Hepatic nuclear factor 4 alpha and the Ca-channel TRPC1 are novel disease
candidate genes in diabetic nephropathy
Monika Niehof and Jürgen Borlak
Online Appendix
Online Appendix Table 1. Shift-probes sequences.
Online Appendix Table 2. ChIP-PCR primer sequences.
Online Appendix Table 3. Real-Time PCR primer sequences and amplification settings.
Online Appendix Table 4. Representative data of obese Zucker diabetic fatty (ZDF) rats and
of +/fa lean nondiabetic control rats.
Online Appendix Table 5. Human kidney biopsies for immunohistochemistry, detailed patient
information.
Online Appendix Table 6. Target gene enrichment in ChIP-DNA.
Online Appendix Table 7. PhastCons17way Wiggle Summary Statistics.
Online Appendix Table 8. PLCB1 gene expression in STZ-induced diabetic rats.
Online Appendix Table 9. Gene expression of potential TRCP1 interaction partners in
kidneys of Zucker diabetic fatty (ZDF) rats.
Online Appendix Fig. 1
Online Appendix Fig. 2
Online Appendix Fig. 3
Online Appendix Fig. 4
Online Appendix Fig. 5
1
Stable gene expression of mitATPase6 in liver and kidney of
Zucker diabetic fatty (ZDF) and STZ rats.
Aroclor 1254 increased HNF4 protein expression and in vitro
binding to TRPC1 and PLCB1
siRNA transfection efficiency in Caco-2 cells
Gene expression of HNF4 and TRPC1 (A) or PLCB1 (B) in kidney
of Zucker diabetic fatty (ZDF) rat.
Stable gene expression of mitATPase6 HNF4 siRNA transfected
Caco-2 cells
ONLINE APPENDIX TABLE 1. Shift-probes sequences.
Gene
Accession
Number
Species
Oligo Name
Position
Sequence
huHNF1
NM_000545
human
HNF1pro
-273 to -247
AGGGCTGAAGTCCAAAGTTCAGTCCCTTC
TRPC1
NM_003304
human
probe131
-8189 to -8167
CTAGGAGTTCAAAGGCCAGCCTG
PLCB1
NM_015192
human
probe05
-936 to -914
AACGTAACCGTTTGCCCGGGTTT
TRPC1
NM_053558
rat
probe156
+4329 to +4351
ATCAGTGAACTTTGAGCTCAGGG
PLCB1
NM_001077641
rat
probe152
+310 to +322
CAGGCAGTTCAATGTCCAATTTT
2
ONLINE APPENDIX TABLE 2. ChIP-PCR primer sequences.
PCR
Gene
Accession number Species Position
HNF1 NM_000545
human
TRPC1 NM_003304
human
PLCB1 NM_05192
human
Primer sequence
Fragment length Annealing
-195 to -214
fwd: CATGATGCCCCTACAAGGTT
274bp
60°C
-449 to -468
rev: ATTGGAGCTGGGGAAATTCT
182bp
60°C
110bp
60°C
+1679 to +1698 fwd: TGGGTTGCCTGAGTTCTCTT
+1839 to +1860 rev: TCTGTCCTCAGTTTGACAGGAA
-933 to -952
fwd: TCTGTCTCGAAAGCACAACG
-843 to -862
rev: AGAAGAGCGCAGTTGAGAGG
LC-PCR
Gene
Accession number Species Position
Primer sequence
Fragment length Annealing Extension Fluorescence
HNF1
NM_000545
-322 to -303
fwd: AGGCCATAGCTCCCTGTCC
149bp
66°C
9sec
86°C
-193 to -173
rev: GCCCCTGCCTGTTCTGTTTA
-521 to -501
fwd: GGCACAGAGCAGACAATCAA
169bp
65°C
9sec
83°C
-372 to -352
rev: TTGGCAAAACACCACAGCTA
119bp
67°C
8sec
86°C
110bp
68°C
8sec
87°C
188bp
65°C
8sec
89°C
174bp
66°C
8sec
86°C
PCK1
NM_002591
human
human
(PEPCK)
TRPC1
NM_003304
human
-8263 to -8242 fwd: TTTGGCAGGGTATGATGACTC
-8166 to -8144 rev: CAAGGGGTCACACTATGTTGTC
PLCB1
ACTB
NM_05192
NM_001101
human
human
(ß-actin)
GAPDH
NM_002046.3
human
-933 to -952
fwd: TCTGTCTCGAAAGCACAACG
-843 to -862
rev: AGAAGAGCGCAGTTGAGAGG
+539 to +559
fwd: CTCCTGAGCGCAAGTACTCC
+707 to +727
rev GCGCAAGTTAGGTTTTGTCA
-6236 to -6216 fwd: ATGGTTGCCACTGGGGATCT
-6082 to -6062 rev: TGCCAAAGCCTAGGGGAAGA
3
ONLINE APPENDIX TABLE 3. Real-Time PCR primer sequences and amplification settings.
