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Podocyte-specific Expression of C1q/TNF-α Related Protein 1 in Mice

C1q/TNF-α Related Protein 1 (CTRP1), an adiponectin paralog, is a novel member of the C1q-TNF Related Protein family. CTRP1 is expressed in the kidney, although its localization and role in the kidney have not been studied. This study examined CTRP1 expression and function in the kidney. CTRP1 immunohistochemistry and PAS staining of the kidneys of C57/BL6 and FGS/Nga mice were performed. In situ hybridization for podocin in the kidney was also performed. CTRP1 immunoreactivity was found only in the glomeruli of the kidney. The CTRP1-immunoreactive cells in the glomeruli were identified as podocytes. The number of CTPR1-immunoreactive cells and the intensity of CTRP1 immunoreactivity were lower in the glomeruli of FGS/Nga mice, which develop progressive proteinuria and focal glomerulosclerosis. CTRP1 is a novel protein expressed in podocytes of the mouse kidney and may have a role in podocytes related to glomerular filtration in the kidney.

C57/BL6 mice. A Multiple Tissue Northern (MTN) blot was purchased from Clontech (CA, USA). 32 P-labeled cDNA probes were prepared from the coding region of CTRP1 cDNA using a High Prime DNA labeling kit (Roche, IN, USA) and were used for hybridization. After hybridization, the membrane was washed with 0.1× SSC, 0.05% SDS at 50� C and exposed to X-ray film for 18 h at -80� C with an intensifying screen.

Tissue preparation
Perfusion fixation was performed transcardially in 4month-old male C57/BL6 and FGS/Nga mice, first with 50 mL of 0.05 M phosphate-buffered saline (PBS, pH 7.4) containing heparin (1 IU/mL) at 4� C and then with 50 mL of ice-cold 10% neutral-buffered formalin for 10 min at a flow rate of 30~40 mL/min. The kidneys of the mice were removed immediately and then fixed in the latter solution overnight. The fixed tissues were embedded in paraffin.

Periodic acid Schiff (PAS) stain
To examine glomerulosclerotic lesions in the kidney, paraffin sections of the FGS/Nga mouse kidney were stained with PAS [11,12].

Antibody preparation and CTRP1 immunohistochemistry
Rabbit anti-CTRP1 polyclonal antibody was prepared for immunohistochemistry [13,14]. Briefly, mouse CTRP1 cDNA encoding the collagen repeat domain was introduced into the pET-28a bacterial expression vector (Novagen, Darmstadt, Germany), which was then transformed into E. coli strain BL21(λDE3). The expression of the collagen domain of CTRP1 was induced by the addition of 1 mM IPTG. The collagen domain of CTRP1 was expressed in the form of an inclusion body and was purified according to the full-length protein folding protocol. The purified collagen domain of CTRP1 was injected subcutaneously into rabbits in one dose. Four months later, the rabbits were sacrificed to obtain rabbit anti-CTRP1 polyclonal antibody.
The deparaffinized sections were heated for 4 min in a pressure cooker containing 10 mM citrate buffer (pH 6.0) for antigen retrieval. Subsequent procedures were conducted at room temperature. The sections were pretreated with 3% H 2 O 2 in 0.1 M PBS (pH 7.4) for 30 min to quench endogenous peroxidase. The sections were incubated for 1 h at room temperature (RT) in the polyclonal CTRP1 antibody in 0.1 M PBS (pH 7.4) containing 0.1% Triton X-100, 1.5% bovine serum albumin (BSA), and 1 : 200 normal goat serum (NGS), and then incubated for 1 h at RT in 1 : 200 biotinylated goat anti-rabbit IgG (Vector, CA, USA) and 1 : 200 NGS in PBS. The immunoreactions were visualized by incubation for 1 h at RT in avidin-biotinperoxidase complex (1 : 100, ABC kit, Vector) in PBS and for 5~10 min in 0.05% 3,3′-diaminobenzidine and 0.01% H 2 O 2 in 0.1 M PBS. Immunolabeled sections were dehydrated in a graded ethanol series, defatted in xylene, and mounted. A similar procedure was used in control experiments, except the sections were processed either in the presence of antibody that had been pre-adsorbed overnight with an excess of immunizing recombinant protein or in the absence of primary CTRP1 antibody.

In situ hybridization for podocin
Immonohistochemistry for podocin, a specific marker for podocytes, with commercially available antibodies showed nonspecific labeling pattern in the kidney. To evaluate whether the CTRP1 immunoreactive cells were podocytes, in situ hybridization for the podocin gene was performed with digoxigenin-labeled riboprobes. The podocin antisense and sense riboprobes were synthesized from mouse cDNA fragments of the coding region cloned into pGEM-T vector (Promega, CA, USA) flanked by the Sp6 and T7 promoters. The paraffin sections that were adjacent to the sections used for CTRP1 immunohistochemistry were used for in situ hybridization. The deparaffinized sections were treated with proteinase K, acetylated with 0.25% acetic anhydride, prehybridized, hybridized with probe at a concentration of 0.5 μg/mL, treated with RNase A, and washed with SSC. The washed tissues were then incubated with anti-digoxigenin alkaline phosphate-conjugated serum (diluted 1 : 500, Roche). The final coloring reaction was performed using nitroblue tetrazolium/5bromo-4-chloro-3-indolyl-phosphate solution. Each sample section was dehydrated and covered with a coverslip, using Permount for viewing.

