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NDT Advance Access published online on October 16, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn570
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Rab 23 is expressed in the glomerulus and plays a role in the development of focal segmental glomerulosclerosis

Tzu-Hao Huang1, Hao-Ai Shui1, Shuk-Man Ka2, Bor-Luen Tang3, Tai-Kuang Chao2, Jin-Shuen Chen4, Yuh-Feng Lin4 and Ann Chen2

1 Graduate Institute of Medical Sciences 2 Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597 4 Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC

Correspondence and offprint requests to: Ann Chen, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC. Tel: +886-2-8792-7008; Fax: +886-2-8792-7009; E-mail: doc31717{at}ndmctsgh.edu.tw and Dr. Hao-Ai Shui, Graduate Institute of Medical Sciences, National Defense Medical Center, 161, Min-Chuan East Road, 6th Section, Taipei, Taiwan, ROC. E-mail: haoai{at}ndmctsgh.edu.tw



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Conflict of interest statement
 References
 
Background. Rab23, a member of the Rab family of small GTPase, has a function in antagonizing sonic hedgehog signal transduction. Both Rab-family and hedgehog-related proteins are involved in sclerosis and fibrosis in certain pathological states, but their roles in focal segmental glomerulosclerosis (FSGS) remain unclear.

Methods. The FSGS model was established in Balb/c mice by a single injection of adriamycin. Serum, urine and mice kidneys were collected on Days 0, 7, 15 and 20. Western blot analysis was performed to detect the levels of Rab23 in the samples. Immunohistochemistry was used to examine the expressional profiles of Rab23 in kidneys. The expressions of transcripts of Rab23, extracellular matrix (ECM) proteins, and various hedgehog signalling pathway genes in kidneys or mesangial cells were evaluated by real-time RT-PCR. The effect of Rab23 on ECM protein expressions was evaluated by the knockdown or overexpression of Rab23 in mesangial cells.

Results. Our results show that elevations of Rab23 were observed in the urine, but not in the serum, of the FSGS mice. Rab23 and hedgehog signalling pathway genes were constitutively expressed in normal kidneys and were significantly up-regulated in the kidneys of FSGS mice. The basal expression of Rab23 was identified in glomeruli, and mesangial cells displayed obvious elevation of Rab23 in the FSGS state. The knockdown or overexpression of Rab23 affected the collagen expression in cultured mesangial cells.

Conclusions. An autocrine loop of hedgehog signalling could be activated in mesangial cells in the FSGS state, and Rab23 may be elevated to suppress hedgehog signalling and/or influence collagen synthesis. Importantly, Rab23 could serve as a biomarker that indicates the severity of FSGS.

Keywords: biomarker; focal segmental glomerulosclerosis; hedgehog signal transduction; mesangial cells; Rab23



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Conflict of interest statement
 References
 
Focal segmental glomerulosclerosis (FSGS) is a form of chronic nephropathy characterized by scattered sclerosis of glomeruli in which only a segment of the capillary is affected [1,2]. The molecules involved in FSGS pathogenesis are being gradually uncovered. These include proteins that cause oxidative stress, podocyte damage, hyperfiltration and sclerosis, which lead to marked proteinuria and scattered glomerular sclerosis seen in the disease [2,3]. Occasionally, some proteins that were originally not considered to play any role in FSGS were demonstrated to be involved in the pathogenesis of the disease. For example, osteopontin, a matrixcellular protein with a chemoattractive property for monocytes, has been shown to play a role in epithelial hyperplasia lesion in FSGS [4]. Recently, in our proteomic profiling of FSGS, we have identified Rab23 in the urine of an FSGS mouse model (unpublished data). However, the changes in expressional profiles of Rab23 and its associated genes in FSGS remain unknown.

Rab23 is a member of the large Rab family of small GTPase, which has about 60 members that play varying roles in the intracellular traffic of proteins and membrane vesicles [5–7]. The expression and function of Rab23 in kidneys are unclear. In contrast to Rab23, many other members of the Rab family have been identified in the kidney. Rabs 4, 5, 7, 11, 13, 18, 20, 25, 34 and 38 are located in tubular cells [8–11], while Rab 3A is located in podocytes [12]. Rab11 and Rab20 co-localize with vacuolar H+-ATPase in tubular and intercalated cells and play roles in regulating proton secretion by transferring the H+-ATPase to the plasma membrane [9]. Rab11 also mediates the vasopressin-induced transport of aquaporin-2 water channels to the cell membrane, and therefore plays a role in water re-absorption in collecting duct cells [8]. In particular, Rab3A and Rab38 are two known factors associated with sclerosis and proteinuria in FSGS. Rab3A is expressed in podocytes and changes in its levels correlate with FSGS in mice and proteinuria in patients [12]. A Rab38 mutation causes proteinuria in fawn-hooded hypertensive rats and is associated with a familial form of FSGS in human [13,14].

