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Four-and-a-Half LIM Domains 2 Is a Coactivator of Wnt Signaling in Diabetic Kidney Disease

† ‡ | Szu-Yuan Li,* Po-Hsun Huang, Der-Cherng Tarng, Tzu-Ping Lin,§ Wu-Chang Yang, | †† Yen-Hwa Chang,§ An-Hang Yang,¶ Chih-Ching Lin, Muh-Hwa Yang,** Jaw-Wen Chen, ‡‡ || †† Geert W. Schmid-Schönbein, Shu Chien,§§ Pao-Hsien Chu, and Shing-Jong Lin

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/b-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 -knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-b1, activated FHL2 protein and Wnt/b-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/b-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2+/+ mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2+/+ mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with di- abetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both struc- tural and functional protein-protein interactions with b-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/b-catenin–induced podocytopathy.

J Am Soc Nephrol 26: 3072–3084, 2015. doi: 10.1681/ASN.2014100989

Diabetic kidney disease (DKD) is the leading cause glomerular filtration barrier. Podocytes are highly of ESRD, affecting 10%–40% of diabetic patients, specialized, terminally differentiated cells that can- and the cost of RRT remains a large economic bur- not proliferate.2 Recently, hyperactive podocyte den for the health care system. Proteinuria is a major and primary clinical aspect of DKD and causes Received October 13, 2014. Accepted February 23, 2015. tubulointerstitial lesions that lead to renal dysfunc- 1 Published online ahead of print. Publication date available at tion. Reducing proteinuria may therefore be a princi- www.jasn.org. pal therapeutic target to improve renal outcome in patients with DKD. However, the pathophysiologic Correspondence: Dr. Po-Hsun-Huang, Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital and mechanism of DKD is multifactorial, and some patients Institute of Clinical Medicine, and Cardiovascular Research develop treatment-resistant proteinuria that results in Center, National Yang-Ming University, No 201, Sec 2, SHih-Pai ESRD despite intensive BP and glycemia control. Thus, Road, Taipei, Taiwan, or Dr. Pao-Hsien Chu, Division of Cardi- fi ology, Department of Internal Medicine; Healthcare Center; identi cation of major DKD mechanisms and develop- Heart Failure Center, Chang Gung Memorial Hospital, Chang ment of new therapeutic options are needed. Gung University, College of Medicine, Taipei, Taiwan. Email: Glomerularepithelial cells, also calledpodocytes, [email protected] or [email protected] are predominantly responsible for maintaining the Copyright © 2015 by the American Society of Nephrology

