Expression of Novel Podocyte-Associated Proteins sult1b1 and ankrd25Xu X.a · Patrakka J.a, b · Sistani L.a · Uhlen M.c · Jalanko H.e · Betsholtz C.a, d · Tryggvason K.a
aDivision of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, bDepartment of Renal Medicine, Karolinska University Hospital, cDepartment of Biotechnology, Royal Institute of Technology, and dDepartment of Medicine, Karolinska Institute, Stockholm, Sweden; eHospital for Children and Adolescence, University of Helsinki, Helsinki, Finland Corresponding Author
Background/Aims: Podocytes have a unique function in the renal ultrafiltration that is achieved by expressing proteins that are highly specific to podocytes. In this study, we identified two novel podocyte-associated proteins. Methods: The expression of sult1b1 and ankrd25 in mouse tissues was studied by RT-PCR. The protein expression was studied by generating polyclonal antibodies that were used in Western blotting and immunohistochemistry. Results: By RT-PCR we detected sult1b1 expression only in glomerular, liver and brain tissues. By immunohistochemistry, sult1b1 was detected in the kidney exclusively in the Golgi apparatus of the podocyte. No expression outside the glomerulus was observed in the kidney. The ankrd25 transcript was detected in most mouse tissues analyzed by RT-PCR. In the kidney, however, immunohistochemistry showed that this protein was expressed only by podocyte, mesangial, and smooth muscle cells. In podocytes, ankrd25 was localized to foot processes. Conclusions: Identification of these two novel glomerulus-associated proteins opens up possibilities to investigate their role in the renal filter physiology and diseases. We speculate that sult1b1 may be involved in the sulfonylation of podocyte protein podocalyxin, whereas ankrd25 may contribute to controlling actin dynamics in podocyte foot processes.
Copyright © 2010 S. Karger AG, Basel
The renal glomerulus is responsible for the ultrafiltration of plasma in the kidney. The filtration barrier has three distinct layers: the endothelial layer, the glomerular basement membrane, and the outer layer composed of podocyte foot processes connected by slit diaphragms. The filter is injured in numerous diseases resulting in proteinuria and progressive renal disease. During the last decade, the podocyte has taken the central stage in research on the pathogenesis of proteinuria . This has been promoted by discoveries in hereditary proteinuria diseases as mutations in several podocyte proteins have been identified to be the cause of human proteinuric kidney diseases [2,3,4,5,6,7]. Genes mutated in hereditary proteinuria syndromes encode for proteins that are expressed highly by podocytes, and they often show very restricted expression pattern elsewhere in the body. This specific expression pattern reflects the unique function of the renal glomerulus and podocytes.
Previously, we have identified many glomerulus-enriched transcripts by analyzing the mouse glomerular transcriptome through large-scale sequencing and microarray profiling [8,9,10]. Importantly, many of these glomerulus-associated transcripts encode for proteins that are previously unidentified in the kidney. In the present study, two of these glomerulus-enriched transcripts, sult1b1 and ankrd25, were analyzed in more detail. sult1b1 shows a very restricted expression profile in mouse tissues and by immunohistochemistry we localized this protein to the Golgi apparatus of podocytes. ankrd25 showed wider expression in mouse tissues. In the kidney, however, this protein showed high expression in podocytes and mesangial cells. Identification of these two glomerulus-associated proteins gives a novel insight into the biology of the glomerulus, and opens up possibilities to study further their role in the glomerulus filtration barrier.
Materials and Methods
The expression of ankrd15 and sult1b1 transcript in the various mouse tissues was analyzed using RT-PCR. The gene-specific primers (sult1b1: left 5′-CTA CAC ATT CCC CAG CAG GT-3′, right 5′-CAG CAC TTG GGA GGT AGA GG-3′; ankrd25: 5′-CAG TGC TCC AGG AGG AAA AG-3′, right 5′-TGA TCT TCT TGG CTG TGT GC-3′) for PCR analysis were designed to amplify 595 bp (sult1b1) and 567 bp (ankrd25) products. As a template for PCR analysis, we used cDNA libraries generated from mRNAs isolated from various mouse tissues (Clontech, Palo Alto, Calif., USA) and from kidney fractions composed of glomerular tufts or the kidney fraction lacking glomeruli. PCR amplification reactions were carried out with Hot Start Taq DNA polymerase (Invitrogen, Carlsbad, Calf., USA) using standard procedures, and the amplified fragments were analyzed on 1.5% agarose gel. Mouse glomerular tufts were isolated from the rest of the kidney as described previously .
