To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach.
Description
The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen.
Conclusion
The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.
Background
Renal glomeruli are highly specialized capillary tufts that produce a nearly protein-free ultrafiltrate of plasma at a rate of about 30 plasma volumes daily. Several hereditary, immune-mediated and metabolic disorders cause glomerular injury, proteinuria, and can lead to renal failure. The three intrinsic glomerular cell types, podocytes, mesangial cells, and glomerular endothelial cells (EC) are highly specialized. Podocytes extend an elaborate array of actin-rich foot processes around the exterior of the glomerular capillary loops, forming a scaffold with nephrin-based filtration slit diaphragms spanning the space between adjacent foot processes [1]. Mesangial cells are pericyte-like cells that, unlike most other pericytes, form an interstitium within the intracapillary space [2]. Glomerular EC are packed with transcellular fenestrae ringed by actin [3, 4]. The fenestrae serve the high glomerular capillary wall hydraulic conductivity [5], while a glycocalyx covering the glomerular EC and podocytes together with the podocyte filtration slit diaphragm impede the movement of plasma proteins [6–8] across the glomerular capillary wall.
Transcriptome and proteomic approaches are helping to define genes highly expressed and/or enriched in glomeruli [9–12]. For instance, Sau3A-anchored SAGE databases have been built with RNA extracted from microdissected nephron segments, and enrichment of several glomerular transcripts relative to other nephron segments has been reported [9]. Furthermore, many proteins uniquely expressed by, or enriched in podocytes have been identified over the past decade and their specific functions are increasingly well defined [13]. Finally, by analysis of ESTs enriched in glomeruli, ehd3 was shown to be the first transcript expressed exclusively by glomerular EC [12].
The current study sought to extend previous transcriptome-based work by building a human glomerular LongSAGE database that can be interrogated directly online. SAGE is based on the principal that a small (here 17 bp) tag sequence immediately 3' of an "anchoring" restriction site is a unique identifier of each transcript [14]. The frequency of specific SAGE tags relative to the total pool of tags reflects their abundance in the source mRNA. In silico comparison of SAGE libraries from diverse tissues can then be used to discover differential expression of transcripts [15]. The level of expression of any specific transcript can also be probed in silico by interrogating SAGE libraries with the transcript's unique SAGE tag sequence.
We report here the gene expression profile of transcripts for human glomeruli and compare them to pooled SAGE libraries for non-glomerular tissues and cells. Many of the most highly enriched glomerular transcripts reported here were previously found in a Sau3A-anchored glomerular library [9]. Nonetheless, the current SAGE database contains additional glomerulus-enriched transcript tags, and since it is NlaIII-anchored it now allows direct comparison with many non-renal SAGE libraries. The data should serve as a useful resource for investigators studying glomerular gene expression.
Construction and content
Cells and Tissues
Human kidney tissue was obtained from the uninvolved portion of tumor nephrectomy specimens (Human Subjects Protocols: #6196 University of Alberta and #155/97 University of Frankfurt). Patient 1 was a 45 year old Caucasian female, patient 2 a 72 yo Caucasian male. Renal tissue was collected only from patients in whom the serum creatinine was within normal limits, and in whom diabetes mellitus, hypertension, and proteinuria were absent. Specific parameters were not collected for individual patients. The relative purity of isolated glomeruli and the normal histological appearance of kidney cortex used in this study are shown in Figure 1. That the cDNA template used for SAGE contained mRNA representing glomerular capillary endothelium is shown by RT-PCR amplification of PECAM-1 (836 bp) and the non-integrin laminin receptor LAMR1 (256 bp). Greater synaptopodin transcript abundance in glomerular, compared to whole kidney cortex mRNA from the same specimen also shows appropriate enrichment of the source mRNA in glomerular podocyte transcripts (Figure 1). Amplification of the long PECAM-1 sequence furthermore shows that the source mRNA was intact. Sufficient mRNA for construction of the SAGE library was only obtained from patient 2. The integrity of this source material was also verified by Agilent 2100 microfluidics analysis (data not shown).
Figure 1
Human Kidney Source Material. Isolated glomeruli (A, D), Hematoxylin & Eosin Stain (B, E) and Masson Trichrome Stain (C, F) for patient 1 (A, B, C) and patient 2 (D, E, F). RT-PCR for PECAM-1 and the 67 kDa non-integrin laminin receptor LAMR1 for patient 1 (pt 1) and patient 2 (pt2) (G). Enrichment of the synaptopodin mRNA abundance, determined by RT-PCR, in glomeruli relative to whole kidney cortex from patient 2 (H).
Human Glomerular SAGE Library Construction
For SAGE, human glomeruli were isolated by sieving in ice-cold phosphate buffered saline (PBS) from the kidney of a 72-year old male using minor modifications of the protocol for rat glomeruli [16, 17]. Glomeruli were immediately placed into RNA-Protect (Qiagen, Valencia, CA) followed by isolation of 4.7 μg total RNA with the RNeasy kit (Qiagen). A SAGE library was then custom-constructed by Genzyme Corporation (Framingham, MA) using the "long" SAGE protocol, producing 17 bp SAGE tags with the CATG (NlaIII) anchoring restriction site [18]. A total of 2304 clones containing concatenated ditags were sequenced, resulting in 48,926 tags. Of these, 1,361 were derived from duplicate ditags. Tags from duplicate ditags were not removed [19]. Twenty-one tags were removed as they contained ambiguous (N) nucleotides leaving 48,905 tags and 22,907 unique long SAGE tags for analysis.