Gene
Accession number Species Position
Primer sequence
Fragment length Annealing Extension Fluorescence
HNF4
NM_022180
fwd: GCCTGCCTCAAAGCCATCAT
275bp
55°C
11sec
88°C
301bp
68°C
12sec
85°C
324bp
68°C
13sec
83°C
213bp
60°C
9sec
87°C
291bp
68°C
12sec
87°C
236bp
60°C
10sec
84°C
152bp
60°C
8sec
87°C
193bp
60°C
7sec
87°C
213bp
60°C
9sec
85°C
rat
+875 to +892
+1130 to +1149 rev: GACCCTCCAAGCAGCATCTC
TRPC1
NM_053558
rat
+1613 to +1632 fwd: CCAAAGAGCAGAAGGACTGC
+1894 to +1913 rev: GCTCGAGCAAACTTCCATTC
PLCB1
XM_342524
rat
+2011 to +2033 fwd: GATGGGATAGTGGCCAACAC
+2315 to +2334 rev: ATTCCGCAGGCAGATGTAGT
TRPC3
TRPC4
TRPC5
NM_021771
NM_001083115
NM_080898
rat
rat
rat
+387 to +406
fwd: CAATGCCTACAAGGGACTGG
+580 to +599
rev: ATGGCTTCCACCTCTTCTGA
+158 to +178
fwd: TGGAGGAAGCCGAGATTTATT
+428 to +448
rev: CCAAGATGATAGGCGTGATGT
+1639 to +1658 fwd: CGCTCTTTGAAACCCTTCAG
+1855 to +1874 rev: CTTCGTTCTCGCAAACTTCC
TRPC7
PKD2
CAV1
EF673687
NW_047421
NM_133651
MitATPase6 AF115770
HNF4
NM_000457
rat
rat
rat
rat
human
+2 to +21
fwd: TCAATTTCAACTGCGTGGAC
+136 to +153
rev: CACGTAGCCCTTGCTGATG
+764 to +783
fwd: CGAGGACTGCGAGGTAAGAT
+937 to +956
rev: GTGAAATTCGGAGCTGGGTA
+4 to +23
fwd: GCAGACGAGGTGAATGAGAAG
+197 to +216
rev: CAGCAAGCGGTAAAACCAAT
+188 to +204
fwd: CTAAAGGACGAACCTGA
315bp
55°C
13sec
83°C
+487 to +503
rev: TGGCCTGCAGTAATGTT
+774 to +780
fwd: CTGCTCGGAGCCACCAAGAGATCCATG 371bp
60°C
15sec
88°C
325bp
60°C
13sec
82°C
315bp
55°C
13sec
83°C
+1120 to +1144 rev: ATCATCTGCCAGGTGATGCTCTGCA
TRPC1
NM_003304
MitATPase6 NC_001807
4
human
human
+507 to +526
fwd: TGGATGTTGCACCTGTCATT
+812 to +831
rev: TTACATTGCCGGGCTAGTTC
+188 to +204
fwd: CTAAAGGACGAACCTGA
+487 to +503
rev: TGGCCTGCAGTAATGTT
ONLINE APPENDIX TABLE 4.
Representative data of obese Zucker diabetic fatty
(ZDF) rats and of +/fa lean nondiabetic control rats.
