECs than pericytes. N=3.

Supplemental Table 1. Primers for qPCR

Gene Product Size (bp) GeneBank number Forward primer Reverse primer GAPDH 133 NM_008084 ctggagaaacctgccaagta aagagtgggagttgctgttg Epha1 131 NM_023580 tgaggggtctaccatctgt agagttgagtccctgatgtg Epha2 126 NM_010139 gccagtttagccaccacaata ttcatgttggccaggtacttc Epha3 97 NM_010140 cacaccaatcaggacttacc cttctgagctgagtttctgg Epha4 120 NM_007936 ctggcagaaagagagaagtg gtgtttggtctggaactctc Epha5 143 NM_007937 caaggtgtctgactttggac agacatcactggaagaggtg Epha6 94 NM_007938 cagaacagccttgctctatc gctcatgctctttctcgtag Epha7 126 NM_010141 gtctccagtgaacagaatcc gtcagccttgctataaccac Epha8 137 NM_007939 aggactcggatgaagagaag acctggttcctcataggtgt Epha10 149 NM_177671 ataccactctgccttgctct tcatccacgccactaatc Efna1 163 NM_010107 tcaatggcaaaatcactcataatc agcagcagtggtaggagcaatact Efna2 92 NM_007909 actacatctctgccacacct ctcatacagggtctcattgg Efna3 139 NM_010108 agtgtctgaggatgaaggtg ggattctctccctcaaagtc Efna4 95 NM_007910 cacccaatctactggaactc gggcagaagatgtctaggta Efna5 140 NM_207654 gaaggtcctgtctaaagctc ctcatgtacggtgtcatctg Ephb1 150 NM_023580 cacgtctctgtcaacatcac gtaccggatctcatagtcca Ephb2 132 NM_010142 cggactacaccagctttaac gaatgtcctccatcatcatc Ephb3 95 NM_010143 acatctcatccagagagtgg gactcacgcacattacacac Ephb4 160 NM_010144 ccaggagcatcacagtcaga aacttccctcccattgctct Ephb6 127 NM_001146351 gctacacagaacaactgcaa tatgtagggtcaacctctcg Efnb1 149 NM_010110 gggcctggaattcaaaaagt tcacagcatttggatcttgc Efnb2 104 NM_010111 aggaatcacggtccaacaag tcggtgctagaacctggatt Efnb3 92 NM_007911 gcctaaccagaggcatgaag catgggcatttcagacacag Cxcl12 130 NM_021704 ctctgcatcagtgacggtaaac atctgaagggcacagtttgg Cxcr4 149 NM_009911 tgaggcgtttggtgctccggtaac tcccggaagcagggttccttgttg Vegfa 122 NM_001025250 tacctccaccatgccaagtggtccc agggtctcaatcggacggcagtagc Acta2 160 NM_007392 ctgacagaggcaccactgaa catctccagagtccagcaca Col1a1 142 NM_007742 gagcggagagtactggatcg gttcgggctgatgtaccagt Col3a1 110 NM_009930 accaaaaggtgatgctggac gacctcgtgctccagttagc Trem1 217 NM_021406 ctgtgcgtgttctttgtctc atgtggacttcactgggtct Il10 117 NM_010548 aagtgatgccccaggcagagaagc aggggagaaatcgatgacagcgcc Pdgfrb 128 NM_001146268 cttcaaagtggtggtgatct cagactcaatgaccttccat Pecam 136 NM_008816 gatttctgagcctagtgtgg ggacacttccacttctgtgt