Tag sequences and their absolute counts in 115 distinct "long" SAGE libraries were retrieved from cgap.nci.nih.gov/SAGE (98,944,923 tags). All "long" SAGE Libraries available before July 1, 2008 were included without selection. Tissues and cells represented include normal brain, breast, skin, pancreas, bladder, gallbladder, uterus, vein, testis, white blood cells, lung macrophages, embryonic stem cells as well as malignant tumors including colon and lung adenocarcinoma, melanoma, among others. The frequency of each tag (count/total tag number) was calculated for each of the 115 libraries and expressed as tags per million (TPM). The mean TPM for the non-glomerular libraries (Pool TPM) is reported. The frequency ratio of the glomerular: Pool TPM was then calculated to establish degree of enrichment of specific tags in glomeruli (Ratio G: P). Statistical comparison of the pooled libraries with the current glomerular SAGE library was based on Chi-square analysis using absolute tag counts [20]. Comparison to a human kidney SAGE library (SAGE_Kidney_normal_B_1, from cgap.nci.nih.gov/SAGE) was based on the short (10 bp) tag sequences.
For each SAGE tag, identification was based on the "Hs_long.best_gene.gz" database found at ftp://ftp1.nci.nih.gov/pub/SAGE/HUMAN/. The SAGE Genie algorithm for identifying the best gene match for SAGE tags was reported by Boon et al. [21]. For some tags, the Blast n algorithm at http://blast.ncbi.nlm.nih.gov/Blast.cgi, was used to match tag sequences that could not be assigned by the "Hs_long.best_gene.gz" database. For these, the SAGE tag had to be in the +/+ orientation with the corresponding mRNA or EST, and fully match the 17 bp sequence immediately 3' of the NlaIII site nearest the Poly(A)+ tail or a stretch of > 8 A's as previously reported [22]. Positive identification based on this latter search strategy is indicated in additional file 1 by asterisks.
RT-PCR Analysis
For quantitative RT-PCR, glomeruli were microdissected from distinct pre-transplant kidney biopsy specimens obtained from three separate donors aged 57, 59 and 63 at the University of Göteborg (Human Subjects Protocol #653-05). Immediately after biopsy, one half of one biopsy core was placed into 0.5 ml of ice-cold PBS containing 100 U RNAse inhibitor (RNAsin) (Applied Biosystems, Foster City, CA, USA). Four to fifteen glomeruli were isolated using a stereomicroscope (Zeiss, Jena, Germany) followed by extraction of total RNA. cDNA was generated from glomerular RNA with SuperScript™ III RT (Invitrogen, Carlsbad, CA, USA). Human kidney, spleen, lung and liver mRNA was purchased from Invitrogen/Ambion (Carlsbad, CA). Reactions without RT for each primer set served as controls. PCR cycling was performed with 100 ng template (94°C – 3 min; 35 cycles: 94°C – 30 sec; 55°C – 30 sec; 68°C – 30 sec plus 1 min for each kilobase pair (kbp) of PCR product to be amplified; 72°C – 7 min). Quantification of gene expression was performed according to the delta Ct method (DeltaCt2/DeltaCt1), as described by others [23], and by this laboratory [22].
Human Glomerular SAGE Database Content
The complete human glomerular SAGE library was deposited in the Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/ repository (record GSE8114, Accession # GSM199994) and in the SAGE Genie collection http://cgap.nci.nih.gov/SAGE as "LSAGE_Kidney_Glomeruli_Normal_B_bjballer1". It consists of 22,907, unique 17 bp tag sequences and the absolute tag count for each sequence. The total tag count in the library is 48,905. The library is also appended in spreadsheet format with tag identification (additional file 1).
Utility
Retrieval of Highly Enriched Glomerular Transcripts
The transcripts most highly enriched in human glomeruli identified by this study are shown in Tables 1 and 2 and Additional files 3 and 4. Of the 22,907 tags, 291 were observed with an absolute count of 4 (81 TPM) or greater and enriched more than 10-fold relative to pooled non-kidney SAGE libraries. For 84 of these no reliable match to a known cDNA sequence was found, and a match to incompletely defined ESTs was observed for 8 others. The tags representing Aldolase B, uromodulin, glutamyl aminopeptidase, glutathione peroxidase, and SLC25A45 were excluded from this set because they were not enriched relative to whole kidney. They likely represent transcripts expressed at very high levels in contaminating tubules. Several highly expressed transcripts produced more than one unique tag, which is common and usually reflects priming from internal poly A(+) runs or alternatively spliced transcripts. After removal of such redundant tags, 133 well-characterized tags highly enriched in glomeruli were established (Tables 1 and 2 and Additional files 3 &4).
