Supplemental Figure 1.

F2R S1PR1

GPR160 ELTD1

CD97 Supplemental Figure 2.

A Human Protein Atlas RNAseq database

B Expression in mouse ssues

lung glom brain heart liver kidney spleen testis skm rok

Gprc5a Gapdh Supplemental Figure 3.

A Gprc5a Pdgfrb Merged

CL

CL

Gprc5a CD31 Merged CL

CL

B C 1.4 * 1.2 mc 1 matrix 0.8

0.6

0.4 matrix 0.2

0 pod end 1 2 mes3

D Vector Gprc5a E

40 kD - - Gprc5a

- acn

Supplemental Figure 4.

Control 12 month-old KO 12 month-old A B

C D

E F 1 2 0.8 * 1.5 score 0.6 m µ 1 0.4

0.2 Slits/ 0.5

Mesangial 0 0 Ctrl KO Ctrl KO Supplemental Figure 5. A

B 1.2 Vector Gprc5a 1 * 0.8 0.6 * 0.4 * NormalizedDensity 0.2 0 pEGFR/tEGR pSmad/tSmad TGF-β1

C 1.8 siCON 1.6 siGprc5a * 1.4 * * 1.2 1 0.8 0.6

NormalizedDensity 0.4 0.2 0 pEGFR/tEGR pSmad/tSmad TGF-β1 Supplemental table 1. List of glomerulus-expressed GPCRs as detected by qPCR. Data shown as mean ± standard deviation (Glom=glomerulus, Rok=rest of kidney).