Unlike the general trafficking function of other Rab family members, Rab23 plays a unique role in regulating the signal transduction of sonic hedgehog, a secreted morphogen that controls the development of multiple organs during embryogenesis [5,15–19]. Hedgehog signalling is initiated by binding of the ligand sonic hedgehog (Shh), desert hedgehog (Dhh) or Indian hedgehog (Ihh) to the receptor patched (PTCH, including PTCH1 and PTCH2). This disinhibits the activity of smoothened (SMO), which then activates glioma-associated oncogene homologue (Gli) transcription factors (including Gli1, Gli2 and Gli3) to turn on the expressions of their target genes [20]. Rab23 appears to antagonize hedgehog signalling, and mutation of Rab23 gene causes a severe defect in the developing neural tube in mice [15,18] and abnormalities of multiple organs in patients of two congenital diseases—Carpenter and Gorlin syndromes [21,22]. In addition, since hedgehog genes, such as shh, possess oncogenic properties [20], Rab23 has been implicated to be involved in carcinogenesis. Indeed, the abnormal expression of Rab23 has been observed in hepatocellular as well as thyroid carcinoma [23,24]; the overexpression of Rab23 has been reported in atrophic gastritis, a pre-stage of gastric cancer [25]. However, although hedgehog-related genes also play roles in fibrosis and/or sclerosis in brain, lung and liver [26–28], the expressional profiles of Rab23 under both of the pathological conditions are still unclear. Similarly, although hedgehog signal transduction participates in kidney development [29,30], the expression of Rab23 in the adult kidney is unknown, especially in the kidney that shows glomerular sclerosis.

Although Rab23 was considered to play a major role in embryogenesis, Rab23 is also found to be highly expressed in adult rodent brain and expressed in low levels in multiple tissues [5,19]. Rab23 may, therefore, have a postnatal physiological function beyond its role in embryonic development. We demonstrated in the present study that Rab23 is elevated in the urine of FSGS mice. Using specific antibodies [19] against Rab23, the expression levels of the protein in normal and FSGS-associated mice were investigated, in conjunction with other hedgehog signalling pathway genes. Our results suggest that Rab23 could play a role in the development of FSGS and could be a marker of FSGS pathology.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Conflict of interest statement
 References
 
FSGS animal model and sample collections
The experiments were performed using 8-week-old BALB/c mice with the approval of the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan, and were consistent with the NIH Guide for the Care and Use of Laboratory Animals. The mice were injected intravenously with a single dose of adriamycin (AD) (0.1 mg/10 g body weight). Days 0, 7, 15 and 20, which cover different severities of FSGS in the mouse model [3,4,31,32], were chosen for collection of blood and urine samples and for sacrifice of the animals for harvesting of kidneys. Blood was collected through the retro-orbital venous plexus [4,31]. To prevent the degradation of urinary protein and contamination by cellular proteins due to cell lysis, long-period collection of urine was avoided; instead, spot urine was collected at the same time of day on Days 0, 7, 15 and 20. To avoid contamination by food, water and faeces that can happen in using metabolic cages, mouse urine was obtained by gentle bladder massage and collected on a Parafilm film [3]. Serum creatinine, blood urea nitrogen (BUN) and urine protein concentration were then measured.

Measurement of serum creatinine, BUN and urine protein concentration
Samples of serum or urine were cleared by centrifugation and stored in liquid nitrogen until analysis. A modified Bradford method that can minimize interference from urea in urine was used to measure urine protein concentration [33]; Serum creatinine was measured using a picric acid method and BUN was measured using a urease assay as previously described [34].

Histopathology and immunohistochemistry
Renal tissues were fixed in 10% buffered formalin and embedded in paraffin for routine histopathology. Sections of the formalin-fixed renal tissues were immersed in xylene to remove paraffin, rehydrated in graded ethanol, stained with haematoxylin and eosin, and examined under a light microscope (Olympus, Tokyo, Japan). Semi-quantitative analysis of sclerosis was performed with an optical microscope according to our previously published criterion [3,4,31,32].