Table 1
Transcripts represented by SAGE tags enriched 30 fold or more in glomeruli
Tubulin polymerization-promoting protein family member 3
GTGACCCCAAGGCCAGT
82
1
0
57
TPPP3
C4orf31
Chromosome 4 open reading frame 31
TACATAAAATTAAAGAG
TAATCTAAGT
226
429
8
0
57
C4orf31
GJA5
Gap junction protein, alpha 5, 40kDa
GACCATTCCTCGGAGTA
AATCTTTGAT
203
122
2
24
55
5
GJA5
RefGJA5
ITGB8
Integrin, beta 8
TCTTGTATCAATGGCAG
633
12
0
52
ITGB8
Ref:ITGB8
TSPAN2
Tetraspanin 2
CCAAGGCACTGAATTAA
143
3
0
49
TSPAN2
WT1
Wilms tumor 1
CTGGTATATGGCTTCAA
TTACAAGATA
406
143
3
0
49
WT1
Ref:WT1
COL4A3
Collagen, type IV, alpha 3 (Goodpasture antigen)
TGCATTATTTTCCAGAT
122
3
0
48
COL4A3
REF:COL4A3
ALS2CL
ALS2 C-terminal like
CGATGCTGACGGGACCC
796
17
0
47
ALS2CL
PLCE1
Phospholipase C, epsilon 1
AACGAACGTGGCTGTAT
163
4
0
46
PLCE1
Ref:PLCE1
WFS1
Wolfram syndrome 1 (wolframin)
ACCCTCCTGTCCAGCAG
204
4
0
46
WFS1
PCOLCE2
Procollagen C-endopeptidase enhancer 2
ATGGAGGTATGAGGCCT
TATGTTCTCT
271
408
9
24
45
17
PCOLCE2
MRGPRF
MAS-related GPR, member F
AGGACCCACTGGGCAGC
CTCTTAAGGC
316
327
8
0
43
MRGPRF
SPTB
Spectrin, beta, erythrocytic
CAATCTGGGGCTGGCCC
82
2
0
42
SPTB
Ref:SPTB
ATP10A
ATPase, class V, type 10A
TCCTCTGCGCCAGGGGA
204
5
0
41
ATP10A
USHBP1
Usher syndrome 1C binding protein 1
TCATAAACTGTCCTGGA
122
3
0
40
USHBP1
MAP6
Microtubule-associated protein 6
TACAGTAGTCTTGCTGG
327
8
0
39
MAP6
NFASC
Neurofascin homolog (chicken)
AGCAATGAAAAGGCCAA
184
5
0
39
NFASC
PLA2R1
Phospholipase A2 receptor 1, 180kDa
AATTTTGCAAAAAGGAA
82
2
0
39
PLA2R1
C1QL1
Complement component 1, q subcomponent-like 1
CGCGGCGGCGACGGCAC
184
5
24
39
8
C1QL1
TMEM178
Transmembrane protein 178
CTTGTTAAATTTTAATG
102
3
24
39
4
TMEM178
TP53I11
Tumor protein p53 inducible protein 11
TACCCCAAGGCCTGATG
102
3
0
37
TP53I11
SYNPO
Synaptopodin
ATATTAGGAAGTCGGGG
CATTTCTACC
519
592
16
0
37
SYNPO
Ref:SYNPO
DACH1
Dachshund homolog 1 (Drosophila)
TAGGACCTATGAAAATT
82
2
0
37
DACH1
TTMA
Two transmembrane domain family member A
CTTTATTGAGTGTTATC
225
6
0
37
TTMA
FABP1
Fatty acid binding protein 1, liver
ACATTGGGTGACATTGT
102
3
24
35
4
FABP1
RAMP3
Receptor (G protein-coupled) activity modifying protein 3
AGCTTGTGGCCTCTATC
1,000
29
0
34
RAMP3
EMCN
Endomucin
CTACTTTGTACATATAA
TTTTCTTTAA
361
449
13
0
34
EMCN
RAPGEF3
Rap guanine nucleotide exchange factor (GEF) 3
AGGAGGGGCTGGGACTG
184
6
72
33
3
RAPGEF3
FAM20B
Major histocompatibility complex, class I, B
TCCCGGCCCGGCCGCGG
82
2
0
33
FAM20B
NPNT
Nephronectin
GTAAAGGTATAAGCCTT
CATTTTTAAT
383
286
9
0
32
NPNT
Ref:NPNT
UACA
Uveal autoantigen with coiled-coil domains and ankyrin repeats
AGTTCTGTTTCACAAGT
82
3
0
32
UACA
KLK7
Kallikrein-related peptidase 7
AGCCACCACGGCCAGCC
592
19
96
31
6
KLK7
LIMS2
LIM and senescent cell antigen-like domains 2
CAGATGGAGGCCTCTGG
776
25
24
31
32
LIMS2
TYRO3
TYRO3 protein tyrosine kinase
GGGCGGGTCCTAGCTGT
1,143
37
72
31
16
TYRO3
Transcripts represented by SAGE tags enriched 30 fold or more in glomeruli compared to pooled SAGE libraries from diverse tissues and cells. The gene symbol and gene name are shown for each tag sequence. Where available, the corresponding short Sau3A SAGE tag (Ref 9) is shown. SAGE tag frequencies are shown as Tags per Million (TPM). Sau3A TPM is derived from Ref 9. Enrichment in glomeruli relative to pooled libraries (G:P) and relative to whole kidney (G:K) is shown for each tag. This table with embedded links to SAGE Genie and links to references showing expression and/or function in glomeruli is provided as additional file 3