GPCR Glom Rok Glom/Rok LPAR6 41892,11 ± 38478,89 1040,06 ± 1370,12 39,28 ELTD1 30275,64 ± 14085,26 33,23 ± 46,99 910,13 GPR116 24020,06 ± 7789,84 55,1 ± 47,75 434,96 PTH1R 15402,81 ± 17644,32 7521,01 ± 3264,57 1,05 CALCRL 14096,09 ± 3854,84 199,06 ± 222,52 69,81 HPRT1 13342,04 ± 10677,69 1824,77 ± 1767,23 6,31 S1PR5 9474,7 ± 10124,3 110,84 ± 29,54 84,48 LPHN2 8645,89 ± 914,74 256,04 ± 293,87 32,77 FZD1 8176,2 ± 4947,45 1321,97 ± 1311,91 5,18 CXCR4 7097,31 ± 4388,91 535,98 ± 640,28 12,24 GPR160 6446,59 ± 1550,07 4816,3 ± 5918,99 0,34 NPY1R 6177,74 ± 6282,76 209,64 ± 296,48 28,47 PTGER4 5323,66 ± 3789,51 179,13 ± 210 28,72 RXFP1 4785,68 ± 1531,16 1,13 ± 1,6 4234,74 ADRB1 4404,61 ± 6229,06 307,91 ± 348,03 13,3 F2R 4140,07 ± 1977,93 167,62 ± 174,95 23,7 GPR4 3969,84 ± 833,92 91,04 ± 112,2 42,61 NPR1 3934,21 ± 3350,55 95,16 ± 45,01 40,34 S1PR1 3456,24 ± 275,58 18,02 ± 20,35 190,78 P2RY13 2889 ± 1976,44 300,27 ± 334,26 8,62 GPR18 2638,88 ± 55,61 274,22 ± 387,81 8,62 CD97 2473,76 ± 1908,21 81,07 ± 22,28 29,51 FZD4 2276,04 ± 1152,38 133,56 ± 118,89 16,04 LGR4 2066,89 ± 917,68 636,44 ± 782,06 2,25 EDNRB 1968,09 ± 1987,51 175,56 ± 203,42 10,21 FZD8 1883,8 ± 1522,78 286,02 ± 164,32 5,59 CMKLR1 1843,35 ± 1319,13 24,54 ± 20,92 74,12 FZD7 1812,88 ± 909,53 334,15 ± 417,1 4,43 CRCP 1618,04 ± 927,95 886,3 ± 190,45 0,83 GPRC5B 1585,22 ± 371,1 43,6 ± 3,61 35,36 APLNR 1437,43 ± 1565,72 17,14 ± 12,4 82,88 P2RY8 1396,83 ± 492,35 116,37 ± 116,17 11 CCRL2 1334,77 ± 648,96 27,91 ± 35,84 46,83 TBP 1306,6 ± 143 201,97 ± 132,33 5,47 LTB4R2 1255,64 ± 913,91 31,48 ± 35,77 38,88 GPR56 1216,47 ± 42,82 707,7 ± 478,7 0,72 NPR2 1208,39 ± 764,5 82,62 ± 83,16 13,63 CYSLTR1 1173,21 ± 16,82 26,73 ± 21,52 42,89 SSTR1 1098,23 ± 906,65 600,49 ± 680,75 0,83 XPR1 1071,68 ± 136,26 861,57 ± 892,77 0,24 GPBAR1 1053,72 ± 873,55 87,5 ± 27,04 11,04 GPR34 1037,58 ± 4,29 456,5 ± 606,21 1,27 TAS2R13 1027,12 ± 214,18 340,33 ± 481,29 2,02 FPR1 1001,44 ± 1104,68 48,7 ± 54,58 19,57 VIPR1 957,99 ± 516,73 1,36 ± 0,22 702,22 GPR65 948,64 ± 345,01 37,8 ± 43,95 24,1 CHRM3 929,86 ± 118,76 3,65 ± 2,57 253,9 DRD5 921,67 ± 1168,33 441,64 ± 624,57 1,09 AGTR1 920,76 ± 1302,15 151,15 ± 205,5 5,09 CX3CR1 873,69 ± 145,36 49,2 ± 46,77 16,76 F2RL2 869,62 ± 159,18 99,39 ± 132,67 7,75 TAS2R14 832,8 ± 731,27 165,07 ± 217,96 4,05 GABBR1 828,79 ± 283 9,51 ± 13,45 86,17 CXCR2 825,56 ± 752,67 61,89 ± 54,6 12,34 TBXA2R 821,6 ± 93,03 0 ± 0 - TAS2R10 773,93 ± 567,76 280,27 ± 396,37 1,76 P2RY1 747,01 ± 345,04 352,9 ± 480,6 1,12 NPY5R 743,98 ± 35,34 372,79 ± 527,21 1 LANCL1 743,76 ± 113,51 320,23 ± 256,55 1,32 S1PR3 720 ± 15,8 36,34 ± 24,18 18,81 TAAR1 709,53 ± 873,23 291,99 ± 393,48 1,43 CCR2 679,16 ± 594,11 83,9 ± 97,54 7,1 EDNRA 678,55 ± 384,23 156,17 ± 169,42 3,35 TAS2R4 632,56 ± 386,52 78,5 ± 95,15 7,06 TAS2R50 620,65 ± 656 254,29 ± 359,62 1,44 DRD4 614,74 ± 725,98 26,47 ± 9,55 22,23 GPR173 597,31 ± 337,05 22,35 ± 15,48 25,73 FPR2 585,44 ± 649,47 29,28 ± 41,41 18,99 PTGER2 564,8 ± 15,77 20,19 ± 14,01 26,97 SORT1 555,94 ± 510,36 202,98 ± 178,16 1,74 HCAR3 548,79 ± 387,63 139,57 ± 128,38 2,93 HTR2B 525,1 ± 250,96 2,72 ± 1,27 191,86 GPR125 517,17 ± 38,73 176,27 ± 185,54 1,93 GPRC5A 504,97 ± 100,44 30,38 ± 42,97 15,62 OPN3 502,64 ± 266,96 497,29 ± 540,34 0,01 GPR180 498,51 ± 99,04 139,09 ± 156,46 2,58 CYSLTR2 497,64 ± 408,53 84,87 ± 112,11 4,86 CXCR7 461,02 ± 70,52 142,67 ± 141,63 2,23 C3AR1 457,53 ± 380,95 56,79 ± 63,58 7,06 P2RY10 456,65 ± 374,09 73,95 ± 104,58 5,18 C5AR1 454,69 ± 13,83 62,87 ± 19,31 6,23 GNRHR 432,27 ± 347,56 125,42 ± 177,37 2,45 CHRM2 414,87 ± 488,42 71,04 ± 100,46 4,84 ADORA1 403,89 ± 418,78 18,65 ± 4,59 20,66 VN1R1 397,47 ± 37,81 52,85 ± 74,74 6,52 TACR1 387,12 ± 315,38 0 ± 0 - GPR182 383,91 ± 53,67 25,6 ± 32,14 14 FZD3 382,1 ± 342,42 198,69 ± 160,84 0,92 MRGPRX1 377,21 ± 533,45 43,62 ± 54,99 7,65 TAS2R1 368,95 ± 454,33 84,16 ± 119,01 3,38 GPRC5D 368,66 ± 6,33 38,84 ± 45,44 8,49 GPR17 363,04 ± 146,32 39,34 ± 24,72 8,23 CCR5 362,98 ± 416,77 63,96 ± 90,45 4,68 GPR22 357,86 ± 