1. Leprecan distribution in the developing and adult kidney
M. Lauer, B. Scruggs, S. Chen, D. Wassenhove-McCarthy, K.J. McCarthy 
Kidney International 
Volume 72, Issue 1, Pages (July 2007)
DOI: /sj.ki
Copyright © 2007 International Society of Nephrology Terms and Conditions

2. Figure 1
Basic organization of the leprecan core protein.(a) A cartoon that shows the domain organization of the leprecan core protein. Leprecan is tentatively a bifunctional molecule, with a PH domain at the carboxyl end of the molecule. A leucine zipper motif is adjacent to the PH domain and may function in dimerization. At the amino end are four tetratricopeptide motifs (TPR) that may impart a protein docking function to the amino end of the molecule. There are also four CXXXC motifs present at the amino end of the molecule, four glycosaminoglycan acceptor sites, one RGD motif, and four N-glycosylation sites present in the molecule. The location of the recognition region for Rb6480 (short black bar) and Rb2096 (long black bar) are indicated above the protein construct. The mRNA probe was developed from a region of mRNA that encodes a portion of the protein shown indicated by the appropriate bar. (b) The alignment of a portion of leprecan and its homologous family members – the dark shaded boxes indicate regions of homology. The black bar above the sequence shows the region of the protein that was used to develop the Rb6480 antiserum. (c) Is the exon organization of the leprecan gene superimposed above the leprecan cDNA sequence. The leprecan mRNA probe spans exons 9–11.
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

3. Figure 2
A graph of the results of an ELISA that used the peptide T71ELPWAPDLDLGPASSLNHDPGAAALHD98 as the antigen. Only the Rb6480 antiserum recognizes the peptide in the ELISA assay, the preimmune antisera, Rb2096, and the secondary antibody all fail to give a signal significantly greater than the background signal. The Western blot adjacent to the graph shows that the antibody recognizes the sequence contained in the expressed leprecan fusion protein representing M1-Q399 of the leprecan sequence whereas the preimmune serum does not.
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Copyright © 2007 International Society of Nephrology Terms and Conditions

4. Figure 3
The photomicrographs are from a 10-μm-thick frozen section of adult rat kidney. (a, b) Widefield fluorescent microscopy images and (c, d) deconvolved images were digitized from double-label immunostained with monoclonal antibodies directed against (a, c) a BM-CSPG and (b, d) a polyclonal antiserum (Rb2096) against leprecan. In previous studies, BM-CSPG was localized to the mesangial matrix of the glomerulus. Although the widefield images show overlap of distribution in some areas of the glomerulus between the two molecules, the deconvolved images better show that leprecan has both an extracellular and intracellular distribution in the kidney as indicated by the arrows in (b, d) showing intracellular staining in a mesangial cell and colocalization in the mesangial matrix (cf. arrows in a and c). Final magnification × 400.
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Copyright © 2007 International Society of Nephrology Terms and Conditions

5. Figure 4
Immunohistochemistry and immunoassay for leprecan using Rb 6480 and Rb The micrographs are of 10-μm-thick frozen sections of adult rat kidney immunostained with (a) Rb2096 and (b) Rb6480 antisera. Both images are the result of digital deconvolution of image stacks of widefield fluorescence images acquired at 0.1-μm steps. Both antibodies globally stain glomerular cells, including the parietal epithelial cells of Bowman's capsule and the othermost lining fibroblast cells. (b) Rb6480 fluorescence shows a focal localization in podocytes (arrows), which is not readily discernable by Rb2096 staining (Final magnfication × 400). (c) The results of a Western blot immunoassay of a 4–12% SDS-polyacrylamide gel through which mesangial cell or podocyte cell lysates (NR=nonreduced; R=reduced) were electrophoresed. The respective blots are presented as mirror images of one another. In podocytes, both antisera recognize a 80 kDa band in the nonreduced (NR) extract lane; Rb6480 also recognizes a second band at 100 kDa whereas Rb2096 shows minimal, if any immunoreactivity for the 100 kDa band. In the corresponding reduced (R) lanes Rb6480 gives the same immunoreactivity pattern as in the previous lane, Rb2096 antiserum recognizes a 90 kDa band. In nonreduced mesangial cell extracts, both antisera show a banding pattern similar to that seen in the nonreduced podocyte extract lanes. In reduced mesangial cell lysates, both Rb6480 and Rb2096 also gave differential banding patterns, with Rb6480 recognizing 95, 80, and 78 kDa bands and Rb2096 recognizing a broad band approximately 82 kDa in size.
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