For single immunostaining of Rab23, OCT-embedded tissues were cut into 5-µm-thick sections. After immersion in acetone for 5 min and air drying, the sections were then incubated with Tris-buffered saline, pH 7.4, containing 0.05% Tween 20 (TBST) and 2% bovine serum albumin (BSA) at room temperature for 30 min for the blocking step. TBST was used for all of the following washing steps. The sections were incubated with a rabbit anti-mouse Rab23 antibody (1:200 dilution) at 4°C overnight, washed with TBST, and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:200) (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h. Bound antibodies were visualized with 3,3'-diaminobenzidine (DAKO, Carpinteria, CA, USA), and the slides were lightly counterstained with haematoxylin. Semi-quantitative analysis of immunohistochemistry (IHC) sections was performed by optical microscope as previously described [4,31,35]. Briefly, fifty glomeruli were examined on each slide, and the Rab23 intensity of glomeruli, podocytes or mesangial cells was assigned a score from 0 to 3. The total intensity score for counted glomeruli, podocytes or mesangial cells was calculated according to the following equation for each specimen: total intensity score = (% intensity negative x 0) + (% intensity trace intensity x 0.5) + (% 1 + intensity x 1) + (% 2 + intensity x 2) + (% 3 + intensity x 3). The values ranged from 0 to a maximum of 300.

For double immunostaining for Rab23/nephrin, the procedure is similar to single immunostaining except that primary antibodies used were rabbit anti-Rab23 (1:150)/goat anti-nephrin (1:20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (1:500) (Jackson ImmunoResearch, West Grove, PA, USA)/Alexa Fluor 594-conjugated donkey anti-goat IgG (1:500) (Invitrogen, CA, USA). Semi-quantification of total Rab23 fluorescence intensity was performed using the same equation described above [4,31,35]. For evaluation of the Rab23 expression in podocytes, the area of overlap of the two fluorophores was estimated and presented as a percentage of the total area of glomeruli.

Proteomics analysis
The urine collected by bladder massage was desalted and concentrated by ultrafiltration as previously described [3]. Urinary proteins (400 µg) from the control or FSGS mice were loaded onto an IPG strip (Immobiline DryStrip 3-10, GE Healthcare, NJ, USA) for simultaneous rehydration. Isoelectric focusing and SDS–PAGE were performed according to the protocols previously described [3,36]. The spots were digested with trypsin and subjected to direct mass spectrometry protein identification. The mass spectrometer used for protein analysis was a Bruker Biflex IV MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). Peptide mass fingerprint (PMF) was obtained by averaging signals generated from at least 500 laser shots. The mass spectra were processed using FlexanalysisTM and BiotoolsTM software (Bruker Daltonics, Bremen, Germany) and the data subjected to a search against the UniProt database (http://www.pir.uniprot.org) by the MS-Fit database searching engine (http://prospector. ucsf.edu/ucsfhtml4.0/msfit.htm). For each PMF search, the mass tolerance was set to 100 ppm and one missed tryptic cleavage was allowed.

Cell culture and adriamycin treatment
Mouse mesangial cells CRL-1927 were obtained from the American Type Culture Collection (Rockville, MD, USA) and routinely maintained in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 5% fetal bovine serum and 14 mM HEPES as previously described [37]. Cultured mesangial cells (6 x 105 cells/plate) were seeded onto 10-cm diameter plastic dishes before the experiments. After cell attachment, mesangial cells were treated with adriamycin (0–0.2 µg/ml) for 0, 12 or 24 h according to our previous protocol [37]. Then, the cells were harvested for immunocytochemistry, real-time RT-PCR and western blot analysis.

Immunocytochemistry
Harvested CRL-1927 cells were attached onto slide glasses by Cytospin (Cytospin 3, Shandon), and then fixed in 2% paraformaldehyde for 15 min. After blocking with 2% BSA in Tris-buffered saline, cells were treated with an anti-Rab23 antibody (1:200) for 1 h and then incubated with HRP-conjugated goat anti-rabbit IgG (1:200) (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. Bound antibodies were visualized with 3,3'-diaminobenzidine (DAKO).

Western blot analysis
Urine samples (15 µl for each), serum samples (15 µl for each) or kidney proteins (50 µg) from adriamycin-treated mice (Days 0, 7, 15 and 20) or proteins (50 µg) from cultured mesangial cells were subjected to 10% SDS–PAGE. Proteins were then transferred to a Hybond PVDF membrane (GE Healthcare). The membrane was blocked in a 20 ml blocking buffer (Tris-buffered saline, pH 8.0, containing 0.05% Tween 20 and 5% skimmed milk) at room temperature for 1 h, incubated with a rabbit anti-Rab23 antibody (1:1000) at room temperature for 1 h, and then washed with TBST three times, followed by incubation with the goat anti-rabbit secondary antibody for 1 h at room temperature. The proteins on the membrane were visualized with an ECL detection kit, and protein intensity was quantified by a densitometer. For tissue or cell samples, Rab23 intensity was normalized by the intensity of the internal control GAPDH.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR)
Kidney tissues or cultured mesangial cells were subjected to total RNA extraction using Trizol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer's instruction. For first-strand cDNA synthesis, 3 µg of total RNA was used in a single-round RT reaction, together with 200 ng of random hexamer primer, 0.5 mM dNTPs, 1x first-strand buffer, 5 mM DTT, 10 units of superscript III reverse transcriptase (Invitrogen) in a total volume of 20 µl. PCR was performed using 1 µl of the RT reaction mixture, 0.1 µM of specific primers (Table 1), 1x PCR buffer, 50 µM dNTPs and one unit of KlenTag DNA polymerase in a total volume of 25 µl.