387,31 272,84 ± 379,27 0,31 GRM3 356,14 ± 426,03 135,92 ± 192,23 1,62 TAS2R3 354,53 ± 319,34 38,11 ± 53,89 8,3 MC4R 352,89 ± 455,32 28,4 ± 40,17 11,43 CHRM5 351,54 ± 289,38 64,06 ± 79,25 4,49 TAS2R38 345,33 ± 411,82 34,93 ± 49,4 8,89 TAS2R16 341,94 ± 408,13 102,01 ± 143,66 2,35 NPY2R 333,89 ± 401,95 34,59 ± 44,44 8,65 GPR174 331,94 ± 304,56 50,64 ± 62,28 5,55 GPR21 330,87 ± 201,16 22,17 ± 31,35 13,93 HCAR1 328,57 ± 38,97 32,52 ± 7,04 9,11 MRGPRF 324,32 ± 8,37 34 ± 31,08 8,54 GRM7 322,15 ± 414,84 100,95 ± 132,24 2,19 TAAR6 318,12 ± 439,81 143,85 ± 190,89 1,21 TRHR 310,23 ± 364,33 109,31 ± 154,59 1,84 LTB4R 307,81 ± 103,47 53,55 ± 26,61 4,75 PTGFR 304,11 ± 361,65 204 ± 288,5 0,49 CHRM1 304,01 ± 40,17 25,64 ± 23,98 10,86 GPR63 301,93 ± 163,86 77,84 ± 96,33 2,88 GPR77 301,08 ± 10,09 114,49 ± 137,33 1,63 CCR1 296,15 ± 259,11 44,6 ± 60,08 5,64 HRH1 294,74 ± 132,58 14,06 ± 19,89 19,96 TAAR8 294,33 ± 294,18 131,53 ± 186,01 1,24 CCBP2 288,98 ± 79,88 43,81 ± 29,52 5,6 MRGPRD 287,27 ± 110,03 16,43 ± 4,18 16,48 HTR1F 286,18 ± 313,64 74,35 ± 105,14 2,85 CNR1 274,6 ± 289,21 28,29 ± 32,82 8,71 LPAR4 271,22 ± 379,55 53,53 ± 75,7 4,07 SIGMAR1 264,55 ± 83,92 89,27 ± 92,51 1,96 CXCR6 263,58 ± 184,94 21,16 ± 18,62 11,45 HTR2A 262,4 ± 331,83 75,59 ± 79,94 2,47 CRHR1 262,06 ± 203,66 46,65 ± 14,84 4,62 GPR133 261,06 ± 150,82 13,61 ± 5,87 18,18 OXTR 259,08 ± 125,38 174,66 ± 247 0,48 TAAR5 253,51 ± 288,62 13,28 ± 18,78 18,09 LPHN1 253,39 ± 141,85 8,64 ± 1,89 28,32 FFAR3 248,12 ± 43,77 19,57 ± 11,02 11,68 CCR3 246,09 ± 231,83 19,26 ± 18,36 11,78 GPR148 243,33 ± 182,91 43,23 ± 47,31 4,63 SMO 242,8 ± 54,27 120,53 ± 109,58 1,01 GPR135 242,24 ± 63,81 139,66 ± 29,34 0,73 CCR6 240,99 ± 259,32 103,99 ± 125,83 1,32 CCR9 239,91 ± 202,09 27,06 ± 38,27 7,87 HRH4 239,04 ± 271,63 65,48 ± 86,85 2,65 XCR1 236,17 ± 296,39 17,81 ± 11,59 12,26 HCAR2 234,9 ± 240,19 19,82 ± 11,32 10,85 GPR101 234,28 ± 283,61 27,14 ± 31,22 7,63 TAS2R5 233,81 ± 62,51 66,22 ± 93,64 2,53 P2RY4 232,72 ± 155,12 11,93 ± 16,87 18,51 FFAR2 231,93 ± 1,68 24,31 ± 28,17 8,54 GPER 230,11 ± 124,41 81,1 ± 21,48 1,84 GP1BA 226,76 ± 36,22 60,93 ± 70,9 2,72 PROKR1 220,45 ± 201,84 21,55 ± 30,47 9,23 MC2R 216,25 ± 257,15 62,26 ± 88,05 2,47 GPR111 213,32 ± 52,92 0 ± 0 - OPN4 204,93 ± 166,61 11,79 ± 4,99 16,38 P2RY2 201,19 ± 1,34 18,27 ± 14,68 10,01 GPR55 199,48 ± 83,54 10,77 ± 15,24 17,51 TAAR2 197,67 ± 250,16 75,54 ± 96,42 1,62 LGR5 196,84 ± 266,28 33,68 ± 47,63 4,84 HRH2 194,7 ± 64,78 18,79 ± 14,31 9,36 PTAFR 194,34 ± 2,19 28,85 ± 13,65 5,74 MLNR 191,37 ± 200,51 30,62 ± 43,3 5,25 GPR15 190,84 ± 247,23 29,86 ± 31,5 5,39 HTR1E 190,33 ± 239,58 12,52 ± 17,71 14,2 MAS1 189,34 ± 213,7 42,13 ± 59,58 3,49 BAI3 187,98 ± 205,11 1,73 ± 0,69 107,35 FZD6 182,78 ± 99,26 98,54 ± 107,33 0,85 GPR152 178,37 ± 113,66 70,7 ± 80,38 1,52 CCKBR 178,04 ± 243,5 17,89 ± 25,29 8,95 GPR61 176,47 ± 38,93 62,61 ± 88,54 1,82 GPR84 176,27 ± 123,87 14,71 ± 20,81 10,98 GPR119 175,94 ± 190,27 7,25 ± 10,25 23,28 MTNR1B 175,73 ± 207,88 9,6 ± 13,57 17,31 GRM8 173,63 ± 226,38 39,47 ± 45,15 3,4 RXFP4 171,85 ± 74,77 14,47 ± 9,67 10,88 OGFR 169,46 ± 109,84 80,55 ± 27,87 1,1 GRM2 169,07 ± 59,58 13,78 ± 13,66 11,27 GHSR 166,68 ± 169,44 18,77 ± 3,36 7,88 LPAR5 162,57 ± 124,99 40,58 ± 36,86 3,01 FZD10 161,37 ± 51,15 71,26 ± 96,86 1,26 HCRTR1 159,08 ± 33,45 16,84 ± 8,37 8,45 AVPR2 156,8 ± 178,87 48,82 ± 42,82 2,21 MC3R 155,41 ± 175,64 9,33 ± 13,2 15,65 TPRA1 155,37 ± 59,24 48,26 ± 65,01 2,22 BDKRB1 154,82 ± 18,21 24,03 ± 17,34 5,44 DARC 153,22 ± 160,04 9,89 ± 13,99 14,49 MC5R 152,1 ± 201,46 34,39 ± 36,86 3,42 GPR45 148,4 ± 132,96 20,85 ± 22,94 6,12 CXCR1 148,02 ± 39,73 3,04 ± 4,3 47,71 EMR3 146,19 ± 22,84 3,52 ± 4,98 40,49 GPR151 144,81 ± 64,8 44,32 ± 51,35 2,27 RGR 143,86 ± 98,89 26,49 ± 37,46 4,43 GPR161 142,66 ± 30,59 0 ± 0 - BDKRB2 137,96 ± 71,68 78,04 ± 58,34 0,77 AVPR1A 134,18 ± 158,56 14,83 ± 4,43 8,05 CNR2 133,25 ± 80,12 16,99 ± 16,51 6,84 GPR6 132,89 ± 178,58 21,92 ± 22,53 5,06 HTR1B 131,64 ± 135,78 9,49 ± 13,42 12,87 GPR113 127,85 ± 81,53 27,5 ± 13,61 3,65 LPAR2 124,31 ± 81,06 21,88 ± 9,6 4,68 CALCR 123,07 ± 44,31 6,26 ± 8,85 18,66 GPR32 122,37 ± 57,27 10,47 ± 3,69 10,69 MCHR1 120,55 ± 48,19 17,68 ± 14,64 5,82 GPR171 120,54 ± 31,36 26,51 ± 19,32 3,55 MC1R 120,43 ± 27,51 41,37 ± 29,77 1,91 HTR1D 118,9 ± 80,43 7,36 ± 2,86 15,15 CELSR3 118,22 ± 17,4 27,33 ± 15,17 3,33 DRD1 117,96 ± 138,52 9,93 ± 14,04 10,88 HTR1A 113,9 ± 96,34 14,64 ± 12,65 6,78 GNRHR2 113,13 ± 81,34 21,82 ± 2,88 4,19 GPR3 112,36 ± 83,66 32,91 ± 38,6 2,41 PTGDR 112,09 ± 70,35 6,72 ± 0,36 15,67 GPR68 111,25 ± 50,67 28,17 ± 2,93 2,95 GPR39 108,77 ± 101,5 29,51 ± 14,39 2,69 PROKR2 103,62 ± 110,51 8,63 ± 12,2 11,01 OXER1 102,84 ± 12,81 51,84 ± 0,62 0,98 TACR3 101,86 ± 22,89 1,86 ± 2,63 53,87 GPR12 101,46 ± 119,46 10,22 ± 14,45 8,93 CCR10 100,19 ± 7,59 21,44 ± 3,68 3,67 ADRA2B 100,08 ± 141,54 6,9 ± 9,75 13,51 CXCR3 98,01 ± 24,72 15,41 ± 4,54 5,36 GALR2 95,6 ± 10,66 46,08 ± 56,43 1,07 EMR2 95,51 ± 29,83 0,87 ± 1,24 108,16 SCARF2 95,22 ± 68,04 3,69 ± 5,22 24,81 FZD2 92,77 ± 42,17 19,99 ± 18,6 3,64 GPR31 92,41 ± 45,2 10,31 ± 3,16 7,96 GPR78 91,78 ± 37,18 25,59 ± 8,29 2,59 GPR124 89,58 ± 17,92 2,83 ± 4,01 30,61 GPR153 89,18 ± 7 43,2 ± 12,48 1,06 CHRM4 88,15 ± 7,66 26,72 ± 25,43 2,3 GALR3 87,01 ± 11,41 13,34 ± 3,2 5,52 ADORA2A 84,88 ± 120,04 32,79 ± 15,47 1,59 PRLHR 83,9 ± 19,77 58,36 ± 82,54 0,44 S1PR4 82,74 ± 10,19 51,78 ± 66,95 0,6 MRGPRE 81,71 ± 8,33 13,1 ± 5,29 5,24 S1PR2 81,14 ± 46,36 16,18 ± 12,06 4,02 RXFP3 77,7 ± 66,69 13,15 ± 5,11 4,91 P2RY6 76,78 ± 48,3 18,23 ± 7,73 3,21 FPR3 74,98 ± 30,43 8,9 ± 12,59 7,42 GPR1 74,41 ± 29,23 0 ± 0 - SSTR4 72,57 ± 62,15 11,37 ± 0,93 5,38 SSTR3 71,97 ± 27,75 3,76 ± 5,32 18,15 GPR27 70,08 ± 64,84 13,75 ± 15,03 4,1 BAI2 65,84 ± 30,39 23,84 ± 16,95 1,76 FFAR1 65,61 ± 15,82 18,58 ± 14,7 2,53 GPR143 59,51 ± 27,72 19,19 ± 3,21 2,1 P2RY14 55,48 ± 24,68 9,73 ± 13,76 4,7 GRM6 54,95 ± 58,21 13,16 ± 8,16 3,18 TAS1R3 49,87 ± 0,26 15,74 ± 16,46 2,17 BAI1 47,64 ± 52,15 24,81 ± 26,43 0,92 GRPR 47,48 ± 38,44 0 ± 0 - TMEM11 47,07 ± 6,85 25,03 ± 0,74 0,88 NPBWR2 46,93 ± 22,73 13,04 ± 12,9 2,6 EMR1 43,74 ± 40,75 7,92 ± 4,36 4,52 HTR6 43,73 ± 55,25 23,54 ± 6,11 0,86 GPR162 42,66 ± 0,59 10,93 ± 1,72 2,9 CCR4 37,09 ± 29,8 2,55 ± 3,61 13,54 NPBWR1 36,43 ± 48 25,89 ± 24,71 0,41 FZD9 34,76 ± 13,04 12,62 ± 4,37 1,75 OPRL1 30,49 ± 27,68 2,65 ± 0,01 10,51 NMUR1 29,43 ± 4,97 0 ± 0 - ADORA3 29,17 ± 41,26 11,17 ± 12,82 1,61 GPR35 28,44 ± 28,79 16,63 ± 6,3 0,71 UTS2R 26,92 ± 13,87 8,45 ± 6,99 2,19 GPR98 26,1 ± 33,49 0,87 ± 1,22 29,17 PDGFRL 24,68 ± 8,92 2,36 ± 3,33 9,48 P2RY12 23,86 ± 9,39 2,33 ± 3,3 9,22 TACR2 18,61 ± 16,35 2,66 ± 1,89 5,98 CCR7 17,61 ± 23,07 3,71 ± 5,25 3,74 GPR114 16,54 ± 7,29 1,79 ± 2,53 8,24 ADORA2B 16,3 ± 23,05 0,53 ± 0,75 29,59 CCRL1 16,06 ± 10,79 0 ± 0 - OPCML 15,79 ± 17,97 6,22 ± 8,79 1,54 CELSR2 15,79 ± 5,38 2,88 ± 4,08 4,48 GPR85 15,46 ± 0,72 1,77 ± 2,5 7,75 GPR123 15,28 ± 13,6 5,19 ± 0,81 1,95 GPR19 12,73 ± 6,65 0 ± 0 - GPR75 11,85 ± 10,02 0,42 ± 0,6 27,1 PTGIR 11,32 ± 7,81 3,8 ± 2,29 1,98 RRH 10,06 ± 3,16 7,76 ± 10,97 0,3 GPR97 10,02 ± 14,17 2,1 ± 2,97 3,77 PTH2R 6,64 ± 0,07 3,42 ± 4,84 0,94 GRM5 6,59 ± 6,22 4,04 ± 0,48 0,63 LHCGR 5,73 ± 7,41 3,58 ± 2,93 0,6 GIPR 5,32 ± 1,11 0,65 ± 0,92 7,17 HTR7 4,7 ± 6,65 0 ± 0 - TSHR 4,65 ± 0,81 0 ± 0 - GPR83 3,9 ± 5,51 0 ± 0 - ADRA1B 3,69 ± 5,22 1,38 ± 1,38 1,67 CCR8 3,46 ± 3,34 0 ± 0 - KISS1R 2,84 ± 3,89 0,6 ± 0,86 3,7 OPN5 2,61 ± 3,69 0 ± 0 - CASR 2,41 ± 2,37 2,16 ± 3,05 0,12 LGR6 2,16 ± 3,06 0,92 ± 1,3 1,36 P2RY11 2,12 ± 0,66 0,61 ± 0,39 2,45 OPRK1 1,77 ± 2,01 0,88 ± 1,24 1,01 GPR37 1,2 ± 0,58 0,6 ± 0,85 1,01 NMUR2 1,2 ± 1,7 0 ± 0 - VIPR2 0,7 ± 0,99 0 ± 0 - GRM4 0,62 ± 0,88 0,52 ± 0,74 0,19 GPR44 0,62 ± 0,88 0 ± 0 - RXFP2 0,53 ± 0,75 0,29 ± 0,41 0,81 NTSR1 0,48 ± 0,69 0 ± 0 - GHRHR 0,48 ± 0,68 0 ± 0 - HCRTR2 0,42 ± 0,59 0 ± 0 - NPFFR2 0,3 ± 0,42 0 ± 0 - HTR4 0,26 ± 0,37 0 ± 0 - LPAR3 0,21 ± 0,29 0 ± 0 - PCR 0,18 ± 0,25 0 ± 0 - GALR1 0,15 ± 0,21 0 ± 0 - F2RL3 0,14 ± 0,2 0 ± 0 - GLP2R 0,03 ± 0,04 0 ± 0 -

