FULL PAPER Laboratory Animal Science

Decreases in Podocin, CD2-Associated Protein (CD2AP) and Tensin2 May Be Involved in Albuminuria during Septic Acute Renal Failure

Takashi KATO1,2), Yoko MIZUNO-HORIKAWA3) and Shinya MIZUNO1,4)*

1)Division of Molecular Regenerative Medicine, Department of Biochemistry and Molecular Biology, Osaka University Graduate School of Medicine, 2–2–B7 Yamadaoka, Suita, Osaka 565–0871, 2)Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565–0871, 3)Protein Research Institute, Osaka University, 1–2 Yamadaoka, Suita, Osaka 565–0871 and 4)Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2–2–B7 Yamadaoka, Suita, Osaka 565–0871, Japan

(Received 27 April 2011/Accepted 15 July 2011/Published online in J-STAGE 29 July 2011)

ABSTRACT. Podocytes have a peculiar structure constituting slit diaphragm (SD) and foot process (FP), and play essential roles in the glom- erular filtration barrier. There is now ample evidence that SD- and FP-associated molecules, such as podocin and CD2-associated protein (CD2AP), are down-regulated during albuminuria of chronic kidney disease. However, it is still unclear whether these molecules are altered during acute renal failure (ARF) with albuminuria. Using lipopolysaccharide (LPS)-treated mice as a model of septic ARF, we provide evidence that the expression of SD- and FP-associated molecules becomes faint, along with albuminuria. In the LPS-treated mice, urinary albumin levels gradually increased, associated with the elevation of blood urea nitrogen levels, indicating the successful induction of albuminuria during septic ARF. In this pathological process, glomerular podocin expression became faint, especially at 36 hr post-LPS challenge (i.e., a peak of albuminuria). Likewise, LPS treatment led to a significant decrease in CD2AP, an anchorage between podocin and F-actin. With regard to this, tensin2 is a novel molecule that stabilizes F-actin extension. Interestingly, glomerular tensin2 expression levels were also decreased during the albuminuric phase, associated with losses of glomerular F-actin and synapto- podin under septic states. As a result, there were some lesions of podocytic FP effacement, as shown by electron microscopy. Based on these data, we emphasize the importance of concomitant decreases in podocin, CD2AP and tensin2 during septic ARF-associated pro- teinuria. KEY WORDS: albuminuria, CD2AP, podocin, sepsis, tensin2. J. Vet. Med. Sci. 73(12): 1579–1584, 2011