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  		Table 1 Primers and gene information for amplification in PCR

 
Real-time PCR was carried out by the iCycler real-time PCR instrument (Bio-Rad, Hercules, CA, USA), using 10 µl of the RT reaction mixture, 0.07 µM gene-specific primers and 12.5 µl ABsoluteTM QPCR SYBR Green Fluorescein Mix (AB gene, Epsom, UK), in a total volume of 25 µl. The gene-specific primers are listed in Table 1. The thermal cycler conditions were as follows: real-time PCR was initiated by enzyme activation at 95°C for 15 min, followed by 50 cycles of 94°C for 15 s, 55°C for 35 s and 72°C for 60 s and a final extension at 72°C for 10 min. Levels of test genes were normalized to GAPDH mRNA levels by a comparative threshold cycle (2–[delta] Ct) method that converts differences of the cycle numbers to Test gene/GAPDH ratios. Fold changes of mRNA were calculated by a (2–[delta] [delta] Ct) method.

Overexpression and knockdown of Rab23
CRL-1927 mesangial cells were seeded onto 6-cm diameter plastic dishes. The overexpression of Rab23 was performed as previously reported [19]. Briefly, the pCIneo construct carrying full-length Rab23 was transfected into cells using Lipofectamine 2000TM (Invitrogen), and cells were harvested 48 h after the transfection. Knockdown of Rab23 was performed by transfection with a double-stranded small interfering RNA (siRNA) that was made by hybridization of a sense (5'-GAUGCAUGA- AUCAUCCAGCAGAUCGAU-3') RNA oligo and an anti-sense (5'-CGAUCUGCUGGAUGAUUCAAUGCATC-3') RNA oligo.

Statistics
All data are presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA by using the SPSS statistics analysis software program. A Newman–Keuls test was carried out when the ANOVA comparisons gave a significant result. Differences were considered significant at P < 0.05.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Conflict of interest statement
 References
 
Pathophysiological manifestations exhibited by mice in the FSGS model
Consistent with our previous reports [3,4,31,32], severe proteinuria and deteriorated renal clearance function were exhibited by the adriamycin-treated mice. Compared to basal levels (0.59 ± 0.082 mg/ml), the urine protein levels were significantly elevated on Day 15 (16.84 ± 3.13 mg/ ml) (F = 13.81, P < 0.01) (Figure 1A). Serum creatinine and BUN, two general parameters for evaluating renal function, were also higher in adriamycin-treated mice compared to control mice. Serum creatinine showed significant elevations on Day 15 (2.02 ± 0.17 mg/dl versus the basal levels of 0.48 ± 0.03 mg/dl; F = 25.17, P < 0.01) (Figure 1B), and BUN also showed significant increases on Day 15 (71.42 ± 8.09 mg/dl versus the basal levels of 21.85 ± 1.99 mg/dl; F = 38.79, P < 0.01) (Figure 1C).


Figure 1
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  		Fig. 1 Pathophysiological manifestations of FSGS mice. Three typical parameters for evaluation of renal function, (A) urine protein, (B) serum creatinine and (C) BUN, were measured at the three time points after adriamycin treatment. n = 6 for each day, **P < 0.01 compared to Day 0. The dashed line shows the mean values of normal mice.

 
Glomerular sclerosis in the FSGS model
Histopathological examination was performed on kidney sections obtained on Days 0, 7, 15 and 20. As shown in Figure 2A, expansion of the extracellular matrix (ECM) and deposition of hyaline mass in the glomeruli were obvious in FSGS mice in comparison to the control mice. Sclerosis scores were significantly higher in FSGS mice than in control mice on Day 7 (13.17 ± 2.06 compared to basal levels of 0.0 ± 0.0; F = 74.48, P < 0.01) and continued to elevate on Day 15 (63.83 ± 6.20 compared to the basal levels; F = 74.48, P < 0.01) and Day 20 (76.83 ± 5.75 compared to the basal levels; F = 74.48, P < 0.01) (Figure 2B). The extent of sclerosis correlated well with the severity of the clinical manifestations as shown in Figure 1.


Figure 2
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  		Fig. 2 Glomerular histopathology in FSGS mice. (A) Kidney tissue histopathological sections on Days 0, 7, 15 or 20, showing a gradual increase of sclerosis in the glomeruli (haematoxylin and eosin staining). Original magnification, x400. (B) A semi-quantitative plot showing the sclerosis scores for the glomeruli in the tissue sections. The sclerosis scores were calculated as described in the Subjects and methods section. n = 6 for each day, *P < 0.05 or **P < 0.01 compared to Day 0.