Supplemental Table 2. Parameters at the time of biopsy for DN patients and for control patients included in immunohistochemical studies. NA = not available. As controls we used tissue from kidneys nephrectomized due to renal cancer. ______Histological Age Gender P-Crea U-Alb Ischemia time Other relevant clinical diagnoses diagnosis (µmol/mol) (mg/L) (min) and histological findings ______DN 50 Male 626 3436 <5 retinopathy DN 68 Male 367 16 <5 hypertension, congestive heart failure DN 55 Male 131 NA1 <5 hypertension, congestive heart failure DN 63 Male 189 3576 <5 hypertension, retinopathy DN 54 Female 161 NA2 <5 MGUS3 (no amyloidosis in biopsy) Control 48 Male 96 NA 40 none Control 58 Male 84 NA <5 none Control 59 Male 83 NA 45 MALT lymphoma Control 80 Male 77 NA 30 hypertension Control 52 Male 52 NA <5 hypertension ______1U-Albumin/creatinine 328 mg/mmol 2U-Albumin/creatinine 470 mg/mmol 3Monoclonal gammopathy of undetermined significance Supplemental Table 3. Parameters at the time of biopsy for DN patients and for controls included in immunoelectron microscopic studies. DN=diabetic nephropathy; NA = not available. As controls we used biopsies from living related donors. ______Histological Age Gender P-Crea U-Alb/Cre Ischemia time Other relevant clinical diagnoses diagnosis (µmol/mol) (µg/mg) (min) and histological findings ______DN 64 Female 118 NA* <5 hypertension DN 64 Male 183 479 <5 hypertension, ischemic heart disease DN 70 Male 142 163 <5 hypertension DN 68 Female 93 9 <5 none DN 68 Male 75 224 <5 hypertension Control 52 Female 72 0.3 <5 none Control 43 Female 58 0.7 <5 none Control 42 Female 61 <5.0 <5 none Control 43 Female 73 <5.0 <5 none Control 49 Female 79 <5.0 <5 none ______*Albuminuria 8.1g/24h Supplemental Table 4. Parameters for Gprc5a KO mice at 12 months of age. No significant difference was observed between KO animals and litter-mate controls. ______Mouse Weight Kidney Blood urea nitrogen U-Alb/Crea (n) (g) (g) (mg/dl) ______KO (6) 32.72±1.32 0.51±0.09 22.08±1.31 32.04±15.41