We raised polyclonal antibodies directed against the human sult1b1 and ankrd25 proteins by producing and purifying recombinant proteins with affinity tags. Residues 30–133 for sult1b1 and 568–685 for ankrd25 were expressed as recombinant proteins with a dual tag – a hexahistidine tag that enabled purification of the expressed antigen by immobilized metal ion affinity chromatography and an albumin-binding protein fragment of Streptococcus protein G with immunopotentiating capabilities. Antibodies were raised by immunization of New Zealand White rabbits, and obtained sera were used for purification of monospecific antibodies. Purification was performed using a two-step purification procedure including depletion of tag-specific antibodies and subsequent affinity purification.
In Western blotting, the protein expression between isolated glomerular tufts and the kidney extract lacking glomeruli was compared. Glomeruli were isolated from human cadaver kidneys unsuitable for transplantation because of vascular abnormalities (from the Fourth Department of Surgery of Helsinki, Finland). The kidney fractions were freeze-dried overnight and the dried samples were solubilized in 9 M urea, 0.5% (v/v) NP-40, 1.5% (w/v) CHAPS, 65 mM DTT and 35 mM NaOH. Then, 10 µg of total protein were separated on polyacrylamide gel and transferred into polyvinyl difluoride membrane. After 1 h incubation at room temperature in blocking solution (5% dry milk powder, 3% casein enzymatic hydrolysate, 1% BSA, 0.1% Tween-phoshate-buffered saline (PBS)), the membrane was incubated overnight with the primary antibody (diluted in blocking solution) at 4°C. The membrane was then washed in 0.1% Tween-PBS for 60 min at room temperature followed by incubation in HRP-conjugated goat anti-rabbit-IgG (Amersham Biosciences, Little Chalfont, UK). Peroxidase activity was detected with Western chemiluminescence reagent (Amersham Biosciences). As a positive loading control, we immunodetected β-actin using polyclonal anti-β-actin antibody (purchased from Abcam, Cambridge, UK).
Immunofluorescence stainings were performed on normal adult human kidneys. Normal kidneys were collected from cadaver donors whose kidneys were unsuitable for transplantation because of vascular abnormalities (from the Fourth Department of Surgery of Helsinki, Finland). The kidney samples were snap-frozen in liquid nitrogen and embedded in OCT (Sakura, Zoeterwoude, The Netherlands). For the stainings, cryosections (10 µm) were first washed briefly in PBS, post-fixed with cold acetone (–20°C) followed by three 5-min PBS washes and blocking with 5% normal goat serum. The primary antibodies were then diluted to 0.5% normal goat serum and incubated overnight at 4°C, followed by three 5-min PBS washes and 1 h incubation with secondary antibody. As a negative control, we incubated sections with a non-immune rabbit serum instead of primary antibodies. For double labeling experiments, the sections were prepared in the identical way, and the incubations were performed sequentially. Double labeling experiments were performed with a monoclonal anti-mouse CD31 antibody (Pharmingen Inc., San Diego, Calif., USA), a monoclonal anti-human nephrin antibody 50A9 , and a monoclonal anti-GM130 antibody (BD Transduction Laboratories, San Jose, Calif., USA). Fluorescence-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, Oreg., USA). The collection and usage of kidney material for these experiments was accepted by local ethical committees.
In our previous microarray analyses, we identified genes enriched in the glomerular transcriptome . sult1b1 and ankrd25 were among the genes enriched in the glomerulus fraction of the kidney. As neither of these proteins had been described previously in the glomerulus, we decided to study their expression in more detail.