Plasma ultra-filtration occurs through the glomerular fil- ciated with sporadic nephrotic syndrome and focal and tration barrier, which consists of highly specialized visceral segmental glomerulosclerosis [11, 32]. These genomic epithelial cells called podocytes, fenestrated capillary endo- studies lead to the determination of a molecular topology for thelium, and an intervening glomerular basement membrane SD, required for maintaining the homeostasis of podocytes- (GBM). Among them, podocytes serve as a molecular sieve podocytes [21]. Notably, the decreases of SD-related mole- that selectively restricts the filtration of different molecules cules (such as podocin and CD2AP) are observed in many on the basis of their size and charge [14]. Podocytes have types of CKD, regardless of the primary etiology [1, 19]. octopus-like cells comprising a cell body and cytoplasmic In contrast to CKD, little is known about the molecular extensions called major processes, which divide into actin- basis of albuminuria during acute renal failure (ARF), rich foot process (FP) interdigitating over each capillary including sepsis. Sepsis-mediated renal injury is a typical loop and counteracting the distensive forces [9, 12]. Each phenotype of ARF that is associated with albuminuria [2, foot process is attached to its neighbor along the length by 33]. During the sepsis, glomerular thrombosis becomes evi- an intercellular adherent-type junction, the slit diaphragm dent via up-regulations of inflammatory cytokines, and then (SD), which is localized just above the GBM. local permeability and hypoxia enhance renal damage, asso- There is a closed correlation between the degree of pro- ciated with a loss in renal blood flow [26, 30]. Even under teinuria and the prognosis of chronic kidney disease (CKD) low glomerular filtration, albuminuria is often noted in sep- [36]. Therefore, it is important to elucidate the mechanisms tic patients [2, 33] and in rodents [31]. Thus, we hypothe- whereby proteinuria occurs and develops at genetic and size that albuminuria may occur during sepsis via the molecular levels. Recent genetic studies identified podocin, possible down-regulation of SD-related proteins, such as encoded by NPHS2, as a mutant gene that causes autosomal podocin and CD2AP, but it is still unclear whether these recessive steroid-resistant nephritic syndrome [3, 29]. Like- molecules are involved in albuminuria during septic ARF. wise, CD2-associated protein (CD2AP) mutations are asso- To test this hypothesis, we used lipopolysaccharide (LPS)-treated mice as a model of septic ARF, because LPS- *CORRESPONDENCE TO: MIZUNO, S., Division of Virology, Depart- ment of Microbiology and Immunology, Osaka University Grad- treated mice manifest albuminuria, as reported [31]. Using uate School of Medicine, 2–2–B7 Yamadaoka, Suita, Osaka this mouse model, we provided evidence that expressions of 565–0871, Japan. podocin and CD2AP by podocytes becomes faint, concomi- e-mail: [email protected] tantly with a peak of urinary albumin excretion. The “syn- 1580 T. KATO, Y. MIZUNO-HORIKAWA AND S. MIZUNO chronized” decreases of cytoskeletal components, such as night at 4°C with goat anti-podocin antibody. The com- synaptopodin, F-actin and tensin2, are also discussed here, plexes were precipitated with protein A/G sepharose. These all of which could be responsible for the onset of albumin- immuno-precipitates were washed three times and dissolved uria during sepsis. in sample buffer. The samples were separated by SDS- PAGE and electroblotted onto PVDF membranes. The MATERIALS AND METHODS membranes were blotted with anti-podocin goat IgG and held overnight at 4°C, then subsequently labeled with per- Animal models: Eight-week-old female mice (C57BL/6J oxidase-conjugated antibody against goat IgG (Santa Cruz). strain) were purchased from SLC (Hamamatsu, Japan). LPS The resultant signals were detected using ECL-plus-chemi- (E. coli O111:B4) was obtained from Sigma (St. Louis, luminescence (Amersham, Little Chalfront, U.K.). MO., U.S.A.). Sepsis was induced via a single injection of Statistical analysis: Data were expressed as mean ± SD. LPS at a dose of 8 mg/kg (i.p.), based on a recent report [15]. A Student’s t-test, ANOVA analysis or Mann Whitney U- Mice were sacrificed at 0, 12, 24, 36 and 48 hr post-LPS test was used to compare the group means. P-values <0.05 challenge. All the