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Wnt/b-catenin signaling has been shown to play an essential RESULTS role in the development of DKD. The Wnt family of signaling participates in multiple developmental events during FHL2 Is Abundantly Expressed in Mature Podocytes embryogenesis and has been implicated in adult tissue homeo- We used three different methods to identify the expression and stasis. Wnt signals modulate pleiotropic effects, such as in- distribution of FHL2 in kidney tissues. In human kidney clude mitogenic stimulation and cell-fate specification. samples, immunohistochemical (IHC) staining with a mono- An important component of the Wnt pathway is b-catenin, clonal antibody showed that FHL2 is highly expressed in which resides in the in a multiprotein complex. glomerular cells (Figure 1, A and B). We further used immu- One component of this complex, glycogen synthase kinase 3 nofluorescence double-staining and confirmed that FHL2 is (GSK3), phosphorylates b-catenin in the absence of Wnt abundantly expressed in podocytes (Figure 1C, Supplemental signaling, and phosphorylated b-catenin is subsequently Material). To avoid the potential antibody cross-reactivity in ubiquitinated and degraded via proteasomes. Upon Wnt sig- IHC staining, we introduced the LacZ gene in the FHL2 locus naling stimulation, b-catenin is stabilized and translocated by homologous recombination, and LacZ staining confirmed to the . As a transcriptional activator, b-catenin that FHL2 is expressed in mouse glomeruli (Figure 1D). binds the of T cell–specific transcription A previous report demonstrated that cultured human podocytes factor (TCF) in the cell nucleus and starts target gene transcrip- have abundant FHL2 mRNA expression in nonpermissive tion.3,4 In clinical studies, aberrant Wnt signaling has been shown conditions.25 Here, we used Western blot to demonstrate to be responsible for various malignant and metabolic diseases.5–8 thatFHL2proteinexpressionismodestinthepermissivepro- As a developmental signaling pathway, the Wnt/b-catenin liferating stage but becomes abundant in nonpermissive well system is essentially silenced in differentiated podocytes, but differentiated podocytes (Figure 1E). activated Wnt/b-catenin signaling has been found in various types of proteinuric kidney disease,9–11 particularly in Diabetes Mellitus–Related Cytokines Activate the Wnt DKD.10,12,13 Wnt/b-catenin is a key element of podocyte dys- Pathway and Upregulate FHL2 Expression in Human function by downregulating nephrin via a Snail-dependent Podocytes mechanism.10 Target inhibition of podocyte Wnt signaling To investigate the effect of high glucose conditions and does not alter kidney microstructure and function but pre- diabetes-related cytokines on FHL2 regulation, differentiated vents podocytopathy in doxorubicin- induced nephropathy.10 human podocytes were exposed to various stimulators. As On the contrary, activation of b-catenin in podocytes results shown in Figure 2, A and B, high glucose, TGF-b,andangio- in podocyte foot process effacement, glomerular basement tensin II stimulate Wnt/b-catenin signaling in podocytes. membrane (GBM) thickening, and significant albumin- Western blot revealed increased active b-catenin protein level, uria,14 implying that the Wnt pathway is not necessary for and importantly, that these stimulators also upregulated FHL2 normal podocyte function, but its activation during renal in- protein expression. The GSK-3 inhibitor, a direct Wnt path- jury might reflect a harmful signaling. way activator, markedly increased active b-catenin but did not The LIM domain is a cysteine-rich motif that has been pro- affect FHL2 protein expression, which suggests FHL2 is not a posed to direct protein-protein interaction. A diverse group of Wnt targeted–protein. proteins containing LIM domains have been identified, which Several groups have addressed the possibility that various display various functions.15 A concept is emerging that the LIM stimuli modulate subcellular distribution of FHL2. To domains can associate with both the and the explore and test this hypothesis in podocytes, we used transcriptional machinery. The functions of LIM domain pro- nuclear/cytoplasmic protein extraction and immunofluo- teins in the nucleus are mainly in tissue-specificgeneregulation, rescencestaining to evaluateFHL2 subcellulardistributionin whereas cytoplasmic LIM domain proteins are mainly involved cultured podocytes. Under normal culture conditions, FHL2 in cytoskeleton organization.16 Four-and-a-half LIM domains was mainly expressed in podocyte cytosol, located on the protein (FHL) contains four-and-a-half LIM domain–binding podocyte cytoskeleton, and translocated to the cell nucleus proteins. Proteins in this family function as adaptors or scaffolds after stimulation by high glucose and diabetes mellitus to support the assembly of metameric protein complexes for (DM)–related cytokines (Figure 2C). The b-catenin was dis- critical cellular processes.17 The best-studied member of this tributed in the cell-cell junction, and no protein-protein family, FHL2, is highly expressed in cardiovascular and urogenital interactions were found between FHL2 and b-catenin under systems. The role of FHL2 has been investigated intensively in normal conditions. However, under high glucose condi- cardiovascular diseases over the past decade,18–20 but its role in tions, both FHL2 and b-catenin simultaneously translocated kidney disease remains unknown. Interestingly, FHL2, but not to the cell nucleus and are costained in the nucleus (Figure FHL1 or FHL3, is involved in the Wnt/b-catenin signaling path- 2D). We further extracted cell nuclear protein for a coim- way.21–23 Here,weusedanFHL2geneknockoutinadiabetic munoprecipitation experiment and confirmed that FHL2 nephropathy mouse model,24 combined with in vitro experiments has a structural protein-protein interaction with activated and clinical data, to clarify the possible role of FHL2 in DKD. b-catenin (Figure 2E).