Control (5) 32.73±0.82 0.49±0.11 22.61±1.41 30.69±27.05 ______

Supplemental Table 5. Parameters for mice treated with STZ to induce diabetes. ______Mouse Weight Kidney weight B-Gluc 7 days HbA1c 24 weeks BUN 24w Mortality (n) (g) (g) after STZ (%) (mg/dl) (n) (mg/dl) ______KO (7) 27.79±2.75 0.595±0.208 >2001 7.91±0.552 22.19±1.48 0

Control (5) 26.75± 2.54 0.546±0.201 >2001 8.34±0.872 23.15± 1.43 0 ______1Animals that did not show diabetic levels of blood glucose were sacrificed and not included in the study. 2Non-diabetic values in mouse (n=5) using this method were <5.00 Supplemental table 6. Primers used in this study.

Gene Species Primer sequences (Forward; Reverse) ACTA2 Human 5’-AGACCCTGTTCCAGCCATC-3’; 5’-GCTAGGGCCGTGATCTCC-3’ ACTA2 Mouse 5’-TGCTGTCCCTCTATGCCTCT-3’; 5’-GAAGGAATAGCCACGCTCAG-3’ Col1a1 Human 5’-GTGCTAAAGGTGCCAATGGT-3’; 5’-GGGTCCTTGAACACCAACAG-3’ Col1a1 Mouse 5’-TGACTGGAAGAGCGGAGAGT-3’; 5’-GAATCCATCGGTCATGCTCT-3’ EGFR Mouse 5’-CTTGCATGTTTGCACTCGTT-3’; 5’-GAGGCAAGAGAAGCAGATGG-3’ Fibronectin Human 5’- AAACCTCGGCTTCCTCCAT-3’; 5’-CGGTGGCTGTCAGTCAAAG-3’ Gapdh Mouse 5’-TGTTCCTACCCCCAATGTGT R-TGTGAGGAGATGCTCAGTG-3’ Gprc5a Human 5’-GCTGCTCACAAAGCAACGAA R-ATAGAGCGTGTCCCCTGTCT-3’ Gprc5a Mouse 5’-CTTCTTCGCATCCTTCTTGG R-AGTGTGTCCCCCATCTCAAG-3’ Gprc5a (genotyping) Mouse 5’-TTGCGGGGTATAGGTGTGT R-GCATATGTGGAACCCGTGTC-3’ Nephrin Human 5’-GAGAGCCCCATTCAAAGGCT R-AGAAGGAGCTCACGGTTTCG-3’ TGF-β Mouse 5’-GGCTACCATGCCAACTTCTG-3’; 5’-GTTGGACAACTGCTCCACCT-3’ 28S Human 5’-TTGAAATCCGGGGGAGAG R-AGATTGTTCCAACATGCCAG-3’ ______

SUPPLEMENTAL MATERIAL AND METHODS

Human kidney tissues and study approval

Control human tissue for stainings and Western blotting was collected from normal parts of kidneys removed due to renal carcinoma at Karolinska

University Hospital (Stockholm, Sweden). For immunoelectron microscopy studies, biopsy material from living kidney donors was used. Renal biopsies from diabetic patients showing DN were collected from archives of Karolinska

University Hospital (Stockholm, Sweden). The biopsies were evaluated using light microscopy, electron microscopy and immunofluorescence following standard procedures. Clinical data of these patients can be found in supplemental Tables 2 and 3. The collection of human tissue was accepted by the local ethical committee.