experiments were performed, according were considered significant. to the regulations on animal experiments at Osaka Univer- sity, with an approval by Animal Research Committee of RESULTS Osaka University Graduate School of Medicine. Determination of urinary albumin levels by SDS-PAGE: Induction of albuminuria and ARF in LPS-treated mice: The samples were collected as spot urine at each time-point The changes in BUN and urinary albuminuria were checked to be examined. The albuminuria was evaluated using SDS- after LPS treatment. A single injection of LPS (8 mg/kg, PAGE, where its concentration (mg/ml) was determined, i.p.) resulted in the apparent increases in BUN levels (Table based on the band size of bovine serum albumin under den- 1), indicating the onset and progression of septic ARF, as sitometry [16]. reported [15]. As expected, albumin levels gradually Blood chemistry: At the scheduled autopsy, blood was increased in the urine samples, with a peak at 36 hr post-LPS collected from the submaxillary artery, and plasma was challenge (Table 1). These clinico-biochemical findings obtained at 0, 12, 24, 36 and 48 hr post-LPS challenge (n=6 indicated the successful induction of septic ARF and albu- per each time point). To evaluate the renal dysfunction, minuria in our mouse model. blood urea nitrogen (BUN) levels were measured by a ure- Decrease in podocin expression in the renal glomuruli of ase indophenol method with a kit (urea nitrogen-B test, septic mice: Although albuminuria became evident 24–48 hr Wako, Osaka, Japan) [25]. post-LPS challenge, there were no significant differences in Immunohistochemistry: Right kidneys were fixed in 10% the number of podocytes between saline- and LPS-treated formalin and 70% ethanol for 24 hr, and embedded in paraf- groups (not shown). Thus, we focused on the degree of fin. The kidneys were cut into 4-µm sections and were sub- podocin, an SD-associated molecule [9]. Immune fluores- jected to the histological processes. To investigate the level cence analysis revealed that podocin was extensively noted of a slit diaphragm protein, the sections were identified along GBM (i.e., linear pattern) in the saline group (Fig. 1A, using an anti-podocin goat IgG (sc-22298, Santa Cruz, left). On the other hand, LPS treatment apparently reduced Santa Cruz, CA, U.S.A.). To identify FP proteins, anti- the glomerular podocin expression, with a loss of linearly CD2AP rabbit IgG (sc-9137, Santa Cruz), anti-synaptopo- positive signals (i.e., granular pattern) (Fig. 1A, right). Con- din mouse IgG (65194, Progene, Heidelberg, Germany), or sistently, there was a difference in the podocin staining anti-tensin2 rabbit IgG (Transgenic, Kobe, Japan) was score between both groups (36 hr: saline; 3.76 ± 0.14 vs. applied onto renal sections, as the primary antibodies. For LPS; 2.10 ± 0.04, P<0.01). antigen retrieval, the sections were treated with citrate Immune blot analysis of podocin strengthened this histo- buffer (pH 6.0). The sections were incubated at 4°C for logical finding: a podocin-positive band, with a molecular overnight, washed with phosphate-buffered saline followed size of 45-kD, was detected by immune precipitation and by detection of fluorescence with Alexa546- or Alexa488- blotting before LPS challenge. The molecular size of the labeled antibodies (Invitrogen, Carlsbad, CA, U.S.A.). podocin-positive band was not altered by saline, but its Morphometry: We quantified the glomerular expressions of podocin, CD2AP and tensin2 according to a staining Table 1. Renal dysfunction and albuminuria after LPS injection score [16]. Briefly, >20 glomeruli were randomly chosen, and expression levels in each glomerulus were scored based Time (hr) BUN (mg/ml)uAlb (g/ml) on the fluorescence extent, as followed: 0; none, 1; faint, 2; 0 20.9  1.88 9.2  11.4 mild, 3; moderate, and 4; strong. The extent of molecular 12 63.3  11.3 57.1  43.4 expressions was expressed as the individual index (i.e., total 24 116.9  19.5 494.7  139.6 score/glomerular number examined) [16]. 36 132.3  32.9 981.4  186.4 Immunoprecipitation and Western blotting: Kidney tissue 48 63.9  18.7 546.1  226.8 homogenates were prepared from mice in lysis buffer, as uAlb: urinary albumin. BUN: blood urea nitrogen. Data are reported [16, 25]. The renal extraction was incubated over- expressed as a mean  SD (n=6). LOSSES OF PODOCIN AND TENSIN2 DURING SEPSIS 1581