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littermates after the development of diabetes by streptozotocin administration. Mice were divided into four groups: FHL2+/+ control 2 2 group, FHL2+/+ DM group, FHL2 / con- 2 2 trol group, and FHL2 / control group. Blood glucose and hemoglobin A1c levels were similarly elevated in the two diabetic groups. Induction of diabetes produced glo- merular hyperfiltration in this DM model, and the two diabetic groups had similar ele- vation in GFR. Urine albumin levels and GFR did not differ between the nondiabetic 2 2 FHL2 / and FHL2+/+ mice throughout the Figure 1. FHL2 is abundantly expressed in glomerular podocytes. (A) IHC staining study period (Figure 4, A–C). Urinary albu- shows FHL2 protein is abundantly expressed in glomeruli in normal human kidney fi fi 3 min was signi cantly elevated in diabetic tissue (original magni cation, 100). (B) With high-power view, the staining showed +/+ a typical podocyte distribution pattern (original magnification, 3400). (C) Double FHL2 mice starting from the second , immunofluorescence staining illustrates that FHL2 colocalized with podocyte marker month of diabetes (P 0.05), and the degree nephrin. (D) Lac-Z staining shows a clear glomerular distribution in the FHL2 null of urinary albumin progressively increased mouse kidney because Lac-Z is introduced in the FHL2 locus, indicating that FHL2 through the fourth month. However, the di- 2 2 transcription is highly activated in glomerular cells. (E) FHL2 protein expression in abetic FHL2 / mice had significantly less cultured human podocytes under permissive and nonpermissive conditions. urinary albumin throughout the whole study period (Figure 4D). At the end of the FHL2 Is a Coactivator of b-catenin and Knockdown of study, the glomerular mesangial volume from kidney tissues FHL2 Attenuates Wnt-Induced Podocytopathy In Vitro of study mice was determined. Diabetic FHL2+/+ mice had To test the possibility that FHL2 facilitates translocation of glomerular hypertrophy and mesangial expansion (P,0.01) b-catenin to the cell nucleus through the Wnt pathway, we compared with nondiabetic FHL2+/+ mice, and the diabetic 2 2 analyzed the b-catenin expression in the cell nucleus with and FHL2 / mice had significantly less mesangial expansion com- without FHL2. As illustrated in Figure 3A, nuclear b-catenin pared with diabetic FHL2+/+ mice (P=0.03) (Figure 4, E and F). was significantly upregulated in response to Wnt stimulation, The thickness of GBM was further determined by electron mi- and knockdown of FHL2 did not alter nuclear b-catenin croscopy. Induction of diabetes increased GBM thickness in aggregation. We then used a TCF/LEF reporter system and FHL2+/+ mice, and deletion of FHL2 significantly attenuated dual-luciferase assay to test the hypothesis that FHL2 is a cofactor GBM thickening in DKD (P,0.05) (Figure 4, G and H). In of b-catenin in podocytes. After 18-hour exposure to GSK3 agreement with a previous study, we found that induction of inhibitor, the relative luciferase units markedly increased. diabetes in mice moderately increases podocyte apoptosis but Administration of FHL2 small interfering RNA (siRNA), only modestly affects podocyte number.14 The two diabetic but not scrambled siRNA, significantly attenuated the TCF groups had similar podocyte numbers and apoptosis ratios transduction activity (Figure 3B), suggesting FHL2 protein (Supplemental Material). is a coactivator of b-catenin in podocytes. Mouse glomeruli were obtained by series sieving of kidney With regard to protein levels, Wnt signaling activates cortex with triple sets of sieving mesh (100 mm, 75 mm, and b-catenin and Snail and represses the critical slit-diaphragm 53 mm), as described previously26; the sieved glomeruli were protein nephrin in podocytes. Under normal culture condi- retrieved from the third mesh and the glomerular proteins tions, knockdown of the FHL2 protein did not affect b-catenin, were extracted for Western blotting analysis. Compared with Snail, or nephrin expression, but under Wnt-On culture con- glomeruli from nondiabetic mice, glomeruli from diabetic ditions, knockdown of the FHL2 protein significantly atten- FHL2+/+ mice had significantly enhanced expression of active uated Snail upregulation and conserved nephrin expression b-catenin, FHL2, Snail, TGF-b, and vascular endothelial (Figure 3, C–F). growth factor (VEGF), and the nephrin expression was down- On the basis of these experiments, we have demonstrated that regulated. This finding suggests podocyte dedifferentiation af- 2 2 FHL2 has structural and functional protein-protein interactions ter induction of diabetes. Moreover, diabetic FHL2 / mice with b-catenin in podocytes, and the knockdown of FHL2 pro- had similarly high levels of active b-catenin, TGF-b,and tein can reduce Wnt-induced podocyte injury in vitro. VEGF but less affected Snail and nephrin expressions com- pared with those in diabetic FHL2+/+ mice (Figure 5). These FHL2-Deficient Mice Have Attenuated Diabetic data indicate that elimination of FHL2 protein did not affect Nephropathy normal renal function and glomerular microstructure in non- To investigate the role of FHL2 in DKD in vivo, we examined diabetic mice but significantly attenuated the severity of kid- 2 2 the severity of kidney injury in FHL2 / mice and their FHL2+/+ ney injury in diabetic mice.

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Figure 2. Various DM-related cytokines that activate Wnt signaling also upregulate FHL2 expression and promote its nuclear trans- location. (A) TGF-b, high glucose, and angiotensin II activate the Wnt pathway in vitro. Interestingly, they also upregulated FHL2. Selective GSK-3 inhibitor directly activates the Wnt pathway but has no effect on FHL2, suggesting that FHL2 is not a Wnt target protein. (B) Quantification of active b-catenin and FHL2 expression by densitometric analyses. (C) By means of subcellular nuclear and cytosol protein extraction, Western blots showed that TGF-b, high glucose, and angiotensin II increased both cytosol and nuclear FHL2; a selective GSK3 inhibitor induced FHL2 translocation from cytosol to nucleus. (D) Immunofluorescence staining of culture podocytes in control culture condition. FHL2 is located in the cytoskeleton and b-catenin is in the cell-cell junction, and they obviously had no protein-protein interaction. But after 24-hour high glucose stimulation, b-catenin nuclear translocation indicated that Wnt signaling is activated. Interestingly, FHL2 also showed a nuclear staining pattern and costained well with b-catenin in high glucose culture condition. (E) Coimmunoprecipitation of nuclear protein, anti-FHL2 antibody, but not the control IgG pulled down b-catenin, which indicated the structural protein-protein interaction between FHL2 and active b-catenin. *P,0.05 compared with control; #P,0.01 compared with control.

FHL2 Nuclear Translocation Is Associated with control and diabetic kidneys. In control kidney samples, FHL2 is Albuminuria and Predicts Renal Function Deterioration stained within the podocyte cytosol, but in diabetic kidney in Diabetic Patients samples a considerable proportion of podocytes exhibit FHL2 To compare the FHL2 distribution in nondiabetic and diabetic staining in the cell nucleus in addition to the cytosol. Patients patients, kidney tissues from the site opposite the tumor in with DM had a higher proportion of podocytes with FHL2 nu- nephrectomized patients were stained with FHL-2 antibody with clear translocation (Figure 6B), and this proportion is even higher IHC, and double-stained with FHL2 and podocyte nuclear in macroalbuminuric diabetic patients. The 46 patients with DM protein WT-1 by the immunofluorescence method. Weexcluded were followed up for renal outcome, and FHL2 nuclear trans- kidney samples from patients with eGFR,60 ml/min per 1.73 m2 location of .50% was a strong predictor of renal function de- after surgery or interstitial fibrosis .25%. In total, 25 nondiabetic terioration (Figure 6C). In mice, induction of diabetes also and 46 diabetic patients were enrolled; their demographic char- triggered a remarkable FHL2 cytosol–nuclear translocation as acters are listed in Table 1. Figure 6A illustrates FHL2 staining in in human DKD (Supplemental Material).