The expression of sult1b1 and ankrd25 in various mouse tissues was studied by RT-PCR. The RT-PCR experiment amplified the sult1b1-specific product only from liver, brain, and kidney tissues (fig. 1). The signal was strongest in the liver. In the kidney, the signal for sult1b1 was detected only in the glomerular fraction, whereas no signal was observed in the kidney portion devoid of glomeruli. In the brain, we observed several PCR products. A signal for ankrd25, on the other hand, was detected in all the tissues studied (fig. 1). The signal for ankrd25 mRNA was strongest in liver and skeletal muscle tissues. In the kidney, a strong PCR product was detected in the glomerulus fraction, whereas only a weak signal was observed in the kidney fraction devoid of glomeruli. The control RT-PCR experiment using GADPH primers gave a strong signal in all samples included in the study (fig. 1).
|Fig. 1. Expression of sult1b1 and ankrd25 in various mouse tissues as detected by RT-PCR. A signal for the sult1b1 transcript is amplified from liver, brain, and kidney tissues. In the kidney fractions the signal is observed only in the glomerulus, whereas no fragment is detected in the fraction devoid of glomeruli. The size of the detected PCR product (∼600 bp) is in line with the expected fragment size (595 bp). The ankrd25 transcript, on the other hand, is detected in all the organs analyzed. The signal is strongest in liver and skeletal muscle tissues. In the kidney, the signal is much stronger in the glomerulus fraction than in the fraction lacking glomeruli. The amplified fragment size (∼550 bp) is in line with the predicted PCR product size (567 bp). Amplification of GADPH, used as a positive control, gives a strong signal in all the tissues analyzed.|
To study the expression of sult1b1 and ankrd25 proteins in more detail, we raised rabbit polyclonal antibodies directed against the corresponding human proteins. These antibodies were used in Western blotting and immunohistochemical experiments to identify cell types expressing these proteins in the kidney. In Western blotting of human kidney lysates, the polyclonal anti-sult1b1 antibody gave strong immunoreactivity in the glomerular lane (fig. 2). Proteins sized ∼120, ∼65, ∼55, and ∼35 kDa were detected in the glomerulus (fig. 2). The size of the sult1b1 protein is predicted to be ∼35 kDa [www. ensembl.org], which corresponds to the smallest protein recognized by our antibody. This glomerulus-specific expression pattern is in line with our previous microarray data  as well as with the RT-PCR data (see above). The polyclonal anti-ankrd25 antibody recognized a protein of ∼120 kDa in both kidney fractions (fig. 2). No signal or only a very weak signal was observed in the rest of the kidney fraction, which is in line with our previous microarray data . In Western blotting experiments, we used β-actin antibody as a loading control, which gave similar reactivity in both lanes (data not shown).
|Fig. 2. Western blotting for sult1b1 and ankrd25 in the glomerulus and in the kidney fraction lacking glomeruli. a Affinity-purified anti-sult1b1 antibody recognizes several proteins in the glomerular fraction. Proteins sized ∼120, ∼65, ∼55, and ∼35 kDa are observed in the glomerulus lane. No reactivity is observed in the kidney fraction lacking glomeruli. b Affinity-purified anti-ankrd25 antibody recognizes a protein sized ∼120 kDa in the glomerular fraction.|
In the immunofluorescence staining of human kidney sections the sult1b1 antibody gave intense immunoreactivity in the glomerular tufts, whereas no obvious positive signal was observed outside the glomeruli (fig. 3a). In the glomerular tufts, the staining was observed as a strong spotted immunoreactivity in the periphery of the glomerular loops (fig. 3b). A double staining experiment with nephrin revealed that immunoreactivity was localized outside the glomerular capillaries (fig. 3b–d), indicating that podocytes expressed this protein. Staining did not colocalize with nephrin (marker for foot processes) or with DAPI (staining for nuclei; fig. 3d). However, a double labeling experiment with Golgi marker GM130 showed nearly complete overlapping staining patterns (fig. 3e–g). Thus, sult1b1 is a novel specific component of the podocyte Golgi apparatus. Control immunostaining using non-immunized rabbit serum gave no immunoreactivity in the glomerulus (data not shown).
|Fig. 3. Expression of sult1b1 in normal adult human kidney as detected by immunofluorescence staining. a Strong immunoreactivity for sult1b1 is detected in the glomerular tufts and no obvious positive signal is seen in the rest of the kidney. b–d Double labeling experiment with sult1b1 and nephrin shows that the sult1b1-specific staining is located on the outer aspects of glomerular capillary tufts indicating that podocytes express sult1b1. The signal for sult1b1 (green) does not colocalize with the foot process marker nephrin (red) or with the nuclear staining (blue). e–g Double labeling with the Golgi protein GM130, however, shows nearly complete overlapping reactivity (yellow) with sult1b1.|
Immunoreactivity for ankrd25 was detected in the glomeruli as well as around blood vessels in mature human kidney sections (fig. 4a). Double staining with anti-CD31 antibody confirmed that the reactivity outside glomeruli was around capillaries (fig. 4b–d). In the glomerulus, the immunostaining for ankrd25 partially colocalized with that of nephrin (fig. 4e–g), indicating localization to foot processes of podocytes. However, the signal for ankrd25 was also detected in the mesangial cells. Thus, the ankrd25 protein is a new glomerulus-associated protein that is expressed in the kidney by glomerular podocytes and mesangial cells, as well as by probably smooth muscle cells of blood vessels.