the expression of synaptopodin (Fig. 2C and 2G), concomi- tantly with the losses of CD2AP and F-actin. We next focused on tensin2, a novel molecule that is required for stable expression of synaptopodin during con- genital nephrotic syndrome in mice (ICGN strain) [17]. Tensin2 was detected in the extra-capillary areas (i.e., podocyte-localized area) of the saline-injected control mice (36 hr) (Fig. 2D). By contrast, LPS treatment resulted in a significant loss of tensin2 at 36 hr post-LPS challenge (Fig. 2H). Indeed, there was a significant difference in the Fig. 1. Podocin reduction in the glomeruli of LPS-challenged tensin2 staining score between the control and septic groups mice. (A) Typical finding of podocin expression in saline- (36 hr: saline; 2.50 ± 0.29 vs. LPS; 1.69 ± 0.29, P<0.05). treated (left) and LPS-treated (right) groups ( 380). (B) West- This is, to our knowledge, the first report to show a decrease ern blotting for the renal podocin expression at 0 (Pre) and 36 hr post-LPS challenge. For abbreviations see text. in tensin2 expression during acquired kidney diseases. Detection of FP effacement by electron microscopy: Since losses of SD- and FP-associated molecules were asso- expression level was reduced to half of the basal control ciated with the albuminuria, we hypothesized that these (Fig. 1B). These histological and biochemical data demon- molecular changes would lead to a change in the ultra-struc- strated a decrease in podocin during septic ARF. ture of podocytes. To test our hypothesis, the morphology Reduced expression of CD2AP leading to actin-associ- of FP was evaluated by an electron microscopy. In the ated molecules: CD2AP is essential for the stabilization of saline-injected control mice, podocytic FP located on GBM SD structure as a common intra-cellular bridge among neph- seemed normal (Fig. 3A and 3B). By contrast, there were rin, podocin and F-actin [21]. In the saline-injected control effaced findings, with an FP-FP fusion, especially 36 hr group, CD2AP was detected in the extra-capillary areas of post-LPS challenge (i.e., albuminuric stage) (Fig. 3C and glomeruli (i.e., podocyte area) (Fig. 2A). By contrast, 3D). Overall, losses of SD- and FP-associated molecules CD2AP-positive signals became faint at 36 hr after LPS (such as podocin, CD2AP and tensin2) were considered to treatment (Fig. 2E). A significant difference in the CD2AP elicit albuminuria through the induction of FP effacement. staining score was seen between both groups (36 hr: saline; 3.67 ± 0.17 vs. LPS; 2.25 ± 0.63, P<0.05). DISCUSSION The change in F-actin was examined, since it is connected to podocin via CD2AP [9, 21]. Consistent with the Although urinary albumin excretion is an important hall- decreases in podocin and CD2AP, glomerular expression of mark of septic ARF [2, 26, 33], the molecular basis of albu- F-actin in the LPS-treated group (36 hr) was also reduced minuria is not fully understood. In this regard, we reported compared to that of the control (Fig. 2B and 2F). The that the loss of nephrin, another key molecule of SD [9], change in synaptopodin, a key player for actin dynamism of becomes evident during sepsis through a loss in nuclear podocytes [9], was then examined. As a result, LPS reduced localization of WT1, a key transcriptional factor of nephrin