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Figure 3. FHL2 is a b-catenin coactivator and knockdown FHL2 that reduces Wnt-induced podocytopathy in vitro. (A) Knockdown FHL2 does not alter Wnt signaling–induced b-catenin nuclear translocation. (B) Wnt reporter system showed GSK3 inhibitor markedly increased Wnt transcription activity, and FHL2 siRNA significantly attenuated the GSK3 inhibitor–induced Wnt activation. (C) Western blot showed Wnt signaling increased active b-catenin and transcription factor Snail, and downregulated nephrin, while FHL2 siRNA

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DISCUSSION is silenced. Unlike phylogenetically lower species, such as fish, in which Wnt activation following injury plays a key role in the It is widely recognized that proteinuria is one of the earliest repair process,35 regeneration in the mammalian kidney is clinical markers of DKD, and the appearance of albumin in highly restricted.36 Becausematurepodocytesareterminally urine indicates a compromised glomerular filtration barrier. differentiated cells in a state of permanent exit from the cell Recent studies have indicated that activation of Wnt/b-catenin cycle and arrested in a postmitotic state, by the end of mitosis signaling induces podocyte dysfunction and causes proteinuric their cytoskeletal actin forms part of the contractile ring. Thus, kidney disease.10,27 The current results suggest that FHL2, an when podocyte mitosis is attempted, it leads to aberrant mi- abundantly expressed protein in podocytes, is a coactivator of tosis and cell death (mitotic catastrophe).37 Meanwhile, acti- Wnt signaling in DKD. In cultured human podocytes, high vation of Wnt signaling induces podocyte dedifferentiation, glucose and DM-related cytokines not only upregulated FHL2 upregulation of the transcription factor Snail, suppression of expression but also promoted FHL2 translocation from the nephrin expression, and detachment of podocytes, causing cytosol to the nucleus. In in vitro studies, knockdown of glomerulosclerosis.10,36 Wnt-induced maladaptive repair FHL2 mitigated induced podocyte dedifferentiation by acti- and fibrosis does not seem to be restricted to glomerular vated Wnt/b-catenin signaling and reduced albumin influx podocytes. A similar process has been found in the lung, liver, through cell-cell junctions of monolayer podocyte under and even skin, causing fibrosis and organ failure.38–40 Wnt-On, but not under Wnt-Off, conditions. In streptozotocin- This study showed FHL2 interacts with b-catenin and af- induced diabetes, FHL2 knockout in diabetic mice significantly fects b-catenin–mediated transcription in a cell-specificman- decreased albuminuria and attenuated the DKD pathologic ner. These findings are in line with previous reports showing changes. In human data, patients with DM had a higher propor- that FHL2 and b-catenin form a protein complex in cell nu- tion of podocytes with FHL2 nuclear translocation, and this pro- cleus,21,23,41 interestingly, FHL2 could be a coactivator of portionisevenhigherinmacroalbuminuricdiabeticpatients.Of b-catenin–mediated TCF/LEF transcription in HEK293 and note, FHL2 nuclear translocation degree is a predictor of renal UDC-Mel-N melanoma cells,21,41 and also a corepressor in function deterioration in the future. These novel findings provide C2C12 myoblasts and CHO cells.23 Our data indicate that in clear evidence that FHL2 plays an important role in the develop- DKD, FHL2 acts as a coactivator of b-catenin in podocytes. ment of DKD, and FHL2 inhibition can reduce Wnt/b-catenin– Because FHL2 amplifies the podocyte damage caused by the induced podocyte abnormality. reactivation of Wnt signaling, FHL2 inhibition could be a po- Various mechanisms (e.g., activation of the angiotensin II tential therapeutic target in preventing DKD progress. pathway, the for advanced glycation end products Conventional b-catenin knockout is lethal in an early em- pathway, the TGF-b pathway, and an imbalance of VEGF/en- bryonic state, but mice with podocyte-specific b-catenin de- dothelial nitric oxide synthase expression) have been proposed letion have no major histologic changes or albuminuria. In to be involved in the pathophysiologic mechanism of DKD. addition, mice with podocyte-specific b-catenin deletion are However, glycemic control, along with currently available more resistant to proteinuric kidney disease, which suggests pharmacotherapies, cannot completely prevent the progres- the Wnt/b-catenin pathway is not required for podocyte sion of DKD.28,29 Therefore, identifying the key signaling cul- maintenance.10,14,42 Several groups have reported promising prits of DKD to explore novel therapeutic agents demands results in treating podocytopathy by the inhibition of Wnt new attention. In a microarray study performed on micro- signaling. Jiang et al. reported that blocking of Wnt signaling dissected human DKD samples, Wnt/b-catenin signaling prevents angiotensin II–induced nephropathy in mice.43 He was one of the most consistently upregulated pathways,13 et al. further showed that paricalcitol prevents doxorubicin- and, in parallel, overexpression of b-catenin in podocytes in- induced nephropathy by suppressing podocyte Wnt/b-catenin duces albuminuria and GBM abnormality.14 Wnt signaling is activation.11 Tzou et al. reported that systemic injection of tightly controlled under normal physiologic conditions and is monoclonal antibody against Wnt receptor attenuates kidney regulated at several levels: the Wnt ligand secretion, the ligand damage in genetically insulin-deficient Akita mice.12 How- receptor affinity, and the target gene transcriptional activity. ever, not all of the experiments targeting Wnt-signaling inhi- The transcriptional activity of the b-catenin–TCF complex bition were successful in treating DKD. An endogenous Wnt can be modulated by complex coactivators and corepressors, receptor antagonist DKK1 was thought to be a potential phar- such as CBP/p300,30,31 Chibby,32 the inhibitor of b-catenin macologic target for podocytopathy, but systemic DKK1 in- and TCF-4 (called ICAT),33 and TNLK/Nemo.34 jection accelerated mesangial matrix accumulation in DKD.44 Wnts are a family of signaling proteins that play an essential Kato et al. reported podocyte-specific b-catenin knockout role in embryogenesis, but their expression in the adult kidney mice have more severe podocytopenia after streptozotocin