Table 2
Transcripts represented by SAGE tags enriched 10 – 30 fold in glomeruli
Symbol
Gene Name
NlaIII long Tag
Sau3A Tag
G:TPM
Sau3A
G:TPM
NlaIII
P:TPM
NlaIII
K:TPM
NlaIII
Ratio
G: P
Ratio
G: K
Link
SAGE Genie
Ref
Link
COL4A4
Collagen, type IV, alpha 4
TAACTTTTGCAAGATGC
122
4
0
29
COL4A4
Ref:COL4A4
AQP1
Aquaporin 1
AGCTCCTGATCAGAGGC
102
4
0
27
AQP1
AQP1
NPR1
Natriuretic peptide receptor A/guanylate cyclase A
AGCAGAGACAATTAAAA
163
6
24
27
7
NPR1
NPR1
PTGDS
Prostaglandin D2 synthase 21kDa
ACGGAACAATAGGACTC
CCGGCCAGCC
5,323
2,368
88
24
27
99
PTGDS
CRHBP
Corticotropin releasing hormone binding protein
AATAAATACATTCAGAA
ATAGTTCTAA
654
286
11
27
CRHBP
TNNI1
Troponin I type 1 (skeletal, slow)
AGGCACCTGGGGCTTCT
TGCGGGCCAA
158
204
8
24
26
9
TNNI1
ODZ2
Odz, odd Oz/ten-m homolog 2
ACAGTCACCACGAGGAG
122
5
26
ODZ2
EPAS1
Endothelial PAS domain protein 1
GAACTTTTCTGTAATGG
82
3
48
24
2
EPAS1
PODXL
Podocalyxin-like
GAGGACACAGATGACTC
ATATATGTCT
2,323
3,369
141
48
24
70
PODXL
Ref:PODXL
TMEM204
Transmembrane protein 204
CGTGCGAGACACGTGTG
204
9
23
TMEM204
SMAD6
SMAD family member 6
TCTCCGGACGCCACCAA
163
7
23
SMAD6
Ref:Samd6
CLEC3B
C-type lectin domain family 3, member B
ACCGGCGCCCGCATCGC
GTGTAGCCGG
361
367
16
72
23
5
CLEC3B
IFITM3
Interferon induced transmembrane protein 3
AACCCCTGCTGCCTGGG
102
4
23
IFITM3
HOXA7
Homeobox A7
GTATGTTGTCTTGAGTT
82
4
21
HOXA7
CHI3L1
Chitinase 3-like 1 (cartilage glycoprotein-39)
GTATGGGCCCTGGACCT
CCCAAGCCTG
564
1,021
48
21
CHI3L1
FRY
Furry homolog (Drosophila)
TGAACTTGTTGCACTGC
408
19
21
FRY
PEA15
Phosphoprotein enriched in astrocytes 15
TCTGCCCTTTTTTGTGG
125
6
24
21
5
PEA15
ZNF250
Zinc finger protein 250
TGGAACCACAAGCAGCC
143
7
20
ZNF250
TGFBR3
Transforming growth factor, beta receptor III
GCAAATCCTGTCGGTCT
CTCCTGTCTA
180
122
6
20
TGFBR3
IGFBP5
Insulin-like growth factor binding protein 5
GAGTACGTTGACGGGGA
TTTGTCTTTT
3,496
367
18
20
IGFBP5
Ref:IGFBP5
FCN3
Ficolin (collagen/fibrinogen domain containing) 3
GACACCGAGGGGGGCGG
GTCAGCCACC
180
715
36
48
20
15
FCN3
LRRC32
Leucine rich repeat containing 32 (GARP gene)
TTGCATACCCTGACCCC
TTTGAAAACA
180
20
1
19
LRRC32
NOSTRIN
Nitric oxide synthase trafficker
CCACACGCAGATTCACT
TTTGAATGGG
158
61
3
19
NOSTRIN
Ref:Nostrin
HTRA1
HtrA serine peptidase 1
TTTCCCTCAAAGACTCT
1,409
74
24
19
59
HTRA1
ITGA1
Pelota homolog (Drosophila)
TGCCAGGTGCAGTCACA
122
6
24
19
5
ITGA1
TNNC1
Troponin C type 1 (slow)
TCCTCAACCCCAAATCC
184
11
17
TNNC1
DOPEY2
Dopey family member 2
AGAATTGCTTGAACCCA
1,347
79
263
17
5
DOPEY2
FOXC1
Forkhead box C1
AGCCTGTACGCGGCCGG
ATTGTTAAAG
519
245
15
24
17
10
FOXC1
CEP3
CDC42 effector protein (Rho GTPase binding) 3
ATGCTTCTGCAGAGACT
TTGGGCCCTC
226
163
10
17
CEP3
BGN
Biglycan
GCCTGTCCCTCCAAGAC
GAGAACGGGA
158
776
48
72
16
11
BGN
Ref:BGN
MAGI2
Membrane associated guanylate kinase
TATTAATAGTCACAGAA
102
7
16
MAGI2
AIF1L
Allograft inflammatory factor 1-like
GGAGTGTGCGTGGACTG
1,572
102
334
15
5
C9orf58
KCNH3
Potassium voltage-gated channel, subfamily H, member 3
CDC14 cell division cycle 14 homolog A (S. cerevisiae)
TATTTTGTTATGAATAG
143
12
24
12
6
CDC14A
CIRBP
Cold inducible RNA binding protein
TGCCCGGGGAATGTTCC
82
7
12
CIRBP
CSRP1
Cysteine and glycine-rich protein 1
CAGGCGGGGTCCTAGGA
82
7
12
CSRP1
FAM20C
Family with sequence similarity 20, member C
CGCCCGTCGTGAATTCA
367
31
119
12
3
FAM20C
CLEC16A
C-type lectin domain family 16, member A
CTTCGTGGGTACTGAAC
122
10
24
12
5
CLEC16A
INF2
Inverted formin, FH2 and WH2 domain containing