Fig. 2. Immunofluorescence images of podocyte-associated proteins at 36 hr after injection of saline (A-D), or LPS (E-H) ( 380). (A, E) CD2AP, (B, F) F-actin, (C, G) Synaptopodin (SynaPD) and (D, H) tensin2. Red signal: Alexa-546 for CD2AP, F-actin and tensin2. Green signal: Alexa 488 for SynaPD. 1582 T. KATO, Y. MIZUNO-HORIKAWA AND S. MIZUNO

pathway), and we are now investigating the roles of these cytokines in the regulation of podocin. Our next attention was paid to the change in CD2AP, an adaptor molecule that provides a bridge between podocin and F-actin [9, 21]. Indeed, CD2AP directly interacts with podocin, or with F-actin to sustain barrier function and mor- phology of podocyte SD [9, 21]. Of note, mice with CD2AP haplo-insufficiency developed glomerular changes at 9 months of age and had increased susceptibility to glomeru- lar injury by nephrotic antibodies [18]. In addition, human patients with focal segmental glomerulosclerosis possessed a mutation that was predicted to ablate expression of one CD2AP allele (i.e., 50%-reduction) [18]. Thus, we predict that the half-reduction of CD2AP by LPS may be sufficient, in addition to the decreases of other SD-associated mole- cules, to elicit albuminuria in this septic model. Since Fig. 3. Ultrastructural examination by electron microscopy in CD2AP is required for stabilization of podocin via inhibi- saline- and LPS-treated groups. (A, B) saline control group (36 hr). (C, D) LPS-treated group (36 hr). Each frame shows the tion of its degradation [35], the loss of CD2AP may, in part, expanded results of podocyte foot process in the saline group (B) explain the additional mechanism of podocin loss. and LPS -treated group (D). Arrows: foot process effacement of The possible mechanisms for explaining the CD2AP loss podocytes. during septic ARF should be discussed. A promoter assay revealed that Sp1/Sp3-binding sites of CD2AP-promoter regions is required for up-regulation of CD2AP [34], while [16]. In this study, we focused on podocin, because it is a inflammatory cytokines, such as TNF-, suppresses various key anchorage that strengthens FP-FP contact via a bridge genes via inhibiting Sp1/Sp3-DNA binding [6]. Thus, between nephrin and CD2AP [21]. Furthermore, a loss of cytokine-mediated inactivation of Sp1/Sp3-binding pro- podocin, rather than nephrin, seems to be a “strict” marker moter sites may cause the loss of CD2AP during septic to predict proteinuria. Actually, podocin loss is sometimes ARF, and future studies would shed light on this specula- independent of nephrin expression in vivo and in vitro: in tion. The loss of CD2AP was concomitant with that of some types or stages of CKD, podocin loss does not occur podocin in our model. With regard to this, production of during albuminuria, even if nephrin is decreased [10, 22]. In these molecules is controlled by the same transcriptional vitro, knockdown of podocin mRNA by siRNA leads to the factor, Lmx1b [23]. On the other hand, nephrin is up-regu- concomitant loss of nephrin [7], but inversely, nephrin lated by another transcriptional factor, WT-1 [16]. Thus, mRNA knockdown does not elicit podocin down-regulation our model is useful for elucidating the transcriptional mech- [8]. Thus, it is still unclear whether podocin expression is anisms of podocyte-specific gene profiles. altered, or not, during sepsis, even if nephrin is decreased Finally, we focused on a novel protein in nephritic glom- [16], and this background promoted the examination of eruli. Tensin2 is a member of tensin family, a focal-adhe- other SD-associated molecules. As a result, podocin loss sion-molecule that stabilizes F-actin by binding to - became evident, along with the peak of albuminuria, as did integrin [4], as did -actinin4 (ACTN4) [21]. Of interest, a nephrin loss [16]. loss of tensin2 acts as a switch to trigger cell migration [27]. It is important to discuss the molecular mechanism of Tensin2 localizes mainly to podocytes in the kidney [5, 17], podocin loss as seen at 36 hr post-LPS. Inflammatory and notably, tensin2 homozygous mutation (i.e., tensin2- cytokines, such as tumor necrosis factor- (TNF-), are deficiency) in the mice (i.e., ICGN strain with an ICR back- able to down-regulate podocin transcription in a culture of ground) leads to albuminuria within a few weeks after birth immortalized podocytes [13]. It is well known that LPS [5, 17, 24]. By contrast, there was no significant albumin- injection rapidly increases TNF- levels in the blood of uria in the congenic strain carrying the tensin2 mutation on rodents (i.e., innate immune system leading to cytokine the C57BL/6J genetic background [28], which raises a con- storm) [15, 30]. We also detected a dramatic increase in troversial issue about the role of tensin2 during albuminuria. blood TNF- levels (i.e., > 1,000-fold increase) within 2 hr Using a model of an “acquired” kidney disease (i.e., septic post-LPS challenge (not shown). Thus, secretion of TNF- ARF), we found the involvement of decreased tensin2 in from macrophages by LPS in distant organs may lead to septic albuminuria. Tensin2 is required for stability of syn- podocin down-regulation in the glomeruli via an endocrine aptopodin [17]. In turn, synaptopodin is important for pathway. In addition, glomerular mesangial cell-derived podocytes to sustain FP extension (i.e., podocyte outgrowth) cytokines, such as transforming growth factor-, can sup- [9, 21, 31]. Thus, we predict that the loss of synaptopodin press podocin production in a culture of podocytes [20]. via down-regulating tensin2, an anchorage between GBM Thus, such an intra-glomerular local system may also partic- and FP, could enhance the FP effacement and subsequent ipate in the LPS-induced decrease in podocin (i.e., paracrine albuminuria in our septic model. LOSSES OF PODOCIN AND TENSIN2 DURING SEPSIS 1583

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Activated macroph- of albuminuria during septic ARF. The present study pro- ages down-regulate podocyte nephrin and podocin expression vides an in vivo model to elucidate the roles and regulatory via stress-activated protein kinases. Biochem. Biophys. Res. mechanisms of SD- and FP-associated molecules under sep- Commun. 376: 706–711. sis-mediated renal injury. 14. Johnstone, D. B. and Holzman, L. B. 2006. Clinical impact of research on the podocyte slit diaphragm. Nat. Clin. Pract. ACKNOWLEDGMENTS. This work was supported by Nephrol. 2: 271–282. grants from the Ministry of Education, Science, Technol- 15. Kamimoto, M., Mizuno, S., Matsumoto, K. and Nakamura, T. 2009. Hepatocyte growth factor prevents multiple organ inju- ogy, Sports and Culture of Japan (No. 20590398 and No. ries in endotoxemic mice through a heme oxygenase-1 depen- 23590458 to SM). We are grateful to Dr. J. McDonald dent mechanism. Biochem. Biophys. Res. 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