does not affect Snail or nephrin expression under unstimulated conditions; rather, it attenuates protein expression changes caused by Wnt signaling. (D–G) Quantified FHL2, active b-catenin, Snail, and nephrin expression level. *P,0.05 compared with Wnt-Off control, #P,0.05 compared with Wnt-On control.

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injection, and the authors concluded that a balanced Wnt-signaling might be critical for maintaining the glomerular filtration barrier.14 In the current study, the diabetic 2 2 FHL2 / mice had similar podocyte num- bers and apoptosis ratio (determined by WT-1–positive cell and tissue TUNEL staining; Supplemental Material). We suggest the discrepancy might be due to the different suppression levels of Wnt-signaling. While Kato et al. totally blocked the podocyte Wnt-signaling by knocking out b-catenin, in our study we did not manipulate the Wnt/ b-catenin axis directly. We identified a coac- tivator of Wnt signaling which is expressed in podocytes, and genetic deletion of FHL2 did not affect kidney microstructure or al- buminuria. FHL2 is known to be a cofac- tor, and knockout of FHL2 only partially blocks the Wnt signaling (glomeruli from 2 2 diabetic FHL2 / mice still have elevated active b-catenin and Snail expression com- pared with nondiabetic mice). Partial in- hibition of Wnt/b-catenin signaling may achieve an internal balance between podocyte survival and differentiation in proteinuric kidney disease. To our knowledge, this study is the first to explore the role of Wnt-cofactor in proteinuric kidney disease. Because Wnt signalingispleotropicandhasvarious functions in maintaining organ homeosta- sis, systemic pharmacologic Wnt inhibition is impractical and risky. In this study, we used in vitro experiments to demonstrate that FHL2, a protein abundantly expressed in kidney podocytes, is a Wnt-coactivator that amplifies Wnt signaling and magnifies podocytopathy. Our in vivo experiments also confirmed that genetic deletion of FHL2 did not alter normal renal function

mesangial expansion in diabetic groups. The 2 2 FHL2 / mice had less mesangial expansion than did their wild-type littermates. (F) Quan- tification of relative mesangial area of the four groups. (G) Electron microscopic picture of GBM in mice; induction of DM significantly increased GBM thickness in FHL2+/+ mice, but 2 2 the severity is markedly attenuated in FHL2 / Figure 4. Knockout FHL2 attenuated DKD in vivo. (A–C) Blood glucose, hemoglobin mice. Note the foot process effacement A1c, and glomerular hyperfiltration level is comparably elevated in diabetic FHL2+/+ among diabetic FHL2+/+ mice, which is almost 2 2 2 2 and FHL2 / mice. (D) Induction of DM significantly increased urine albumin level with normal among diabetic FHL2 / mice. (H) time, and knockout FHL2 attenuated albuminuria. (E) Periodic acid–Schiff stain of Quantification of GBM thickness in four mouse glomeruli (original magnification, 3400); note the glomeruli hypertrophy and groups. NS, not significant.

