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A human glomerular SAGE transcriptome database

  • Jenny Nyström2,
  • Wolfgang Fierlbeck3,
  • Anna Granqvist2,
  • Stephen C Kulak1 and
  • Barbara J Ballermann1Email author
Contributed equally
BMC Nephrology200910:13

https://doi.org/10.1186/1471-2369-10-13

Received: 03 December 2008

Accepted: 05 June 2009

Published: 05 June 2009

Abstract

Background

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 [68] across the glomerular capillary wall.

Transcriptome and proteomic approaches are helping to define genes highly expressed and/or enriched in glomeruli [912]. 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

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

Link

Ref

NPHS2

Nephrosis 2, idiopathic, steroid-resistant (podocin)

TTTCCGTGACTCATCTA

CCTCACTGAA

1,534

2,287

0

48

Infinity

48

NPHS2

Ref:NPHS2

SOST

Sclerosteosis

ACATATGAAAGCCTGCA

TCGAGGAGAC

158

82

0

0

Infinity

 

SOST

 

MME

Membrane metallo-endopeptidase

CTGCAGTGTTCGAGTGG

TATAAAGCGA

271

102

0

0

4446

 

MME

Ref:MME

CLIC5

Chloride intracellular channel 5

AATCTGAACCAATTACC

CAGTCATCTG

1,038

3,593

5

24

719

150

CLIC5

Ref:CLIC5

NPHS1

Nephrosis 1, congenital, Finnish type (nephrin)

TAAACATAAGTATGCTC

  

1,041

2

0

549

 

NPHS1

Ref:NPHS1

TCF21

Transcription factor 21

CGAGTGCTGAGCAAGGC

ATAGGATAGC

451

817

2

0

514

 

TCF21

Ref:TCF21

CDKN1C

Cyclin-dependent kinase inhibitor 1C (p57, Kip2)

CCGCTGCGGGGCCCTGG

AGCGCCTGAG

383

490

1

0

506

 

CDKN1C

Ref:CDKN1C

DDN

Dendrin

TGAACTTGGCCACATCA

  

306

1

0

373

 

DDN

Ref:DDN

THYMU2010816

CDNA FLJ36413 fis, clone THYMU2010816

TATCACTGGGGAGGGAA

ATTAGTCAAT

203

449

2

0

285

 

THYMU2010816

 

FLJ22271

CDNA: FLJ22271 fis, clone HRC03191

GCTTTGTCGCAACGCTC

  

82

0

0

263

 

FLJ22271

 

PTPRO

Protein tyrosine phosphatase, receptor type, O

GATATACAACAGAAAAC

AACTGTGTAA

226

755

4

0

215

 

PTPRO

Ref:PTPRO

FGF1

Fibroblast growth factor 1 (acidic)

TAAAGGCCTTTAATAAG

TTCTTAGAAG

474

102

0

24

210

4

FGF1

Ref:FGF1

PTHR1

Parathyroid hormone receptor 1

TGACCAGGCGCTGGGGG

  

1,143

8

96

141

12

PTHR1

Ref:PTHR1

CLDN5

Claudin 5

GTAGGCGGCTGCCTCTT

  

82

1

0

127

 

CLDN5

 

LRRC2

Leucine rich repeat containing 2

GGACTGCACTCCAGCCT

  

82

1

0

108

 

LRRC2

 

AL049990

MRNA; cDNA DKFZp564G112 (from clone DKFZp564G112)

ACTTGGAAATAAACAAA

  

143

1

0

105

 

AL049990

 

HTRA1

HtrA serine peptidase 1

ACCGACAGGCCAAAGGA

CGCAGGCAGA

1,398

143

1

0

105

 

HTRA1

 

FAM65A

Family with sequence similarity 65, member A

GCTGCTGTCAGCACCCA

AGGCTGTTGT

316

204

2

0

99

 

FAM65A

 

SEMA3G

Semaphorin 3G

ACTGCCCCTGAGCTCTG

  

674

7

24

94

28

SEMA3G

 

SPOCK1

Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1

AGAATACCTTAATACTG

TTTAATACTT

226

122

1

0

92

 

SPOCK1

 

CRB2

Crumbs homolog 2 (Drosophila)

TGCAGCAGTGGCAGCCT

  

367

4

0

89

 

CRB2

 

ENPEP

Glutamyl aminopeptidase (aminopeptidase A)