Age (weeks)
Blood glucose (mmol/l)
Mean
SD
SEM
N
HbA1c (%)
Mean
SD
SEM
N
Albumin/Creatinine (mg/mg)
Mean
SD
SEM
N
Age (weeks)
Blood glucose (mmol/l)
Mean
SD
SEM
N
HbA1c (%)
Mean
SD
SEM
N
Insulin (ng/ml)
Mean
SD
SEM
N
Albumin (mg/kg/h)
Mean
SD
SEM
N
Clearance Creatinine (ml/kg/min)
Mean
SD
SEM
N
Urine flow (ml/kg/h)
Mean
SD
SEM
N
Obese
12,5
Lean
12,5
20
24
28
27,0
1,4
0,5
9
6,4
0,4
0,1
9
7,1
0,3
0,1
9
6,9
0,4
0,1
9
6,9
0,4
0,1
9
9,7
0,7
0,2
9
11,0
0,7
0,2
9
5,1
0,0
0,0
9
4,6
0,1
0,0
9
4,2
0,1
0,0
9
4,4
0,1
0,0
9
674,97
424,36
141,45
9
714,58
257,63
85,88
9
1207,57
488,62
162,87
9
11,40
4,41
1,47
9
6,05
1,50
0,50
9
7,47
1,73
0,58
9
5,99
2,61
0,87
9
17
27
37
10
17
27
37
11,9
6,7
1,7
15
29,7
5,1
1,3
15
32,5
3,5
1,0
11
44,9
4,2
1,3
11
7,3
0,7
0,1
20
8,0
1,5
0,3
20
7,2
0,9
0,2
16
15,5
3,7
1,0
13
6,1
0,9
0,2
15
10,0
1,6
0,4
15
8,7
1,2
0,4
11
10,1
0,9
0,3
9
4,5
0,1
0,0
20
4,6
0,1
0,0
20
4,1
0,1
0,0
16
4,7
0,1
0,0
12
3,2
1,2
0,3
15
2,2
1,1
0,3
15
1,2
0,6
0,2
11
0,9
0,3
0,1
11
1,2
0,3
0,1
20
1,0
0,2
0,0
20
0,9
0,2
0,1
16
0,8
0,4
0,1
13
0,35
0,24
0,06
15
1,78
1,09
0,28
15
9,24
6,70
2,02
11
14,23
7,69
2,32
11
0,121
0,041
0,009
20
0,090
0,069
0,015
20
0,070
0,033
0,008
16
0,079
0,051
0,014
13
5,4
1,3
0,3
15
4,0
1,7
0,4
15
7,6
0,6
0,2
11
4,5
0,9
0,3
11
6,0
1,0
0,2
20
4,9
1,7
0,4
20
6,7
0,4
0,1
16
4,5
0,8
0,2
13
3,5
1,8
0,5
15
3,3
2,1
0,5
15
14,5
3,6
1,1
11
15,1
4,6
1,4
11
1,0
0,3
0,4
20
1,5
0,6
0,3
20
1,0
0,2
0,4
16
0,8
0,1
0,0
13
20
24
28
20,3
3,9
1,3
9
25,0
3,1
1,0
9
29,0
2,7
0,9
9
8,6
0,5
0,2
9
10,4
1,1
0,4
9
277,35
145,03
48,34
9
Obese
10
Lean
Liver and kidney of fa/fa obese Zucker diabetic fatty (ZDF) rats and of +/fa lean nondiabetic control
rats, aged 14 weeks were kindly provided by W. Linz and H. Ruetten, Sanofi-Aventis, Frankfurt,
Germany (14). Phenotype data of representative studies are provided.
5
ONLINE APPENDIX TABLE 5.
Human kidney biopsies for immunohistochemistry,
detailed patient information.
Patient identification Sex Age Diagnosis
numbers
12823/03
m
42
0-Biopsy, healthy
18305/03
m
40
0-Biopsy, healthy
5635/03
m
34
0-Biopsy, healthy
Patient identification Sex Age Diagnosis
Duration of
Measures of
numbers
diabetes
nephropathy
5 years
Creatin 3 mg%
13914/02
m
73
Diabetes
mellitus
1758/04
m
67
Diabetes
Protein 1 g/d
29 years
mellitus
8414/98
f
67
Diabetes
mellitus
6
Creatin 2.3 mg%
Protein 11 g/d
not available
Creatin 2.5 mg%
ONLINE APPENDIX TABLE 6. Target gene enrichment in ChIP-DNA. Target gene
enrichment versus ß-actin in ChIP-DNA versus total input DNA was analyzed by real-time
PCR. Calculation was performed using the Δ ΔCT-method in three independent experiments.
Gene
Fold change
SD
HNF1
107.6
47.2
PEPCK
6.9
3.9
PLCB1
4.2
2.9
TRPC1
2.4
1.8
7
ONLINE APPENDIX TABLE 7.
PhastCons17way Wiggle Summary Statistics. The
PhastCons program (integrated in the UCSC genome browser) was used to analyze the level
of sequence conservation amongst mammals and PhastCons17way (17 species) Wiggle
Summary Statistics are shown.