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2 2 experiment, diabetic FHL2+/+ and FHL2 / mice had similar severity of hyperfiltration. Third, although we demonstrated that ge- netic FHL2 inhibition attenuates the severity of DKD, we cannot prove the beneficial effect is only on podocytopathy because FHL2 is also expressed in heart, smooth muscle, and even mesangial cells.45 Fourth, with regard to future potential clinical implications, the effect of FHL2 inhibition in other or- gan systems under DM conditions is un- known. In without DM, genetic FHL2 knockout has a beneficial effect on high-fat diet–induced atherosclerosis,46 but it also reduced the ability for neovas- cularization.20,47 Future research is needed to address these unanswered questions. In conclusion, the current study indi- Figure 5. Knockout FHL2 prevents Wnt-induced podocyte dedifferentiation in vivo. cates that FHL2 is abundantly expressed in (A) Western blot of glomeruli lysate from four groups. Induction of diabetes increased the podocyte cytoskeleton, and that high TGF-b,activeb-catenin, VEGF, Snail, FHL2, and downregulated nephrin in glomeruli 2 2 glucose and other DM-related cytokines from FHL2+/+ mice. Diabetic FHL2 / mice had similar elevation of TGF-b, active b-catenin, and VEGF levels but had less Snail upregulation and preserved nephrin upregulate FHL2 and promote its cytosol- expression. (B–D) Quantification of protein expression of b-catenin, Snail, and nephrin. to-nuclear shifting. In the podocyte nu- cleus, FHL2 acts as a coactivator to amplify Wnt/b-catenin signaling. This process is fi Table 1. Patient characteristics bene cial for nephron regeneration in lower species but is harmful for mammal kidney Control DM with ACR <300 DM with ACR >300 Characteristic (n=25) (n=20) (n=26) repair (Figure 7). We demonstrate for the first time that FHL2 plays a critical role in Age (yr) 55.4612.6 53.5612.5 49.6610.8 albuminuria and subsequent kidney damage Men, % (n) 64(16) 80(16) 69(18) Fasting blood glucose (mg/dl) 95.067.4 174.2629.3 183.3635.5 in DM. FHL2 could be a potential therapeu- Hemoglobin A1c (%) 5.460.2 7.460.4 7.860.5 tic target against diabetic kidney damage. BUN (mg/dl) 24.566.1 21.265.6 20.267.8 Creatinine (mg/dl) 0.960.2 1.160.3 1.260.3 Urinary ACR (mg/g) 1865 184675 5486142 CONCISE METHODS Values expressed with a plus/minus sign are the mean6SD. ACR, albumin-to-creatinine ratio. Histology and IHC Staining Paraffin-embedded mouse kidney (3 mmthick- but ameliorated kidney damage caused by diabetes. This ness) was stained with periodic acid-Schiff reagent by standard pro- concept was further demonstrated from clinical pathology tocol. Fifteen cortical glomeruli per mouse with a 403 lens were showing that patients with DM had a higher proportion of digitalized, and the mesangial index were calculated with MetaMorph podocytes with FHL2 nuclear translocation, and increased Imaging System as (area of periodic acid–Schiff staining/total area of FHL2 nuclear translocation was a strong predictor of renal glomerulus) 3100.48 IHC staining was performed using standard function deterioration. On the basis of these findings, we heat-induced epitope retrieval method and routine staining protocol. propose FHL2 may be an efficient therapeutic target for Human kidney specimens were obtained from diagnostic renal bi- the prevention of DKD progression. opsies and nontumor kidney tissue from patients with renal malig- Several limitations in the current study design should be nancy and nephrectomy. Patients with GFR,60 ml/min per 1.73 m2 noted. First, although our clinical data indicated that podocyte after surgery or kidney interstitial fibrosis .25% were excluded. To FHL2 nuclear translocation is strongly associated with creat- calculate the proportion of FHL2 nuclear translocation, human kid- inine doubling and the development of ESRD, we did not ney samples were double-stained with FHL2 (green) and podocyte establish a causal relationship. Second, in a rodent DKD model, nuclear markers WT-1 (red), with the yellowish cell nucleus defined the major deficiency is the absence of renal failure even if as podocyte FHL2 nuclear translocation. For each sample, five non- unilateral nephrectomy is performed to accelerate disease sclerotic glomeruli were selected to obtain a mean ratio. Taipei Veterans progression. Rodents develop the glomerular hyperfiltration General Hospital Institutional Review Board approved the human study, stage only after the development of diabetes.24 In our and all enrolled participants gave their informed consent.

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TCF/LEF Reporter and Coimmunoprecipitation The podocytes’ Wnt signal transcription activity was measured by a commercial TCF/LEF Dual- Luciferase Reporter Assay Kit according to manufacturer’s protocol (Qiagen). Briefly, dif- ferentiated podocytes were cotransfected with inducible Wnt transcription factor–responsive construct expressing firefly luciferase, and a 40:1 constitutively expressing Renilla luciferase construct as transfection control; at the same time, a noninducible firefly luciferase reporter and Renilla luciferase construct was cotransfected in another plate of cells to determining back- ground reporter activity. The Wnt transcription activity was determined by relative luciferase activity between inducible/ noninducible re- porters. For coimmunoprecipitation experi- ments, podocytes were cultured under 25 mM glucose culture medium for 48 hours and the nu- clear proteins were extracted; endogenous FHL2 and active b-catenin were coimmunoprecipitated by a commercial kit (Pierce Biotechnology) ac- cording to the manufacturer’sprotocol.