 
Identification of Rab23 in urine from FSGS mice by proteomic analysis
We have previously reported serial changes of urinary proteome in FSGS mice [3] based on more abundant proteins. However, after the concentration of the urine proteins, we have now identified a new spot corresponding to Rab23 in 2D gels of urine from FSGS mice (white arrow in Figure 3A, right panel) that was absent (undetectable) in gels of control mice (left panel). The peptide mass fingerprint of the identified protein matches 10 peptides that cover 46.4% Rab23 amino acid sequence (Figure 3B). The expression of Rab23 during FSGS has never been identified by our or other labs’ proteomics work. In contrast, most of other spots shown in Figure 3 have been published in our recent paper [3].


Figure 3
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  		Fig. 3 Detection of Rab23 in FSGS mouse urine by proteomic analysis. (A) Magnified image of the areas containing Rab23 in the 2D gels of urine of control and FSGS mice. The position of Rab23 is indicated by a white arrow. The molecular mass and pI ranges are indicated, respectively, at the right and at the top of the images. (B) Peptide mass fingerprint of the identified Rab23 protein. The mass spectrum and identified Rab23 amino acid sequence are shown. The sequences that match the peptide masses derived from the digested protein are indicated (U: unmodified, O: oxidized methionine, C: alkylated cysteine, P: phosphorylated amino acids). There are 10 matched peptides, and the sequence coverage of Rab23 is 46.4%.

 
Serial increase of Rab23 levels in mouse urine during FSGS
In order to confirm the identification of Rab23, we used the specific anti-mouse Rab23 antibody [19] to perform western blot analysis of the concentrated urine proteins. As shown in Figure 4, urine Rab23 protein can be detected by western blot analysis in FSGS mice on Days 7, 15 and 20, but undetectable on Day 0 when FSGS was absent (F = 26.18, P < 0.01). The Rab23 levels increased with the progression and severity of FSGS in mice (Figure 4 versus Figure 2; Figure 4 versus Figure 1). In addition, blood is unlikely to be the source of urine Rab23 protein as Rab23 was not detectable in mouse serum at any stage of FSGS (Figure 4A).


Figure 4
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  		Fig. 4 Western blot analysis of Rab23 in urine and serum of FSGS mice. (A) Representative blots showing Rab23 bands in mouse urine (the top blot) and serum (the bottom blot) at Days 0, 7, 15 and 20. (B) Statistical data based on densitometric quantification for Rab23 concentration. n = 6, **P < 0.01 between the two indicated groups.

 
Increases of Rab23 expression in mouse kidneys during FSGS
In order to delineate the source of urinary Rab23, we checked both mRNA and protein expressions levels of Rab23 in kidneys of the FSGS mice. As shown in Figure 5A, renal Rab23 mRNA levels on Day 7 (0.12 ± 0.019), Day 15 (0.18 ± 0.028) and Day 20 (0.27 ± 0.059) were significantly higher than those on Day 0 (0.013 ± 0.0025) (F = 9.85, P < 0.01), again correlating with the increase in severity of FSGS. Similarly, renal Rab23 protein levels on Day 7 (0.61 ± 0.078), Day 15 (1.02 ± 0.11) and Day 20 (0.93 ± 0.090) were also significantly higher than those on Day 0 (0.24 ± 0.028) (F = 19.18, P < 0.01), indicating that kidney tissues could be a major source of urinary Rab23 protein in FSGS mice.


Figure 5
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  		Fig. 5 Real-time RT-PCR and western blot analyses of Rab23 in kidneys at different FSGS stages. (A) Statistical data of real-time PCR showing the time course of change for Rab23/GAPDH mRNA ratios after adriamycin treatment. (B) Western blots showing the changes in Rab23/GAPDH protein ratios. The upper panels show representative gel images, and the lower panels display the statistical data based on densitometric quantification showing the Rab23/GAPDH ratios. n = 6, *P < 0.05 or **P < 0.01 compared to Day 0.

 
Localizing Rab23 expression in mouse kidneys
In order to determine the tissue/cell location of Rab23 expression in kidneys, we performed single and double immunostaining to respectively evaluate Rab23 expression based on counting colour and fluorescence intensity scores.