Position: chr3:143,917,766-143,917,788 = huTRPC1 -8189 to -8167 (EMSA probe, Promoter)
Total Bases in view: 23
Statistics on: 23 bases (% 100.0000 coverage)
Database: hg18
Data
start
Chrom
chr3
Each data
# of Data
value spans
values
# bases
Data
end
143917766 143917788
23
Bases
covered
Table: phastCons17way
Minimum Maximum Range
1 23 (100.00%)
0
0
Mean
Standard
Variance
PhastCons score
deviation
0
0
Position: chr3:143,927,635-143,927,816 = huTRPC1 +1679 to +1869 (ChIP fragment, Intron 1)
Total Bases in view: 182
Statistics on: 182 bases (% 100.0000 coverage)
Database: hg18
Data
start
Chrom
chr3
Each data
# of Data
value spans
values
# bases
Data
end
143927635 143927816
182
Bases
covered
0 0.732819 0.732819
Chrom
chr3
Data
start
Data
end
143927588 143927921
334
Table: phastCons17way
Bases
covered
0 0.732819 0.732819
Position: chr20:8,060,360-8,060,382 = huPLCB1 -936 to -914 (EMSA probe, Promoter)
Total Bases in view: 23
Statistics on: 23 bases (% 100.0000 coverage)
Database: hg18
Chrom
Data
start
Data
end
chr20 8060360 8060382
23
Bases
covered
1 23 (100.00%)
0.135887 0.0464812 0.215595
Table: phastCons17way
Mean
Standard
Minimum Maximum Range PhastCons Variance
deviation
score
0
0
0
0
Position: chr20:8,060,344-8,060,453 = huPLCB1 -952 to -843 (ChIP fragment, Promoter)
Total Bases in view: 110
Statistics on: 110 bases (% 100.0000 coverage)
Database: hg18
Chrom
Data
start
Data
end
chr20 8060344 8060453
Each data
# of Data
value spans
values
# bases
110
Bases
covered
1 110 (100.00%)
Chrom
Data
end
chr20 8167302 8167477
8
Each data
# of Data
value spans
values
# bases
176
Bases
covered
Minimum Maximum Range
Mean
Variance
PhastCons score
0 0.228346 0.228346
1 176 (100.00%)
Minimum Maximum
0
0
Table: phastCons17way
Range
0 0.0374646 0.0374646
Standard
deviation
0.0338583 0.00385828 0.0621151
Position: chr20:8,167,302-8,167,477 = huPLCB1 +106006 to +106181 (ChIP clone, Intron 2)
Total Bases in view: 176
Statistics on: 176 bases (% 100.0000 coverage)
Database: hg18
Data
start
Standard
deviation
Mean
Standard
Minimum Maximum Range PhastCons Variance
deviation
score
1 334 (100.00%)
Each data
# of Data
value spans
values
# bases
Variance
0.138762 0.0505961 0.224936
Position: chr3:143,927,588-143,927,921 = huTRPC1 +1632 to +1965 (ChIP clone, Intron 1)
Total Bases in view: 334
Statistics on: 334 bases (% 100.0000 coverage)
Database: hg18
Each data
# of Data
value spans
values
# bases
0
Table: phastCons17way
Mean
PhastCons
score
Minimum Maximum Range
1 182 (100.00%)
0
Table: phastCons17way
Mean
PhastCons
score
0.00527255
Variance
Standard
deviation
7.88948e0.00888228
05
ONLINE APPENDIX TABLE 8.
PLCB1 gene expression in STZ-induced diabetic
rats. Gene expression was determined by with real-time RT-PCR and computed relative to
expression of mitATPase6, a housekeeping gene.
Gene
Organ
Treatment
Number of
Mean SD
animals
PLCB1
PLCB1
*A
kidney
liver
[%] of the
p-value*
control
control
n=8
0.11
0.06
STZ
n=7
0.14
0.04
control
n=8
3.12
3.69
STZ
n=9
0.07
0.09
125.7
0.1391
2.4
student's t test was used to compare the STZ group against the control group. The results were
considered significant when the p value was less than 0.05. Values for the liver control group are not
normal distributed, Shapiro-Wilks W test with p-value less than 0.05 (Statistica software, version 7.1,
StatSoft), therefore no student's t-test was performed.
9
ONLINE APPENDIX TABLE 9.
Gene expression of potential TRCP1 interaction
partners in kidneys of Zucker diabetic fatty (ZDF) rats. Gene expression was determined by
real-time RT-PCR in 14 weeks old ZDF rats (n=10 animals) and lean controls (n=10
animals). Gene expression was computed relative to expression of mitATPase6, a
housekeeping gene.