Western Blot Analysis Proteins of glomerular homogeneous or podocyte Figure 6. Podocyte nuclear translocation is associated with proteinuria and kidney cell lysis were analyzed by Western blot as described function deterioration in diabetic nephropathy. (A) FHL2 staining in patients with and 50 without DM. IHC staining showed typical podocyte staining of FHL2 in those without previously. The primary antibodies used were: b b DM; in patients with DM, however, in addition to podocyte cytosol, FHL2 is also stained anti-active -catenin (NonPhospho -catenin, in podocyte nucleus. Using double stain of podocyte nucleus marker WT-1, immu- D13A1; Cell Signaling Technology), anti-nephrin nofluorescence staining illustrated that glomeruli from diabetic kidney have a consid- (Acris, Herford, Germany), anti-snail, anti-VEGF, erably higher ratio of podocytes with FHL2 nuclear translocation. In contrast, only few anti–TGF-b (Abcam, Inc., Cambridge, UK), and podocytes from non-DM kidney showed FHL2 nuclear translocation. (B) Patient without anti-FHL2 (MBL Life Science, Nagoya, Japan). DM has a low ratio of podocyte FHL2 nuclear translocation, and a patient with DM and albuminuria .300 mg has the highest FHL2 nuclear translocation ratio. (C) In patients Immunofluorescence Staining and with DM, podocyte FHL2 nuclear translocation is a strong indicator for renal function Confocal Microscopy deterioration. Kidney cryosections and cultured podocytes were fixed with 4% paraformalin for 15 minuted and in 11% Triton X-100 for 10 minutes. After Podocyte Cell Culture blocking with 10% bovine albumin for 30 minutes, the slides were The conditional immortalized human podocyte line was kindly immunostained with primary antibodies against nephrin, FHL2, total b provided by Dr. Morin Saleem and cultured as previous described.49 -catenin or WT-1 (Abcam, Inc.) overnight. Slides were viewed un- Cells were cultured at 33°C in RPMI 1640 medium supplemented der an Olympus FV10i confocal microscope. with 10% FBS and 1% ITS (Sigma-Aldrich, St. Louis, MO). To induce differentiation, podocytes were moved to 37°C. All experiments were Mouse Model of Diabetes performed 14 days after thermoshift. To test the protein expression To clarify the effects of FHL2 on DKD, we created type 1 DM in 2 2 under different culture conditions, podocytes were treated with FHL2 / mice and their wild-type littermates and evaluated the sub- 2 2 TGF-b (5 ng/ml; R&D Systems), 25 mM glucose, and angiotensin sequent diabetic kidney damages. FHL2 / mice were generated in a B6 2 II (10 6 M; Sigma-Aldrich). A selective GSK3 inhibitor, SB216763 genetic background as previously described, and FHL1 and FHL3 were (5 mM; Sigma-Aldrich) was used to activate Wnt signaling directly in not increased in these FHL2 null mice.51 DM was induced by daily vitro. For some experiments, podocytes were transfected with Wnt intraperitoneal injections of streptozotocin (Sigma-Aldrich), 50 mg/kg TCF/LEF reporter plasmid, scrambled siRNA, or FHL2 siRNA for5days,in8-week-oldmalemice.InductionofDMwasconfirmed with (Santa Cruz Biotechnology) by electroporation (Neon Transfection fasting blood glucose level .300 mg/dl 2 weeks after streptozotocin injec- System; Invitrogen, Carlsbad, CA). tion. Diabetic mice received small-dose sustained-release insulin implants

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nephrectomy was performed 1 week before strepto- zotocin injection to accelerate progression of DKD.52 The mice were divided into four groups: FHL2+/+ control group, FHL2+/+ DM group, 2 2 2 2 FHL2 / control group, and FHL2 / DM group (n=12–15 for each group). All mice were housed under standard conditions with normal food and euthanized 4 months after induction of diabetes. The Institutional Care Committee of Taipei Veterans General Hospital approved all experimental procedures involving animals.

GFR and Urine Albumin Measurement Mouse GFR was assessed by the FITC-inulin clearance method as previously described.53 For measurement of urine albumin, mice were placed in metabolic cages. The 24-hour urine was centrifuged and albumin level was deter- mined using a commercialized ELISA (Albuwell M kit; Exocell Inc., Philadelphia, PA). Because the Jaffe method overestimates creatinine levels in rodents, mouse urine creatinine was deter- mined by HPLC.