Figure 6A shows the semi-quantitative data of single immunohistochemical stain. Total intensity scores of Rab23 in glomeruli were elevated on Days 7, 15 and 20 (139.50 ± 19.93, 202.33 ± 18.20 and 208.50 ± 23.91, respectively), significantly higher than those on Day 0 (61.83 ± 7.86) (F = 14.12, P < 0.01). Similarly, the expression levels of mesangial Rab23 in FSGS mice on Days 7, 15 and 20 (145.33 ± 17.83, 189.33 ± 21.63, and 181.83 ± 25.07, respectively) were significantly higher than those of control mice on Day 0 (67.33 ± 4.72) (F = 8.65, P < 0.01) (Figure 6A), correlating well with the increases of Rab23 transcripts (real-time RT-PCR) and protein (western blot analysis) shown above (Figure 5). The expression of Rab23 was also noticed in the podocytes (black arrows in the tissue sections, Figure 6A), but these did not show a significant elevation in FSGS mice on Day 7, 15 or 20 in comparison to the control mice (39.83 ± 3.95) (F = 1.53, P > 0.05).


Figure 6
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  		Fig. 6 Expression of Rab23 in kidney tissues at different FSGS stages. (A) Representative IHC-stained sections are displayed in the upper panels, showing the basal expression of Rab23 on Day 0 in both podocytes (black arrows) and mesangial cells (white arrow heads), and elevated expressions of Rab23 in the mesangium on Days 7, 15 and 20. The lower panels show the scores of colorimetric intensity of Rab23 in glomeruli (left), as well as the scores of podocytes and mesangial cells (right). n = 6, **P < 0.01 compared to Day 0. (B) Double immnuofluorescent staining of Rab23/nephrin at different FSGS stages. The upper panels show the representative single-fluorescence images of nephrin and Rab23, and the merged images. The lower panels show the scores of total fluorescent intensity of Rab23 in glomeruli (left), and the area of overlapping fluorescence (right).

 
Figure 6B shows the semi-quantitative data of double immunostaining of Rab23/nephrin. Consistent with Figure 6A, counting of the fluorescence intensity of total glomerular Rab23 showed that total Rab23 expression elevated along the course of FSGS disease progression (F = 6.62, P < 0.05). However, the major areas of Rab23 elevation do not coincide with nephrin staining. Indeed, based on calculating the overlapping fluorescent areas of Rab23/nephrin, a mild but not significant (F = 0.514, P > 0.05) elevation of Rab23 was observed in the nephrin-positive area.

Constitutive expression of Rab23 and hedgehog signalling pathway genes in mesangial cells
Since Rab23 elevation can only be observed in mesangial cells in FSGS mice, we wondered whether it is merely a drug response to adriamycin stimulation.

In order to confirm the Rab23 expression in glomeruli mesangial cells, we analysed Rab23 expression in a mesangial cell line. In addition, since Rab23 is known to play a role in regulating hedgehog signal transduction, we, therefore, also checked the expressions of hedgehog signalling pathway genes in the mesangial cell line. As shown in Figure 7, constitutive expression of Rab23 can be observed in the mouse mesangial cell line CRL-1927 (immunocytochemistry in Figure 7A, 0 h bar in Figure 7B, and 0 h lane in Figure 7C) and basal constitutive expression of Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2 and Gli3 can also be detected in these cells (0 hr bars in Figure 7D).


Figure 7
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  		Fig. 7 Expressions of Rab23 and hedgehog signalling pathway genes in cultured mesangial cells. (A) Immunocytochemical staining of Rab23 in cultured mesangial cells. The cells stained with the second antibody are only shown as the negative control. (B) Statistical data of real-time PCR showing the insignificant change of Rab23 expression after adriamycin (0.2 µg/ml) stimulation. (C) Western blot analysis showing insignificant changes in Rab23 expression after adriamycin (0.2 µg/ml) treatment. Typical gels representing Rab23 and GAPDH proteins are shown in the upper panels, and statistical data based on densitometric quantification are shown in the lower panels. P > 0.05 compared to 0 h. (D) Statistical data of real-time PCR showing insignificant changes in the expression of hedgehog signalling pathway genes (Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3) after adriamycin (0.2 µg/ml) treatment.

 
As shown in Figure 7B and C, the expressions of both mRNA and protein of Rab23 did not change significantly after adriamycin (0–0.2 µg/ml) treatment for 24 h (F = 1.315 and 0.029, respectively, P > 0.05). We can, therefore, rule out an immediate and direct drug-elicited effect by adriamycin on Rab23 expression in the cells. Similarly, in Figure 7D, the expressions of hedgehog signalling pathway genes (Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2 and Gli3) were not significantly altered by adriamycin (0–0.2 µg/ml) treatment of the mesangial cell line (F = 0.011, 1.54, 1.79. 1.43, 0.32, 0.97, 0.58, 2.30, and 0.22, respectively, P > 0.05).