TCCAGCCCCTGAAGTTG
184
16
48
12
4
INF2
BMP7
Bone morphogenetic protein 7
TGGAACCCGGTCTTGTG
204
18
12
BMP7
Ref:BPM7
ZDHHC6
Zinc finger, DHHC-type containing 6, transmembrane protein 4
TGGTACTTCTCTTTTCC
AATGGATGTT
1,128
592
51
12
ZDHHC6
PPAP2B
Phosphatidic acid phosphatase type 2B
ATGTAGGTGCCACCCAC
AACCACATGC
654
184
16
119
11
2
PPAP2B
Ref:PPA2B
TXNIP
Thioredoxin interacting protein
AGAAACTAGAGGGCAGG
102
9
11
TXNIP
ITGA3
Integrin, alpha 3
GTACTGTAGCAGGGGAA
CTCCACAGAG
180
817
72
24
11
34
ITGA3
Ref:ITGA3
C19orf63
Chromosome 19 open reading frame 63
AAAGAGTCGGGGCTGGA
82
7
48
11
2
C19orf63
IGFBP2
Insulin-like growth factor binding protein 2, 36kDa
GCCTGTACAACCTCAAA
CAGGGAGCCC
880
1,797
161
96
11
19
IGFBP2
Ref:IGFBP2
PTGER4
Prostaglandin E receptor 4 (subtype EP4)
TTTTGTTGCTCAGTGTT
306
28
11
PTGER4
FLRT3
Fibronectin leucine rich transmembrane protein 3
TATTTTTCTAGGCATAA
82
8
24
11
3
FLRT3
VWA1
Von Willebrand factor A domain containing 1
CCCAGGACACCAGCTGG
531
49
24
11
22
VWA1
LARGE
Like-glycosyltransferase
AAAGCCCAGTTCTGAAG
82
8
24
11
3
LARGE
LINGO1
Leucine rich repeat and Ig domain containing 1
AAGATGATATGAGGCCG
122
12
10
LINGO1
MYO1E
Myosin IE
TATGAATGTACTAAGTA
ATATACTGTA
248
245
26
9
MYO1E
Ref:MYO1E
Transcripts represented by SAGE tags enriched 10 – 30 fold in glomeruli compared to pooled SAGE libraries. The gene symbol and gene name are shown for each unique NlaIII-anchored long SAGE tag sequence. Where available, the corresponding short Sau3A SAGE tag (Ref 9) is shown. SAGE tag frequencies are shown as Tags per Million (TPM). Sau3A TPM is derived from Ref 9. Enrichment in glomeruli relative to pooled libraries (G:P) and relative to whole kidney (G:K) is shown for each tag. This table with embedded links to SAGE Genie and links to references showing expression and/or function in glomeruli is provided as additional file 4
A previously published Sau3A-anchored SAGE library [9] prepared from microdissected human glomeruli contained 184 SAGE tags that were enriched in glomeruli relative to other micro-dissected nephron segments. These represented 156 well-characterized transcripts. As expected, the corresponding NlaIII SAGE tag for 143 of these was also observed in the current glomerular SAGE library (Tables 1 and 2 and Additional files 3 and 4 and additional file 2). For 47 transcripts represented in both libraries a 10-fold or greater enrichment of the NlaIII tag relative to non-glomerular cells and tissues was observed and is shown in Tables 1 and 2 and Additional files 3 and 4. The NlaIII tag corresponding to the remaining 96 transcripts identified in the Sau3A library was enriched relative to whole kidney, in keeping with the previous report [9], but less than 10 fold relative to non-renal tissues (additional file 2).
Many of the highly expressed and highly enriched transcripts observed in this library are encoded by genes already known to be unique or highly enriched in glomerular podocytes, for instance Podocin (NPHS2), Nephrin (NPHS1), transcription factor 21 (Pod1, FLJ35700), Protein Tyrosine Phosphatase Glepp 1 (PTPRO), Synaptopodin (SYNPO), indicating that this SAGE database appropriately represents glomerular transcripts and that it identifies transcripts enriched in glomeruli. Some of the SAGE tags enriched in glomeruli represent known endothelial cell-predominant genes, for instance Endomucin (EMCN), claudin 5 (CLDN5), NOSTRIN and CD34, consistent with abundant EC in glomeruli.