Electron Microscopy Mouse kidney cortex was fixed with 2.5%

glutaraldehyde followed by 0.5% OsO4.These were then series dehydrated and embedded ac- cording to routine procedures. Thin sections were viewed and digitally recorded using a transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan). The GBM thickness was determined by the orthogonal intercept method.54 In essence this applies a correction factor for oblique sectioning to the harmonic mean of a large number of random orthogonal intercept measurements of the GBM of several glomeruli.55,56 Glomerular peripheral capillary loops were identified under electron micros- copy. To eliminate bias in the sectioning area, the capillary loops were photographed. The shortest distance between the endothelial cyto- Figure 7. Schematic representation of proposed pathologic role of FHL2 in diabetic plasmic membrane and the outer lining of the nephropathy. In normal condition, FHL2 is located on the podocyte cytoskeleton and lamina rara externa underneath the cytoplasmic cytosol b-catenin is degraded via proteasomes; they have no protein-protein in- membrane of the podocyte foot processes was teraction. In the diabetic condition, high glucose and other DM-related cytokines measured with a logarithmic ruler. The apparent b activate Wnt signaling in podocytes, increase cytosol -catenin, and promote its nu- harmonic mean thickness (lh) was calculated, clear translocation; on the other hand, these DM-related cytokines also active FHL2, from which the true harmonic mean thickness FHL2 serves as a b-catenin coactivator in podocytes nucleus, and FLH2 amplifies the (Th) was estimated by the following equation: target gene transcription. Wnt-target , such as Snail, cause podocyte de- differentiation and downregulate the important slit-diaphragm protein nephrin. 8 106 Th ¼ 3 3lh; 3p M (half-tablet per mouse) (Linbit; LinShin Canada, Inc., Scarborough, Canada); this dose of insulin does not remarkably affect blood glucose where M represents the final print magnification (15,0003 in current but can prevent ketoacidosis and death in diabetic mice. Left study).57 In the current study, five glomeruli of each mouse were analyzed.

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Statistical Analyses 13. Woroniecka KI, Park AS, Mohtat D, Thomas DB, Pullman JM, Susztak K: All values are expressed as mean6SD unless otherwise specified. Dif- Transcriptome analysis of human diabetic kidney disease. Diabetes 60: ferences between multiple groups were evaluated for significance 2354–2369, 2011 14. Kato H, Gruenwald A, Suh JH, Miner JH, Barisoni-Thomas L, Taketo using ANOVA analysis with a Bonferroni post hoc test. Statistical anal- MM, Faul C, Millar SE, Holzman LB, Susztak K: Wnt/b-catenin pathway yses were performed using GraphPad Prism software, version 5.0 in podocytes integrates cell adhesion, differentiation, and survival. (GraphPad Software, San Diego, CA). A two-tailed P value ,0.05 was J Biol Chem 286: 26003–26015, 2011 considered to represent a statistically significant difference. 15. Chu PH, Ruiz-Lozano P, Zhou Q, Cai C, Chen J: Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system. Mech Dev 95: 259–265, 2000 ACKNOWLEDGMENTS 16. Zheng Q, Zhao Y: The diverse biofunctions of LIM domain proteins: determined by subcellular localization and protein-protein interaction. Biol Cell 99: 489–502, 2007 This study was supported, in part, by the following research grants: 17. Shathasivam T, Kislinger T, Gramolini AO: Genes, proteins and com- NSC 102-2633-B-075-001, NSC 102-2314-B-182A-060-MY2 and plexes: The multifaceted nature of FHL family proteins in diverse tis- 103-2314-B-075-004, UST-UCSD International Centre of Excellence sues. JCellMolMed14: 2702–2720, 2010 in Advanced Bio-engineering, NSC-100-2911-I-009-101-A2 from the 18. Slinin Y, Ishani A, Rector T, Fitzgerald P, MacDonald R, Tacklind J, Rutks I, Wilt TJ: Management of hyperglycemia, dyslipidemia, and Ministry of Science and Technology, VGH-V102B-016 and VGH- albuminuria in patients with diabetes and CKD: a systematic review V102E2-002 from Taipei Veterans General Hospital, and for a KDOQI clinical practice guideline. AmJKidneyDis60: 747–769, CMRPG3B1311 from Chang Gung Memorial Hospital. 2012 19. Welsh GI, Hale LJ, Eremina V, Jeansson M, Maezawa Y, Lennon R, Pons DA, Owen RJ, Satchell SC, Miles MJ, Caunt CJ, McArdle CA, Pavenstädt H, Tavaré JM, Herzenberg AM, Kahn CR, Mathieson PW, DISCLOSURES Quaggin SE, Saleem MA, Coward RJ: Insulin signaling to the glomer- None. ular podocyte is critical for normal kidney function. Cell Metab 12: 329– 340, 2010 20. 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AFFILIATIONS *Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital and Institute of Clinical Medicine, National Yang-Ming † University, Taipei, Taiwan; Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital and Institute of Clinical ‡ Medicine, and Cardiovascular Research Center, National Yang-Ming University, Taipei, Taiwan; Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, and Institute of Physiology, National Yang-Ming University, Taipei, Taiwan; §Department of Urology, Taipei Veterans General Hospital, Department of Urology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; | Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital and School of Medicine, National Yang-Ming University,

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Taipei, Taiwan; ¶Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, and Institute of Anatomy and Cell Biology, National Yang-Ming University, Taipei, Taiwan; **Division of Hematology and Oncology, Department of Medicine, Taipei Veterans †† General Hospital and Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; Department of Medical Research, Taipei Veterans General Hospital, Institute and Department of Pharmacology, and Cardiovascular Research Center, National Yang-Ming University, ‡‡ Taipei, Taiwan; The Institute of Engineering in Medicine, University of California San Diego, La Jolla, California; §§Departments of || Bioengineering, Nanoengineering, Institute of Engineering in Medicine, University of California San Diego, La Jolla, California; and Division of Cardiology, Department of Internal Medicine; Healthcare Center; Heart Failure Center, Chang Gung Memorial Hospital, Chang Gung University, College of Medicine, Taipei, Taiwan

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