Expressional profiles of hedgehog signalling pathway genes in kidneys of FSGS mice
The expression of Rab23 and hedgehog signalling-related genes in adult kidneys has never been studied before. We, therefore, profiled the expression of these hedgehog signalling-related genes in the kidneys of both control and FSGS mice. As shown in Figure 8, the expressions of all genes of the hedgehog signalling pathway can be detected in kidneys of the normal control mice on Day 0, indicating that these genes are constitutively expressed in normal adult mice kidneys. This finding is interesting as the developmentally regulated hedgehog signalling genes were not expected to be expressed in high levels in adult kidneys.


Figure 8
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  		Fig. 8 Real-time RT-PCR analysis of hedgehog signalling pathway genes in kidneys from different FSGS stages. Statistical data of real-time PCR showing the expression of Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3 and GAPDH on the indicated days. n = 6, *P < 0.05 and **P < 0.01 compared to Day 0.

 
Upon induction of FSGS, the expressions of Ihh, SMO and Gli3 were significantly enhanced by Day 15 and Day 20 (F = 7.96, 5.90 and 8.21, respectively, P < 0.01), while the expressions of Shh, Dhh, Gli1, Gli2, PTCH1 and PTCH2 remained unchanged (F = 1.18, 2.66, 0.17, 2.10, 2.59 and 2.33, respectively, P > 0.05) (Figure 8). The changes of the expression of the hedgehog signalling pathway genes indicated that hedgehog signalling could be activated in the FSGS state compared to the control state.

Effects of Rab23 on the expressions of ECM proteins in mesangial cells
To check whether Rab23 can affect ECM protein expression in mesangial cells, we manipulated Rab23 expression in the cells and then checked whether the expressions of fibronectin and collagens can be affected. As shown in Figure 9, the knockdown and overexpression of Rab23 by transfections with siRNA and with a Rab23 expression construct can respectively cause down-regulation and up-regulation of collagen type 1A2 (P < 0.05), whereas the expressions of fibronectin and other types of collagen were not affected (P > 0.05). This result indicates that Rab23 might contribute to FSGS by affecting the synthesis of certain ECM proteins.


Figure 9
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  		Fig. 9 Effects of Rab23 on transcript levels of ECM proteins. The fold changes of mRNA in comparison to the control level are shown at the vertical axis, while the names of ECM proteins are indicated at the horizontal axis.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Conflict of interest statement
 References
 
In our investigations based on a lead target revealed by proteomic analysis of urine, we found that Rab23 is expressed in podocytes and mesangial cells within glomeruli. Rab23 elevation allowed its detection in the urine, probably through release by damage of both cells. We infer that mesangial Rab23 could play a more crucial role in FSGS than podocyte Rab23, as the FSGS-associated change of Rab23 expression is observed only in the mesangial cells, rather than the podocytes for different severities (Days 0, 7, 15 and 20) of FSGS. However, we did not study the very late FSGS stages such as Week 6 shown by some literatures [38–40], as mice in that stage could have significant systemic problems, which change the urine protein profile and lead to misinterpretation of our data [32].

Mesangial cells are known to play significant roles in the sclerosis of FSGS [41]. These cells are located among glomerular capillaries, providing support and regulating glomerular blood flow by their contractile activity [37]. In addition, mesangial cells can perform phagocytosis [42,43], as well as secretion [31] of various ECM proteins (such as collagen, fibronectin and laminin) to affect glomerular ECM compositions. Accordingly, mesangial cells can control glomerular filtration and ECM protein deposition that contribute, respectively, to proteinuria and sclerosis under the pathological condition of FSGS. In the present study, we have made the novel finding that Rab23 is constitutively expressed in mesangial cells and is up-regulated in the FSGS state. The finding suggests that Rab23 might somehow influence the physiological activities of mesangial cells and play roles in the development of FSGS. Although the roles of Rab23 in mesangial cells and in FSGS are still unclear, they can be speculatively deduced from literature addressing Rab23 functions in other cell types.

Rab23, like other members in the Rab family, is likely to function in some aspect of the trafficking of intracellular proteins [5–7] and could be involved in endocytosis or exocytosis [19,44]. Although Rab23 is known to regulate sonic hedgehog signalling during development, the protein that is transported by Rab23 in the hedgehog signalling pathway remains unidentified [5]. Likewise, although Rab23 is expressed in high abundance in neurons of adult mouse brains, transport pathways and cargoes that are regulated by Rab23 in neurons are also unknown [5,19]. However, using HeLa cells’ phagocytosis of mutant S. Typhimurium as a model system, Rab23 has been demonstrated to be involved in the phagocytic membrane traffic [44]. Since Rab23 is constitutively expressed in mesangial cells (Figures 6A and 7A–C), Rab23 could control the traffic of cargo proteins whose physiological functions are essential to mesangial cells. However, since Rab23 expression is up-regulated in mesangial cells in the FSGS state (Figures 5 and 6), some of the proteins that are transported by Rab23 in mesangial cells might also play a role in the development, or disease progress, of FSGS. Mesangial cells have been reported to be capable of phagocytosing many extracellular proteins, including immune complexes, growth factors and ECM proteins [42,43]. Since phagocytosis of ECM proteins can influence the development of sclerosis in FSGS, future investigations should address whether Rab23 is involved in the phagocytosis function of mesangial cells.