To independently demonstrate the utility of this database in defining enrichment of transcripts in glomeruli, RT-PCR comparing the level of expression of 19 transcripts enriched in the glomerular SAGE library with that in lung, spleen and liver was performed. Lung, liver and spleen were not represented in the pooled SAGE libraries used here. For each, glomeruli microdissected from the kidneys of three distinct donors were used. The source mRNA used for RT-PCR was distinct from that used for generation of the SAGE library. Transcripts were chosen to represent a spectrum of glomerular enrichment, and some well-known podocyte-predominant transcripts (TCF21, VEGFA) were included as internal controls. Overall, the degree of glomerular transcript enrichment observed by RT-PCR compared to lung, liver and spleen was similar to that observed by SAGE, though there was variation between lung, spleen and liver (Table 3). The wide range of expression observed in the three non-glomerular tissues was expected, as the pooled SAGE-based comparison does not take into account tissue-to-tissue variation in gene expression.
Table 3
Ratio of mRNA abundance in glomeruli compared to lung, spleen and liver.
SAGE
RT-PCR
Symbol
Name
LongTag
G: TPM
P: TPM
Ratio G: P
G: Lu
G: Sp
G: Li
SOST
Sclerosteosis
ACATATGAAAGCCTGCA
82
0.00
Infinity
130 ± 48
13007 ± 4827
7969 ± 2957
TCF21
Transcription factor 21
CGAGTGCTGAGCAAGGC
817
1.59
514
49 ± 16
27 ± 9
384 ± 123
FGF1
Fibroblast growth factor 1 (acidic)
TAAAGGCCTTTAATAAG
102
0.49
210
490 ± 70
166 ± 23
2565 ± 367
SPOCK2
sparc/osteonectin, cwcv and kazal-like domains
TGTGGAGTGTACTTGTT
245
1.44
170
45 ± 13
39 ± 12
258 ± 75
PTHR1
Parathyroid hormone receptor 1
TGACCAGGCGCTGGGGG
1143
8
141
196 ± 55
51 ± 14
30 ± 8
EFNB1
Ephrin-B1
AGGGAAGAGGAAAGTGC
102
1.50
68
20 ± 4
17 ± 3
30 ± 6
CDKN1C
Cyclin-dependent kinase inhibitor 1C (p57, Kip2)
TAGCAGCAACCGGCGGC
1021
46
22
51 ± 17
45 ± 15
198 ± 66
IGFBP5
Insulin-like growth factor binding protein 5
GAGTACGTTGACGGGGA
367
18
20
10 ± 4
22 ± 9
43 ± 17
CLDN5
Claudin 5
GACCGCGGCTTCCGCCG
715
38
19
5 ± 1
27 ± 7
24 ± 6
FAM65A
Family with sequence similarity 65, member A
GGTTCCTGGTGCCCCTT
755
43
17
18 ± 5
11 ± 3
51 ± 15
FOXC1
Forkhead box C1
AGCCTGTACGCGGCCGG
245
15
17
25 ± 6
302 ± 74
452 ± 110
C9orf58
Chromosome 9 open reading frame 58
GGAGTGTGCGTGGACTG
1572
102
15
36 ± 3
2 ± 0
171 ± 16
NES
Nestin
TGCTGACTCCCCCCATC
2001
138
14
22 ± 6
23 ± 6
339 ± 84
VEGFA
Vascular endothelial growth factor A
TTTCCAATCTCTCTCTC
1450
109
13
16 ± 4
53 ± 12
22 ± 5
ZDHHC6
Zinc finger, DHHC-type containing 6
TGGTACTTCTCTTTTCC
592
51
12
Infinity
60 ± 18
Infinity
MYO1E
Myosin IE
TATGAATGTACTAAGTA
245
26
9
11 ± 3
21 ± 6
13 ± 3
MYL9
Myosin, light chain 9, regulatory
GGAGTGTGCTCAGGAGT
3287
454
7
4 ± 1
15 ± 4
45 ± 11
ITM2B
Integral membrane protein 2B
TCACCTTAGGTAGTAGG
3328
508
7
4 ± 1
4 ± 1
5 ± 1
MYO1D
Myosin ID
ATTGTAGACAATGAGGG
327
82
4
3 ± 0
7 ± 1
7 ± 1
The SAGE tag frequency in glomeruli (G: TPM) and SAGE library pool (P: TPM) as well as the relative SAGE tag enrichment in glomeruli (Ratio G: P) is shown. The transcript abundance relative to lung (G: Lu), spleen (G: Sp) and liver (G: Li) was determined by RT-PCR using distinct sets of microdissected glomeruli from kidneys of three different donors. G: glomeruli, Lu: lung, Sp: spleen, Li: liver. Mean ± SEM.
Finally, it is of note that 117 transcript tags observed 2 or more times and enriched > 500 fold in this glomerular library remain unidentified or poorly characterized (additional file 1). At least some of these will likely prove to be currently unknown glomerulus-predominant transcripts.