|Fig. 4. Expression of ankrd25 in normal adult human kidney as detected by immunofluorescence staining. a Strong immunoreactivity for ankrd25 was detected in glomerular tufts. In addition, the signal was also observed occasionally outside glomeruli (arrows). b–d Double labeling with endothelial marker CD31 shows that ankrd25 is detected not only around glomerular capillary endothelium, but also surrounding blood vessels outside glomeruli (arrows). Staining for ankrd25 surrounds the signal of CD31 but does not overlap with it. e–g Double staining with the podocyte foot process marker nephrin shows that the signal for ankrd25 is located in foot processes. Staining for ankrd25 is also observed in mesangial cells (arrows).|
In this study, we describe two new glomerular proteins – sult1b1 and ankrd25. Both proteins are highly expressed by glomerular cells suggesting that they have a role in the glomerular filtration barrier. sult1b1 is a member of the sulfotransferase protein family. These enzymes transfer sulfate groups to specific molecules. The sult1b1 protein was localized to the cytoplasm of podocyte cell bodies and double staining with Golgi marker GM130 revealed it to be a novel component of the podocyte Golgi apparatus. Thus, sult1b1 seems to be involved in the post-translational modification of podocyte proteins. Previously, the tissue distribution of sult1b1 has been reported by several groups [13,14,15,16,17,18]. The results of these studies vary somewhat but most of the studies have found high expression in the liver and intestine and very weak expression or no expression elsewhere in the body. We found that sult1b1 was expressed only by the liver, the brain, and glomerular podocytes. This very highly restricted expression pattern suggests that this enzyme has a very dedicated role in podocytes. The reason why sult1b1 is so highly expressed by podocytes may be that it is needed for the sulfonylation of one or several podocyte-associated proteins. One obvious candidate is podocalyxin. Although not completely specific to podocytes it is nevertheless very abundantly expressed by podocytes and highly sulfated. To reveal whether sult1b1 is indeed involved in the sulfonylation of podocalyxin, a mouse line lacking sult1b1 should be created. Our laboratory is in process of knocking out sult1b1 in mouse [Patrakka et al., unpubl. data].
ankrd25, also known as Kank2, is a member of a family of proteins (Kank1–4) containing C-terminally ankyrin-repeat domains . Previously, these proteins have been shown to be involved in the regulation of actin cytoskeleton assembly. By RT-PCR, Zhu et al.  detected ankrd25 expression in all human tissues studied. This is in line with our RT-PCR results. In the kidney, on the other hand, the immunoreactivity for ankrd25 was detected only in podocyte, mesangial, and vascular smooth muscle cells. In podocytes, staining for ankrd25 was localized to foot processes. As the assembly of actin cytoskeleton in foot processes needs to be tightly regulated, ankrd25 is an obvious candidate to be a component of the molecular machinery controlling actin dynamics in podocyte foot processes.
To conclude, here we describe the expression of two novel glomerular proteins. Identification of these two proteins opens up possibilities to analyze the role of these molecules in the physiology and diseases of the glomerular filtration barrier.
Research is supported by The Swedish Medical Research Council (J.P. and K.T.), The Swedish Society of Medicine (J.P.), and the Alice and Knut Wallenberg Foundation (K.T.).
Karl Tryggvason, MD, PhD
Division of Matrix Biology, Department of Medical Biochemistry and Biophysics
SE–171 77 Stockholm (Sweden)
Received: July 27, 2009
Accepted: March 24, 2010
Published online: August 18, 2010
Number of Print Pages : 8
Number of Figures : 4, Number of Tables : 0, Number of References : 21
Nephron Experimental Nephrology
Vol. 117, No. 2, Year 2011 (Cover Date: January 2011)
Journal Editor: Hughes J. (Edinburgh)
ISSN: 1660-2129 (Print), eISSN: 1660-2129 (Online)
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