GCCTGGAATTGGATACA

ATATGAATTA

632

143

2

96

79

1

ENPEP

Ref:ENPEP

SLC45A1

Solute carrier family 45, member 1

GGCGTGGACATCTCTCT

  

143

2

0

77

 

SLC45A1

 

TNNT2

Troponin T type 2 (cardiac)

ATGCATTTTGGGGGTTA

TGCTCCTCGC

338

184

2

24

76

8

TNNT2

 

CD34

CD34 molecule

GGACCAGGTCTTGGAGC

  

102

1

24

75

4

CD34

Ref;CD34

EFNB1

Ephrin-B1

AGGGAAGAGGAAAGTGC

  

102

2

0

68

 

EFNB1

Ref:EFNB1

IL1RL1

Interleukin 1 receptor-like 1 (ST2 Protein)

AGGGCAGGGACATCATC

TTTGTAGACT

158

82

1

0

64

 

IL1RL1

 

ST6GALNAC3

ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-

TGATGCCCTTGAACACC

  

286

5

0

63

 

ST6GALNAC3

Ref:ST6GALNAC3

 

acetylgalactosaminide alpha-2,6-sialyltransferase 3

          

NTNG1

Netrin G1

TGTAACAGCCCCCTCTA

  

102

2

0

63

 

NTNG1

 

SPOCK2

Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2

CATAAAGGAAATCAAAT

TGGTAGGTTG

248

1,123

18

0

61

 

SPOCK2

 

ABLIM2

Actin binding LIM protein family, member 2

ACACGCCAAGTCCCGTT

  

122

2

0

60

 

ABLIM2

Ref:ABLIM2

MGC16291

Hypothetical protein MGC16291

TGGGTCAAACTCTGAAA

  

122

2

24

58

5

MGC16291

 

BCAM

Basal cell adhesion molecule (Lutheran blood group)

CCCGCCCCCGCCTTCCC

ACGTGGTATC

632

3,205

55

788

58

4

BCAM

Ref:BCAM

TPPP3

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

TGCCCCTGCCTCTACCT

  

163

11

 

15

 

KCNH3

 

PARD6G

Par-6 partitioning defective 6 homolog gamma (C. elegans)

TCGTTCAGTGCCCCAGC

  

82

5

 

15

 

PARD6G

 

TM4SF18

Transmembrane 4 L six family member 18

CAAGTATACCACCCTTC

  

82

5

 

15

 

TM4SF18

 

TENC1

Tensin like C1 domain containing phosphatase (tensin 2)

AATAGGGGAAAAAAGAG

ACATGAATAG

519

531

37

 

15

 

TENC1

Ref:TENC1

DPP6

Dipeptidyl-peptidase 6

ACATTTGGTTAAAAAAA

  

82

6

 

14

 

DPP6

 

NES

Nestin

TGCTGACTCCCCCCATC

  

2,001

138

72

14

28

NES

Ref:NES

ADORA1

Adenosine A1 receptor

TGACTAATAAAAAACTG

  

82

6

 

14

 

ADORA1

Ref:ADORA1

VEGFA

Vascular endothelial growth factor A

TTTCCAATCTCTCTCTC

CCCTGGCTCC

248

1,450

109

24

13

61

VEGFA

Ref:VEGFA

ZNF135

Zinc finger protein 135

GGGAAACTCCATCTCTA

  

102

8

 

13

 

ZNF135

 

SLC48A1

Solute carrier family 48 (heme transporter), member 1

GTGCATCAGAGCGGGAA

  

82

6

 

13

 

SLC48A1

 

FARP1

FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1

GGGTAGTGTCAGTCGGA

  

122

10

72

13

2

FARP1

 

C1orf115

Chromosome 1 open reading frame 115

TCGAAGATTCACTGGGA

  

102

8

24

12

4

C1orf115

 

DAG1

Dystroglycan 1 (dystrophin-associated glycoprotein 1)

CAGAGACGTGGCTGGCC

  

1,286

104

48

12

27

DAG1

Ref:DAG1

PPAP2A

Phosphatidic acid phosphatase type 2A

AAACACCAACAACTGGG

CAGATTGGTC

248

286

23

 

12

 

PPAP2A

Ref:PPA2A

SEMA3B

Semaphorin 3B

TGCCGCCCGCAGCCTGC

  

612

50

72

12

9

SEMA3B

 

CDC14A

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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