GPCR expression analysis using qPCR

Glomeruli isolated through sieving were used in the analysis. We isolated

RNA using standard methods and analysed the expression of human GPCRs using a pre-designed 384-well plate (GPCR Tier 1-4 H384, Bio-Rad).

Experiment was repeated twice and Gapdh was used for normalization.

RT-PCR and real-time PCR

Total RNA from mouse/human glomeruli, kidney tissue devoid of glomeruli and cultured podocytes was isolated using Trizol reagent (Invitrogen) and

RNA was reverse-transcribed using iScriptTM Reverse Transcription kit (Bio- rad). PCR experiments were performed using standard procedures. Relative mRNA levels of genes were calculated using the ΔΔCt method. Gapdh

(mouse) and 28S (human) were used for normalization. Primer sequences used for amplication are listed in supplemental table 4. Experiments were repeated at least three times, and data are expressed as mean ± S.E.

Statistical analysis was performed using Student's t test or one-way analysis of variance with Tukey's post hoc test. Glomeruli were isolated from human and mouse kidney tissue using standard procedures.

Western blotting

Proteins from kidney fractions and cultured cells were extracted by RIPA buffer and Western blotting was performed as described previously (23).

Antibodies used were directed against Gprc5a (Atlas Antibodies,

HPA007928), nephrin (22), p-EGFR (Tyr1068) (Cell Signaling, #2234), total

EGFR (Cell Signaling, #4627), p-Smad2/3, (Cell signaling, #8828), total

Smad2/3 (Cell signaling, #8685), TGF beta1 (Abcam, ab155264), actin

(Abnova, #DH1003) and calnexin (Abcam, #ab10286).

Immunostaining

Immunofluorescence and immunoperoxidase stainings were performed as previously described (4). Primary antibodies used were the same as used for

Western blotting. Additionally, anti-CD31 (BDbioscience, #560984), anti-

ASMA (Sigma, #H5228), PDGFRB (RandD system, #MAB126) and Gprc5a

(12) antibodies were used. Staining intensity in renal biopsies for Gprc5a and for ASMA in mouse kidneys was scored in each glomeruli as: 3 = strong; 2 = moderate; 1 = weak; 0 = absent. For Gprc5a, 87 glomeruli in 5 DN and 100 glomeruli in 5 control cases were analyzed. For ASMA, 30 glomeruli/mouse were analyzed in 5 -/- and 5 control kidneys.

Immunoelectron microscopy

Immunolabeling was performed as described previously (Wernerson et al,

2003). To semi-quantify labeling in immunoelectron microscopy, 30 images covering podocyte cytoplasm were chosen randomly in each biopsy. The area of the podocyte cytoplasm was calculated by point counting using a 1,5 x1,5 cm square lattice, and expressed as µm2. The number of gold particles was counted in the images and the mean concentration was calculated in each biopsy.

Electron microscopy

For transmission electron microscopy kidney samples were fixed with 2% paraformaldehyde and 2,5% glutaraldehyde. To quantify the thickness of the

GBM and number of slits, we selected 5 random capillaries from each sample.

In each capillary the distance between the podocyte and endothelia in 5 randomly chosen areas were measured and the mean value from the 5 capillaries was calculated and expressed as nm. In each capillary, the length of the GBM was measured and the number of podocyte slits was counted.

Thus from 5 capillaries, the mean value was calculated and expressed as slits/µm.

Mouse models Gprc5a mutant mouse strain in pure FVB background was generated using

TALEN technology (Cyagen). We targeted exon 2 of Gprc5a mouse gene and the mutation was verified using Sanger sequencing. Genotyping was performed by PCR-amplifying a 340 bp fragment around the deletion followed by digestion with Kpn1 enzyme. The amplified product is digested to two

170bp fragments in wild-type alleles, whereas mutant allele lacks the digestion site and thus only 340bp is detected. Inactivation of Gprc5a gene was verified using Western blotting and RT-PCR.

Diabetes was induced in 8-week old littermate control and Gprc5a mutant mice by giving intraperitoneal injections of streptozotocin (50 mg/kg, Sigma-

Aldrich) for 5 consecutive days. Induction of hyperglycemia was confirmed by measuring non-fasting blood glucose 2 weeks after the last injection and by measuring HbA1c at the end of the experiment. Urine was collected every second week starting from 5 weeks after the last injection. Mice were euthanized 24 weeks after injections. Key parameters of these mice are summarized in Table 5. The animal work was accepted by the local ethical committee.