In Silico Interrogation of the Glomerular SAGE Database
The current database can be retrieved directly or interrogated in silico. It may be used to determine whether any specific gene is highly expressed in glomeruli, and to define transcripts that are highly enriched relative to other tissues for which SAGE libraries are available.
To assess whether a specific transcript is expressed in glomeruli, the SAGE tags uniquely identifying the transcript can be found at http://cgap.nci.nih.gov/SAGE/ using the "SAGE Anatomic Viewer" [21]. The "Digital Northern" tool is then used to evaluate the level of expression in the SAGE libraries of the collection, which includes the current library. The collection can also be interrogated using specific NlaIII SAGE tags of cDNA sequences for which a gene symbol may not yet have been assigned. The tag can be retrieved from any cDNA sequence by identifying the 17-nt sequence immediately 3' of the last NlaIII site (CATG) prior to the poly(A+) tail. Its frequency in the glomerular database is an indicator of the level of expression in human glomeruli. The 95% confidence interval for observing any tag with a true count of 4 is ± 3.96. Hence, any transcript producing a tag frequency of 4 per 48,905 (81.8 TPM) or greater has a 95% probability of being represented in this library. Failure to find the SAGE tag representing any specific transcript in this library indicates that its expression level is lower than the limit of detection, or that the transcript does not contain an NlaIII restriction site from which a SAGE tag could be generated.
The "LSAGE_Kidney_Glomeruli_Normal_B_bjballer1" database can also be compared directly to a single, or sets of other SAGE databases in the SAGE Genie collection using the "SAGE Digital Gene Expression Displayer (DGED)" tool at http://cgap.nci.nih.gov/SAGE/. This type of analysis will return data similar to those in additional file 1, though comparison can also be restricted to specific libraries rather than the pool of libraries evaluated here.
Finally, this SAGE library with matching transcript identification, glomerulus to pool ratio and glomerulus to kidney ratio is supplied as additional file 1, where the order is based on tag abundance. This data set contains only 18,152 SAGE tags, as any tag found only once and not in any other library was removed. The table can be retrieved without restriction and, if desired, sorted based on the degree of tag enrichment.
Discussion
This study established a human glomerular SAGE library that can be used for data mining by investigators with an interest in glomerular cell biology and pathophysiology. The library was appropriately enriched in SAGE tags representing transcripts known to be restricted to glomerular podocytes, including nephrin [24], podocin [25], synaptopodin [26], podocalyxin [27], transcription factor 21 [28], the protein tyrosine phosphatase receptor type O GLEPP1 [29], the cyclin dependent kinase inhibitor C1 [30] and nestin [31]. It is therefore likely that other transcripts whose SAGE tags are much more highly represented in this library compared to SAGE libraries from other tissues and cells are also expressed predominantly in glomeruli.
A SAGE library that used Sau3A as the anchoring restriction enzyme was previously produced from human glomerular mRNA [9]. It identified 155 highly expressed transcripts in glomeruli that were enriched in glomeruli when compared to microdissected non-glomerular nephron segments. Since the previously published glomerular SAGE library is based on the Sau3A anchoring restriction site, it does not allow in silico comparison of tag frequencies with the much greater collection of NlaIII-based SAGE libraries. All except 12 transcripts reported to be enriched in glomeruli by Chabardes-Garonne [9] were observed in the current glomerular SAGE library. The corresponding NlaIII tag for a subset of these (47 tags) was enriched > 10 fold when compared to non-renal tissues and cells (Tables 1 and 2 and Additional files 3 &4), providing independent evidence that these represent glomerulus-predominant transcripts.
The current study also identified 86 transcript tags that were enriched more than 10 fold in glomeruli, but which were not represented in the previous Sau3A anchored library (Tables 1 and 2 and Additional files 3 &4). Failure to find a Sau3A SAGE tag for known glomerulus-restricted genes like nephrin, or an NlaIII SAGE tag for endoglin and VCAM1, suggests either that the tag frequency was too low to be detected or that the required restriction site was absent from the transcript. The current study also shows that that several transcripts more highly expressed in glomeruli compared to other nephron segments [9] are not restricted to glomeruli when compared to non-renal tissues or cells (additional file 2). This is not surprising since some transcripts that are not shared between nephron epithelium and glomerular capillary tuft nevertheless may be highly expressed in other tissues.