Gene
Treatment
Mean
SD
TRPC3
control
0.9942
0.2905
ZDF
0.6395
0.2576
control
0.6956
0.3895
ZDF
0.9364
0.4963
control
1.4971
0.3484
ZDF
1.3604
0.6658
control
0.8636
0.6102
ZDF
0.7646
0.6372
control
0.0887
0.0862
ZDF
0.0807
0.0615
control
0.7960
0.2985
ZDF
0.5724
0.1804
TRPC4
TRPC5
TRCP7
PKD2
CAV1
*
p-value*
0.0157
0.2649
0.5974
0.7418
0.8297
0.0816
A student's t test was used to compare the ZDF group against the control group. The results
were considered significant when the p value was less than 0.05.
10
ONLINE APPENDIX FIGURE LEGENDS
ONLINE APPENDIX FIGURE 1. Stable gene expression of mitATPase6 in liver and
kidney of Zucker diabetic fatty (ZDF) and STZ rats.
Gene expression was determined by real-time RT-PCR in kidney and liver of 14
weeks old ZDF rats (n=10) and lean controls (n=10) and in kidney (n=7) and in liver
(n=9) of 2 months old STZ-induced diabetic rats (control n=8, respectively).
ONLINE APPENDIX FIGURE 2. Aroclor 1254 increased HNF4 protein
expression and in vitro binding to TRPC1 and PLCB1. A: HNF4 western blot
analysis (left) of 20 µg nuclear extract of control and Aroclor 1254 treated Caco-2
cells. Electrophoretic mobility shift assay with 2,5 µg nuclear extract of control or
Aroclor 1254 treated Caco-2 cells and oligonucleotides corresponding to the A-site of
the HNF1 promoter (HNF1pro) and to putative HNF4 binding-sites within the
identified promoters (TRPC1, probe 131; PLCB1, probe 05) as 32P labeled probes. In
supershift assays an antibody directed against HNF4 (+) was added. B: HNF4
western blot analysis (left) of 30 µg liver nuclear extract of control and Aroclor 1254
treated rats. Electrophoretic mobility shift assay with 2,5 µg rat liver nuclear extract of
control or Aroclor 1254 treated animals and oligonucleotides corresponding to the Asite of the HNF1 promoter (HNF1pro) and to putative HNF4 binding-sites within
the identified promoters (TRPC1, probe 131; PLCB1, probe 05) as 32P labeled
probes. In supershift assays an antibody directed against HNF4 (+) was added.
ONLINE APPENDIX FIGURE 3. siRNA transfection efficiency in Caco-2 cells.
Caco-2 cells (1,5x105 cells/well in 24-well plate) were transfected in triplicate for 48 h
with 25 nM of Alexa-Fluor488 labeld siRNA using HiPerFect transfection reagent.
ONLINE APPENDIX FIGURE 4. Gene expression of HNF4 and TRPC1 (A) or
PLCB1 (B) in kidney of Zucker diabetic fatty (ZDF) rat. Linear regression of
correlation with 0.95 confidence interval are shown. Gene expression was
determined by real-time PCR in 14 weeks old ZDF rats (n=10) and lean controls
(n=10).
ONLINE APPENDIX FIGURE 5. Stable gene expression of mitATPase6 HNF4
siRNA transfected Caco-2 cells. Caco-2 cells (1,5x105 cells/well in 24-well plate)
were transfected in triplicate for 48 h with 25 nM of HNF4 or Alexa-Fluor488 labeld
siRNA (AF488, negative control) using HiPerFect transfection reagent. Gene
expression in untransfected cells (control) is shown for comparison.
11
ONLINE APPENDIX FIGURE 1
relative units
1500
1000
500
Kidney
12
Liver
Kidney
ZDF
control
ZDF
control
STZ
control
STZ
control
0
Liver
-
+
-
+
-
+
-
+
72h Aroc
control
72h Aroc
control
72h Aroc
control
72h Aroc
control
24h Aroc
A
48h Aroc
ONLINE APPENDIX FIGURE 2
- +
-
+ HNF4
HNF4
68
55
HNF4
HNF4
42
Caco-2
-
+
-
+
-
+
-
+
control
72h Aroc
PLCB1
probe 05
72h Aroc
control
control
Aroclor
control
B
TRPC1
probe 131
72h Aroc
HNF1pro
-
+
-
HNF4
68
55
HNF4
HNF4
42
rat liver
HNF1pro
13
+ HNF4
TRPC1
probe 131
PLCB1
probe 05
ONLINE APPENDIX FIGURE 3
14
ONLINE APPENDIX FIGURE 4
A
B
15
ONLINE APPENDIX FIGURE 5
16