Results
The CTRP1 gene was expressed more abundantly in the mouse heart, liver, and kidney than in the lung, brain, spleen, skeletal muscle, and testis (Fig. 1).
No signal was detected in the absence of primary antibodies in kidney sections. Pre-incubation of the antibody against CTRP1 with the corresponding CRTP1 proteins also completely abolished the immunolabeling in kidney sections (data not shown). CTRP1 immunoreactivity was found in the cells located in the peripheral part of the glomeruli. The location and shape of the CTRP1-immunoreactive cells strongly suggested that CTRP1 is expressed in podocytes ( Fig. 2A). In situ hybridization for podocin in the adjacent tissue section showed that the pattern of podocin-positive cells was very similar to that of CTRP1immunoreactive cells (Fig. 2B). These two stains suggest that CTRP1 is expressed in podocytes. CTRP1 immunoreactivity was detected in the cytoplasm of the podocyte cell body, but may not be on the cell membrane of the foot process. This implies that CTRP1 is not related to the filtration barrier directly, including the foot processes of podocytes and slit diaphragm.
PAS staining showed that the amount of PAS-positive mesangial matrix was increased in the kidneys of FGS/ Nga mice (Fig. 3A, 3B). Increased amounts of PAS-posi-tive mesangial matrix indicate a thickened basement membrane in the glomeruli. This is one of the features of glomerular filtration membrane damage and increased protein permeability. Urine analysis with a urine analysis strip showed proteinuria in the FGS/Nga mice (data not shown). Given that the FGS/Nga mice developed proteinuria and glomerulosclerosis beginning at the age of 3 months, this experimental animal provides an available animal model of glomerulosclerosis. Both the number of CTRP1 immunoreactive cells and the intensity of CTRP1 immunoreactivity were decreased in the sclerotic glomeruli regions, compared with the normal region in the FGS/Nga mice (Fig. 3C, 3D). These findings suggest that CTRP1 is Podocyte-specific Expression of CTRP1 149 Fig. 1. Tissue expression pattern of the CTRP1 gene in the mouse (Northern blot analysis). CTPR1 mRNA is expressed abundantly in the mouse heart, liver, and kidney.

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involved in the normal activity of podocytes related to glomerular filtration.

Discussion
Our data show that CTRP1 is expressed in the podocytes of the mouse kidney, and that CTRP1 expression is downregulated in the glomeruli in the kidney accompanying focal glomerulosclerosis. This study provides the first description of the localization of CTRP1 expression in the normal kidney and in the FGS/Nga mouse, a model of glomerulosclerosis.
Adiponectin has collagen repeat and complement C1q domains [15,16], can bind to collagen, and shows antiinflammatory and vasoprotective activities [17][18][19][20], in addition to an anti-diabetic effect. CTRP1 also has collagen repeat and C1q domains [7], which are features of adiponectin paralogs. Consequently, we expect that CTRP1 has a function similar to that of adiponectin. A recent report showed that CTRP1 prevents collagen-induced platelet aggregation by specifically blocking von Willebrand's factor (vVW) binding to collagen [8]. In this study, we showed that CTRP1 is expressed in podocytes but not in endothelial cells or smooth muscle cells of the intrarenal arteries/arterioles, or in the endothelium of the glomerular and peritubular capillaries in the normal kidney [2]. In addition, CTRP1 expression is attenuated in the podocytes of an animal model of glomerulosclerosis. These facts suggest that CTRP1 is involved in the normal function of the glomerular filtration barrier.
The filtration barrier of the kidney consists of three specialized layers: the capillary endothelium, the glomerular basement membrane, and a single-cell layer of glomerular 150 Keun-Ja Cho, Young Yang, Young Ho Lee, Sooil Kim

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epithelial cells or podocytes. The foot processes of neighboring podocytes interdigitate regularly, leaving filtration slits between them that are bridged by an extracellular structure known as the slit diaphragm. A growing number of proteins, including nephrin, CD2AP, FAT, ZO-1, Pcadherin, podocin, and Neph 1-3 have been shown to be expressed in the slit diaphragm, and some of these molecules play a major role in maintaining the structural and functional integrity of the slit diaphragm [21,22]. However, our data show that CTRP1 is expressed in the cytoplasm of podocyte cell body, not in the cell membrane of foot process or slit diaphragm. Although CTRP1 is not expressed in the slit diaphragm, we cannot rule out the possibility that CTRP1 acts in the slit diaphragm because CTRP1 is a secretory protein [7] released from the podocytes in the glomeruli and can reach the slit diaphragm. Further in vivo and in vitro study is needed to elucidate the role of CTRP1 in the kidney.
In conclusion, CTRP1 is expressed in the podocytes of the mouse kidney and may be involved in the role of podocytes in glomerular filtration in the kidney.