Several transcripts not previously shown to have a specific function in glomeruli were highly expressed and enriched in glomeruli when compared to non-glomerular tissues. Among these, the tag for the chloride intracellular channel 5 (CLIC5) is very abundant in the glomerular transcript pool, and its frequency in glomeruli was more than 800 fold greater than in other tissues. The transcript "DKFZp564B076" whose SAGE tag was previously shown to be enriched in microdissected glomeruli [9] and later in cultured glomerular EC in this laboratory [22] is identical to the 3' end of CLIC5. CLIC5 is an ezrin-binding protein involved in maintaining actin-based microvilli in the placenta and actin-based stereocilia in the inner ear [32]. Its role in glomerular cell function is as yet undefined. The transcript for the basal cell adhesion molecule (BCAM) is also very abundant in glomeruli and enriched approximately 58 fold. BCAM is a glycoprotein that functions as a receptor for alpha5 laminin. BCAM immunoreactivity is observed in both, glomerular podocytes and glomerular EC, and mice deficient in BCAM have significant structural abnormalities of glomeruli [33]. Glomerular expression of the parathyroid hormone receptor 1 (PTHR1) was not expected. PTHR1 is very abundant in renal proximal tubule cells and could therefore represent proximal tubule contamination. However, since the PTHR1 SAGE tag was less abundant in renal cortex than in glomeruli (Table 1 and Additional file 3), its enrichment in this library cannot be due to proximal tubule contamination. Indeed, mesangial cells express PTHR1 [34]. More work is required to define the function of PTHR1 in mesangial cells. In this regard, it is of great interest that Sclerostin, an inhibitor of bone matrix formation whose expression is regulated by PTH, is also expressed at much higher levels in glomeruli than in most non-renal tissues and cells (Table 1 and Additional file 3) or in other nephron segments [9]. While we have no comparison with a bone SAGE library where sclerostin is likely expressed at high levels, the finding nonetheless suggests that it could be involved in regulating extracellular matrix depositon in glomeruli. Nephronectin, a ligand for integrin alpha8beta1 is known to be essential for renal development, and is expressed in renal epithelium. Enrichment of the nephronectin SAGE tag in the glomerular library relative to kidney cortex is in keeping with the observation by Brandenberger et al [35], who observed very strong nephronectin immunoreactivity in differentiating glomeruli. The secreted glycoprotein testican 2 SPARC (SPOCK2) belongs to in the osteonectin/SPARC family [36] is also highly expressed and enriched in glomeruli. Members of this family of proteins regulate cell-cell and cell-matrix interactions, and SPOCK2 is induced after glomerular injury [37]. The other protein in this family is connective tissue growth factor (CTGF). The SAGE tag for CTGF was observed at a high frequency in glomeruli (additional file 1) but it was not highly enriched relative to other tissues. Nonetheless, both SPOCK2 and CTGF likely play a critical role in regulating glomerular remodeling. In 2006 Lakhe-Reddy and coworkers [38] described the localization of beta 8 integrin to glomerular mesangial cells and observed that its expression may suppress mesangial cell dedifferentiation via Rac1 activation. The SAGE tag for integrin beta 8 was highly expressed and enriched in this glomerular library.
While several semaphorins are expressed in renal glomeruli, so far a role for semaphorin 3G, whose SAGE tag is abundant and enriched in this database, has not been described. Still, semphorin 3G, which has repulsive function via neuropilin 2 binding in the CSN neuronal guidance, is also highly expressed in kidney [39], begging the question whether it serves an important function is in glomeruli. Based on this study many other transcripts are highly enriched in glomeruli. It is hoped that other investigators will use this database as a tool to further define the transcriptome of glomerular cells in health and disease.
We did not observe the NlaIII SAGE tag for EHD3, a transcript previously shown to be unique for glomerular endothelial cells [12], in this library. A SAGE tag for EHD3 also is not observed in the previously published Sau3A-anchored library [9]. Failure to observe this tag does not detract from the previous observations but only suggests that the EHD3 transcript abundance was too low to generate a SAGE tag in the two glomerular SAGE libraries.
Finally, not all tags observed in this SAGE library have as yet been matched to a specific gene. For some of these unidentified SAGE tags, matching sequences within the human genome are observed, but whether they represent specific transcripts is currently not known.
Conclusion
We have constructed a new human glomerular SAGE library, based on the NlaIII anchoring restriction site. The database can be searched to determine whether specific transcripts are highly expressed and/or enriched in glomeruli and it can be used a resource to further study transcripts that appear to be glomerulus-enriched but whose function in glomeruli has not been investigated so far.
Availability and requirements
The SAGE database (GEO Accession #GSM199994) described here is available for download from http://www.ncbi.nlm.nih.gov/geo/. It can also be downloaded from, or interrogated in silico at http://cgap.nci.nih.gov/SAGE/ without restriction. The annotated database containing Tag sequences, glomerular frequencies, gene identification, as well as frequency ratios to pooled and kidney libraries is available as additional file 1.
Notes
Abbreviations
SAGE:
Serial Analysis of Gene Expression
TPM:
Tags per million
RT-PCR:
Reverse Transcriptase Polymerase Chain Reaction
WT-1:
Wilms Tumor 1
EC:
Endothelial Cell(s)
EST:
Expressed Sequence Tag
cDNA:
Complementary DNA
bp:
base pair.
Declarations
Acknowledgements
The work was supported by Establishment Grant CEG63108 from the Canadian Institutes of Health Research (CIHR) and by CIHR operating grant MOP641814. B.J. Ballermann holds the Tier 1 Canada Research Chair in Endothelial Cell Biology. J. Sorensson-Nystrom's work was supported by Grant #14764 from the Swedish Research Council. W. Fierlbeck was supported by a fellowship grant from the Deutsche Forschungsgemeinschaft (Fi 829/1-1)
Authors’ Affiliations
(1)
Department of Medicine, University of Alberta
(2)
Department of Nephrology, Göteborg University
(3)
Department of Nephrology, University of Frankfurt/Main
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