Rab23 expression can apparently influence cell proliferation. Recently, some reports have shown that the overexpression of Rab23 can be observed in certain types of cancers [23–25]. Knockdown of Rab23 expression can actually inhibit the growth of hepatocarcinoma cells [23], implicating that Rab23 has a proliferation-promoting effect, at least in hepatomas. However, since Rab23 can block hedgehog signal transduction [5,20] that is growth promoting in certain cancer cells [45–47], Rab23 might also be anti-proliferative for certain cells. Excessive proliferation of mesangial cells is a common phenomenon observed in various types of nephropathy [48,49], which disrupts the delicate structure of glomeruli and affects their filtration function. Although proliferation of mesangial cells in FSGS is not as significant as other forms of acute nephropathies [48,49], it can occasionally be observed in the tissue sections of FSGS kidneys (data not shown). The proliferative state of mesangial cells might affect severity of sclerosis as proliferation can increase the number of the cells that are able to phagocytise and secrete ECM proteins, thus affecting the ECM deposition. Future studies would address whether Rab23 and hedgehog signal transduction can influence the proliferation rate of mesangial cells.

The hedgehog signal transduction is initiated by the binding of Shh, Dhh or Ihh to the receptor PTCH, a repressor of SMO. This results in the disinhibition of SMO, which in turn activates the Gli transcription factors and downstream target genes [20]. Secreted hedgehogs play both autocrine and paracrine roles in embryogenesis and carcinogenesis [45–47] and also play pathogenic roles in fibrosis and/or sclerosis in certain organs [26–28]. Since both the ligands (Shh, Dhh and Ihh) and receptors (PTCH1 and PTCH2) are expressed in mesangial cells (Figure 7D), an autocrine role of the mesangial-derived hedgehog is possible. In addition, since Rab23 is a negative regulator of hedgehog signalling [5,15–19], the constitutive expression of Rab23 in mesangial cells (Figure 7A–C) could play a negative counterbalancing role in modulating hedgehog signalling in these cells. Since Rab23 and some of the hedgehog signalling pathway genes were up-regulated in FSGS kidneys (Figures 5, 6 and 8), the predicted autocrine action of hedgehog in mesangial cells might be activated in the FSGS state and may play a role in the development and progression of the disease.

The exact mechanisms underlying the up-regulation of Rab23 and hedgehog signalling pathway genes in FSGS are still unclear. The elevated proteins could play either a protective or a pathogenic role in FSGS development. What is clear is that the up-regulation of the genes is not caused by adriamycin directly, as the phenomenon was not observed in adriamycin-treated mesangial cell line (Figure 7). Since Rab23 levels in both kidney tissue and urine correlated with the severity of FSGS (Figures 4–6 versus Figures 1 and 2), Rab23 is a potential biomarker of chronic nephropathies. Since the knockdown or overexpression of Rab23 can affect the collagen expression in cultured mesangial cells (Figure 9), Rab23 may contribute to FSGS by influencing ECM protein deposition.

In conclusion, using various approaches, we have demonstrated that Rab23 and hedgehog signalling pathway genes were constitutively expressed in podocytes and mesangial cells and were up-regulated in mesangial cells in the FSGS state. Based on the data, we suggested that an autocrine loop of the hedgehog signalling pathway could be activated in mesangial cells during FSGS in mice, and Rab23 could play a feedback role in suppressing the activated hedgehog signalling transduction. Importantly, Rab23's elevation in FSGS could serve as a biomarker for chronic nephropathies.



   Conflict of interest statement
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Conflict of interest statement
References
 
None declared.



   Acknowledgments
 
This work was supported by grants NSC 97-2320-B-016-010-MY3, NSC 96-2320-B-016-011-MY3 and NSC 96-2320-B-016-012-MY2 from the National Science Council, Taiwan, ROC. B.L.T. is supported by a grant (R-183-000-102-112) from the Ministry of Education, Singapore, and the academic staff research fund, National University of Singapore. T.H.H. is supported by a grant (EZRPF-360221-09511N023) from Chang-Gung Institute of Technology, Taiwan, ROC. H.A.S. is supported by a grant (DOH97-TD-I-111-TM022) from Department of Health, Executive Yuan, Taiwan, ROC.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Conflict of interest statement
 References
 

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Received for publication: 6. 1.08
Accepted in revised form: 18. 9.08


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