Histological analyses

Kidneys for histological analysis were prepared according to standard procedures. Mesangial matrix expansion was evaluated in 12 month-old and

STZ-treated animals using PAS staining. The measurements were done in 5 -

/- and 5 control mice using two methods: 1) By calculating mesangial index

(using standard procedures with ImageJ software) in 5 random glomeruli/mouse; 2) By semi-quantitative scoring of 30 glomeruli/mouse as follows: 0 = no expansion, 1 = mild expansion, 2 = moderate expansion; 3 = severe expansion.

The presence of sclerotic changes in STZ-treated mice was quantified as follows: 30 glomeruli from each mouse was analyzed, score was 0 if no sclerotic lesions was present; 1 if segmental or global sclerosis was detected.

A total of 5 -/- and 5 control mice were analyzed.

Cell culture and generation of stable cell line

Immortalized human podocytes were grown as described previously (5).

Stable Gprc5a overexpressing cells were established by Lipofectamine 2000 transfection of pcDNA3.1-hGprc5a construct using standard procedures.

Silencing of Gprc5a expression was done using siRNA (Invitrogen) according to standard procedures. To study effects of Gprc5a on EGF mediated signaling, we added 25ng/ml of HB-EGF on culture media and collected cell lysates at 0 and 5 minutes. Experiments were repeated at least three times.

Luciferase reporter Assay

Effect of Gprc5a on Smad2/3 mediated target gene transcription was assessed further by using the dual-luciferase reporter system (Promega). The podocityes were transfected with pGL3-CAGA-luc plasmid and with renilla luciferase construct pRL-TK-Rluc. The pGL3-CAGA-luc is a reporter of the

Smad2/3 activation as the CAGA-box in the vector is transcribed by activated

Smad2/3 resulting in luciferase activity. Luciferase assays were performed according to standard procedures in normal culture media and with addition of 25ng/ml HB-EGF. Data is presented as relative luciferase units. Experiments were repeated at least three times.

FIGURE LEGENDS FOR SUPPLEMENTAL FIGURES

Supplemental Figure 1. Immunoperoxidase staining for F2R, S1PR1,

GPR160, ELTD1 and CD97 in human kidney tissue shows clear signal in glomeruli. Data extracted from Human Protein Atlas database

(proteinatlas.org). Magnifications: x400.

Supplemental Figure 2. (A) Expression of Gprc5a mRNA in different human tissues as detected by RNA sequencing (data extracted from www.humanproteinatlas.org). Gprc5a mRNA is highly expressed in lung tissues and only sparsely in other human organs. (B) Expression of Gprc5a mRNA in different mouse tissues as detected by RT-PCR analysis. Gprc5a can be detected only in lung tissue and in isolated glomerular capillary tufts.

Gapdh was used as a loading control.

Supplemental Figure 3. (A) Double labeling experiment with mesangial marker PDGFRB (red) does not show co-localization with Gprc5a (green).

Double staining with endothelial marker CD31 (red) does not show overlapping reactivity with Gprc5a (green). CL=capillary loop. (B)

Immunoelectron microscopy for Gprc5a does not show significant labeling in mesangial cells (mc). (C) Quantification of immune-gold experiment shows significant enrichment of Gprc5a signal in podocytes (pod) in comparison to endothelial (end) and mesangial cells (mes). (D) HEK293 cells transfected with Gprc5a construct show a strong band of about 40kDa whereas cells transfected with a control vector show no reactivity. Actin was used as a loading control. (E) Immunostaining with a previously characterized anti-

Gprc5a antibody (12) shows strong podocyte staining around glomerular capillary loops in both immunofluorescence and immunoperoxidase stainings.

Magnifications: x1000 in A; x200 in E. Data shown as the mean ± s.e in C;

*P<0.05 was considered statistically significant.

Supplemental Figure 4. (Analysis of 12-month old mice. (A-B) Knockout kidneys show normal normal histology in tubulointerstitial space despite obvious glomerular abnormalities. (C-D) In electron microscopy, podocyte slit diaphragms (black arrows) are intact and glomerular endothelial cells show normal morphology (red arrows). (E) Significant mesangial expansion is observed in 12-month old animals by semi-quantitative scoring. (F) No significant foot process effacement is detected as measured by counting podocyte filtration slits / GBM length. Data shown as the mean ± s.e in E.

*P<0.05 was considered statistically significant.

Supplemental Figure 5. Gprc5a in cultured human podocytes. (A)

Immunofluorescence staining for Gprc5a shows immunoreactivity at the plasma membrane (arrows) in the podocyte cell line transfected with Gprc5a.

(B) Normalized densities for pEGFR and pSmad2, as well as density for TGF-

β1 in control and Gprc5a overexpressing cells. The activation of EGFR and

Smad2, as well as expression of TGF-β1, is significantly reduced in overexpressing cells. (C) Normalized densities for pEGFR and pSmad2, as well as density for TGF-β1 in control and Gprc5a silenced cells. The activation of EGFR and Smad2, as well as expression of TGF-β1, is significantly promoted in cells with silenced Gprc5a expression. Data shown as the mean

± s.e in B and C; *P<0.05 was considered statistically significant.