6. Figure 5
Leprecan expression in adult glomeruli. The photomicrographs are of (a, b) 4-μm-thick paraffin-embedded sections or (c–h) 10-μm-thick frozen sections of adult rat kidneys. (a, b) The paraffin-embedded sections were immunostained with a polyclonal antiserum directed against an epitope at the amino end of (b) leprecan (Rb6480) or (a) the preimmune antiserum from the same animal. Leprecan-associated immunostaining is strong in mesangial cells, the mesangial matrix, and in Bowman's capsule and weak in tubular basement membranes. Intracellular staining is evident in the tubular epithelial cells and in podocytes (arrows) associated with the glomerulus. The inset in (b) shows the intracellular staining for leprecan in an enlarged image of the podocyte that was demarcated by the arrows in (b). The preimmune antiserum for Rb6480 is essentially nonreactive (a). The micrographs in (c–e) are high magnification (final magnification × 400) of frozen sections of adult rat kidney probed with the antisense RNA probe for leprecan. (c–e) Images of the same field of view taken with (c) phase-contrast optics, (d) darkfield optics, and (e) a merged image (enlarged) in which the darkfield image has been pseudocolored red. The silver grains indicative of probe hybridization are evenly dispersed throughout the entire glomerulus. (f–h) Images of rat kidney that were probed with the sense RNA probe to demonstrate the extent of nonspecific background binding of the probe on the tissue sections.
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

7. Figure 6
Rb6480 staining in developing kidney. The photomicrographs are of paraffin-embedded sections of day 1 postnatal rat kidney immunostained with Rb6480. Low-magnification micrographs of developing kidney show that leprecan was globally expressed throughout the entire renal parenchyma, but stronger immunofluorescence was found in (b) the renal capsule (arrows) and (a) maturing nephroi (arrows). (b) In mesenchymal aggregates (indicated by the dashed line), the earliest developmental structures of the nephron, leprecan-associated immunofluorescence was found in the cytoplasm of the cells composing the aggregates. Cells composing the ureteric bud (ub) also strongly expressed leprecan. (c) In capillary loop phase glomeruli (indicated by the dashed line), cells of the tail of the comma (arrows) showed strong, focal staining for leprecan as well as the cells destined to develop into the tubular epithelium. (vc=vascular cleft) (d) High magnification (× 400) of a capillary loop phase glomerulus and its associated tubular elements. The mesangial cells and matrix are strongly stained by Rb6480, as well as Bowman's capsule (arrow) and the extraglomerular mesangium (EM). Podocytes are weakly stained by this antibody and there does not appear to be any staining associated with the glomerular basement membrane. The inset in (d) was enlarged from the glomerulus shown in the full-view image. (Final magnifications: (a) × 200, (b–d) × 400.)
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

8. Figure 7
Leprecan expression in developing glomeruli. The photomicrographs were of 10-μm-thick frozen sections of day 1 postnatal rat kidney that were imaged with (a–c) phase-contrast optics, (d–f) darkfield optics, (g–i) epifluorescence illumination, (j–l) represent the merge of corresponding darkfield and phase-contrast images. The darkfield images represented as a pseudocolored red overlay over the phase-contrast image. (d–f) The tissue sections had been probed with antisense leprecan RNA probe and (g–i) counterstained with Hoechst stain to highlight the cellular organization of the corresponding structures. (a, d, g, j) Images of a mesenchymal aggregate and surrounding stromal cells. All cells in the aggregate and the surrounding cells are positive for leprecan expression. (b, e, h, k) Images of a comma-phase glomerulus, showing localized expression of leprecan in cells of the tail of the comma that will develop into the visceral and parietal epithelium (arrows). (c) Cells in the center of the comma show enhanced leprecan expression. (vc=vascular cleft) (c, f, i, l) Images of an early capillary loop stage glomerulus, showing high levels of leprecan expression in cells throughout the entire structure. Also note the high levels of expression in the surrounding stromal tissue. Final magnification × 400. Antisense controls for these images are shown in Figure S1.
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

9. Figure 8
Leprecan expression in hypertrophic glomeruli. The photomicrographs are paraffin-embedded sections of (a) normal adult rat kidney or (b) unilateral nephrectomized kidney immunostained with Rb6480. Leprecan staining is present in the mesangial matrix/mesangial cells and Bowman's capsule in glomeruli from both animals. Staining is also present in the tubular epithelium of both specimens. In the case of the glomerular hypertrophy seen in (b) the unilateral nephrectomized animal there is an apparent increase in the area of leprecan staining compared to the normal animal. Final magnification × 400.
Kidney International  , 82-91DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions