Nephrin Deficiency Activates NF-B and Promotes Glomerular Injury

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Nephrin Deficiency Activates NF-␬B and Promotes Glomerular Injury

Sagair Hussain,* Leile Romio,* Moin Saleem,† Peter Mathieson,† Manuel Serrano,‡ Jorge Moscat,§ Maria Diaz-Meco,§ Peter Scambler,* and Ania Koziell*

*Molecular Medicine Unit, Institute of Child Health, London, and †Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, United Kingdom; ‡Spanish National Cancer Research Centre, Madrid, Spain; and §Department of Genome Science, Genome Research Institute, University of Cincinnati, Cincinnati, Ohio

ABSTRACT Increasing evidence implicates activation of NF-␬B in a variety of glomerular diseases, but the mechanisms involved are unknown. Here, upregulation of NF-␬B in the podocytes of transgenic mice resulted in glomerulosclerosis and proteinuria. Absence of the podocyte protein nephrin resulted in NF-␬B activation, suggesting that nephrin negatively regulates the NF-␬B pathway. Signal transduction assays supported a functional relationship between nephrin and NF-␬B and suggested the involvement of atypical protein kinase C (aPKC␨/␭/␫) as an intermediary. We propose that disruption of the slit diaphragm leads to activation of NF-␬B; subsequent upregulation of NF-␬B-driven genes results in glomerular damage mediated by NF-␬B-dependent pathways. In summary, nephrin may normally limit NF-␬B activity in the podocyte, suggesting a mechanism by which it might discourage the evolution of glomerular disease.

J Am Soc Nephrol 20: 1733–1743, 2009. doi: 10.1681/ASN.2008111219

NF-␬B is a transcription factor activated by cell sur- of aPKC␨/␫ inhibitory proteins such as Par4 can also face receptor signaling to meet stress and inflam- abrogate NF-␬B activation.12,13 matory responses, regulating key cellular processes Glomerular disease manifests as urinary protein such as inflammation, innate and adaptive immu- leak resulting from malfunction of the glomerular nity, and cell growth and survival.1 Five mamma- filtration barrier. Podocytes separated by slit dia- lian NF-␬B proteins share a Rel homology domain phragms are crucial in maintaining barrier integ- with composition of the active dimer dictated by rity.14 Injury disrupts their actin cytoskeleton, caus- cell type and nature of inducing stimulus.2 Inactive ing foot process effacement; detachment from the NF-␬B is sequestered in the cytoplasm bound to glomerular basement membrane (GBM), with sig- I␬B3; phosphorylation of I␬B releases active NF-␬B, nificant molecular reorganization of the slit dia- which translocates to the nucleus to induce an ex- phragms; and urinary protein loss.15 tensive range of target genes.4 RelA dimers are the Nephrin, an Ig superfamily member, is a key slit dia- most abundant and potent gene transactivators phragm component and presumed adhesion molecule within the family.5 that contributes directly to a physical filtration barrier at Induction of NF-␬B signaling and specificity of transcriptional response are dependent on a com- Received November 30, 2008. Accepted March 17, 2009. plex interplay of pathways. Adaptor proteins p626 7 Published online ahead of print. Publication date available at and MyD88 and intracellular messengers such as www.jasn.org. atypical protein kinase C (aPKC␨/␫)8 connect ␬ ␨ ␫ Correspondence: Dr. Ania Koziell, Molecular Medicine Unit, In- NF- B with cell surface receptors. aPKC / acti- stitute of Child Health, 30 Guilford St, London, WC1N 1EH, UK. vates NF-␬B by either release from I␬B9 or direct Phone: 00-44-207-242-9789, ext. 0713, 00-44-783-481-4994; nuclear phosphorylation,10 whereas activation is se- Fax: 00-44-207-905-2609; E-mail: [email protected] verely impaired by aPKC␨ deficiency.11 Expression Copyright ᮊ 2009 by the American Society of Nephrology

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the slit diaphragm.16,17 Nephrin mutations result in disruption of the actin cytoskeleton and severe glomerular disease. Downregulation of nephrin expression unrelated to gene mutations also occurs in podocyte injury,18,19 and mutations that are within the cytosolic tail and do not affect protein expression cause equivalent glomerular disease,20,21 supporting a role in cellular signaling. This is verified by evidence that nephrin serves as a signaling scaffold to recruit other podocyte proteins,22–25 is phosphorylated by Src- family kinases,26,27 and associates with adapters such as Nck to regulate the actin cytoskeleton.28,29 To our knowledge, the mechanism of NF-␬B regulation in glomerular cells is unknown. Evidence for the involvement of NF-␬B activation in glomerular and renal tubular cell injury is pro- vided by correlative studies of human Figure 1. Glomerular disease is present in Par4Ϫ/Ϫ mice. (A) A total of 4 ␮l of urine from kidney disease30,31 and experimental 3-mo-old WT (WT1 and WT2) and Par4Ϫ/Ϫ (KO1 through 6) mice was suspended in Laemmli disease models.31,32 Complement acti- buffer and subjected to SDS-PAGE. Coomassie staining detected proteinuria in Par4Ϫ/Ϫ mice but vates NF-␬B and NF-␬B–dependent not their WT counterparts. A band corresponding to albumin is shown (68 kD; black arrow). The gene transcription in podocytes in variability in proteinuria resolved with increasing age of mice. (B) Hematoxylin and eosin staining of Par4Ϫ/Ϫ and WT glomeruli at 2 wk (a and b), 3 mo (c and d), and 6 mo (e and f). At 2 wk, only vitro,33 and upregulation of IL-1, IL-4, mild mesangial hypercellularity and glomerular enlargement is present in Par4Ϫ/Ϫ glomeruli, with and TNF-␣is detected in injured podo- preservation of the tubulointerstitial compartment (a). At 3 mo, marked mesangial expansion, cytes, supporting a direct role for glomerulosclerosis, and widening of the subcapsular space is evident, with some capsular adhe- ␬ NF- B activation in human glomerular sions (c). Significant disease progression occurs by 6 mo, with widespread scarring and atrophy of disease.30,31 Whether nephrin modu- glomeruli, tubular atrophy, and pseudocysts containing proteinaceous material (arrow; E). In 25% lates NF-␬B has not been investigated, of mice, coexisting renal tubular cysts were lined by simple epithelium (*), which also progressed but regulation of NF-KB activation by in severity with age. Glomeruli and renal tubules were normal at all ages in WT controls (B, D, and other Ig superfamily molecules in- F). (C) Par4Ϫ/Ϫ glomeruli were also abnormal at the ultrastructural level (c through h). In 2-wk-old volved in cell adhesion, namely N-Cam mice (c and d), there is discontinuous podocyte foot process effacement and mild cell body and L1Ig6, is well described.33,34 swelling, but the GBM is normal. By 3 mo, there is marked effacement and swelling with hyaline We investigated a causal link be- deposits and vacuoles, glomerulosclerosis, mesangial expansion, and patchy blebbing of the GBM tween NF-␬B activation and renal (e and f). By 6 mo, podocyte swelling, sclerosis, and foot process fusion is extensive, with diffuse thickening, corrugation, and denudation of the GBM. The mesangial matrix is folded and col- glomerular damage by examining lapsed (g and h). By contrast, normal podocyte and glomerular architecture is seen in WT mice and whether genetic inactivation of an littermates (a and b). Podocyte foot processes are well demarcated, and the mesangium is normal ␬ upstream inhibitor of NF- B acti- with preservation of the urinary space and no tubular cysts. Magnifications: ϫ40 in B; ϫ300 in C vation could result in glomerular in- (a, c, e, and g); ϫ6000 in C (b, d, f, and h). jury independent of immune cell activation. Considering its ubiquitous role in maintaining glo- notype has been reported in conjunction with mouse models merular filtration, we then explored whether the receptor pro- resulting in NF-␬B deficiency, we examined the significance of tein nephrin participates in the regulation of this process. NF-␬B activation in the kidney in vivo, focusing on models with constitutive activation of NF-␬B resulting from removal of natural pathway inhibition. This conventional approach en- ␬ RESULTS ables study of NF- B overexpression while avoiding off-target effects.35 Activation of NF-␬B Results in Glomerulosclerosis and One such model involves genetic inactivation of the prostate Proteinuria In Vivo apoptosis response 4 gene (Par4), which specifically binds and We first tested the hypothesis that NF-␬B activation is a key inhibits aPKC␨/␭/␫ and is a critical negative regulator of the RelA/ response leading to glomerular damage. Because no renal phe- NF-␬B pathway.12,13 Disruption of Par4 through homologous re-

1734 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1733–1743, 2009 www.jasn.org BASIC RESEARCH combination in outbred (CD1) wild-type (WT) mice resulted in de-repression of aPKC␨/␭/␫ and activation of NF-␬B.36 Al- though Par4 is ubiquitously expressed, no clear systemic phenotype was identified. We therefore examined Par4Ϫ/Ϫ mice for renal abnormalities. Par4Ϫ/Ϫ mice were studied at 2 wk, 3 mo, and 6 mo (n ϭ 40). Although mice ini- tially seemed normal, by 3 mo, the majority had developed significant proteinuria (Fig- ure 1A). Analysis of kidneys by light microscopy at 2 wk showed mild mesangial expansion within glomeruli and ultrastructural evidence of foot process fusion indicative of a primary podocyte defect, supported by evidence of some podocyte loss on electron microscopy (Figures 1, Ba and C, c and d). At 3 mo, increasingly severe glomeruloscle- Figure 2. RelA and aPKC␨/␭/␫ are activated in Par4Ϫ/Ϫ podocytes. (A) Double- rosis was apparent with hyalinosis, tubular immunofluorescence staining of WT and Par4Ϫ/Ϫ glomeruli showing expression of atrophy, pseudocysts, and luminal deposi- RelA/NF-␬B (a and b) and acetylated tubulin (c and d) in murine podocytes (white tion of proteinaceous material (Figure 1Bc). arrows), with image merge (e and f). RelA expression is cytoplasmic in WT podocytes At the ultrastructural level (Figure 1C), (a and e), indicative of quiescence, and nuclear in Par4Ϫ/Ϫ podocytes (b and c), light microscopic changes were mirrored by indicative of activation even at 2 wk of age. (B) Double-immunofluorescence staining Ϫ Ϫ ␨ ␭ ␫ progressive disruption of podocyte archi- of WT and Par4 / glomeruli shows phospho-aPKC / / (Thr410/403) (a and b) and tecture and foot process fusion, with glo- acetylated tubulin (c and d) in murine podocytes (white arrows), with image merge (e merular hypertrophy, abnormal duplica- and f). Phospho-aPKC expression is predominately nuclear in WT podocytes with some cytoplasmic staining (a and e), whereas in Par4Ϫ/Ϫ glomeruli, the equilibrium tion of the GBM, and hyaline deposits shifts to cytoplasmic predominance (b and c) even at 2 wk of age. Magnification, ϫ63 clearly apparent (Figure 1C, e and f). By 6 (oil immersion lens). mo of age, glomerular disease was severe, and approximately 30% of animals had developed a coincident sis, we examined the direct NF-␬B target of this pathway, RelA, renal cystic tubular phenotype not dependent on the degree of and phospho-aPKC␨ expression in glomeruli of Par4Ϫ/Ϫ and proteinuria or gender (Figure 1B, g and h). Because crosses WT mice at 2 wk, 3 mo, and 6 mo of age. Because activated RelA were performed on an outbred background, a degree of genetic localizes to the nucleus, we investigated the subcellular localiza- heterogeneity was expected. Ultrastucturally, complete disruption in glomeruli for evidence for NF-␬B activation in situ. Con- tion of podocyte architecture was evident, with extensive foot focal immunofluorescence microscopy of cortical kidney sections process fusion, massive cell body swelling, sclerosis, collapse showed cytoplasmic RelA expression in WT podocytes indicative and folding of the mesangial matrix, and denudation of the of quiescence (Figure 2A, a, c, and e). Conversely, a shift to nuclear diffusely thickened and corrugated GBM. expression signified activation in Par4Ϫ/Ϫ podocytes (Figure 2A, Glomerular disease was detected in Ͼ90% of mice and the b, d, and f). This activation was not detected in other Par4Ϫ/Ϫ concurrent cystic tubular phenotype in approximately 25%. glomerular cells or in WT glomeruli. Both phenotypes became progressively milder with subse- Similarly, we confirmed the activation and localization of quent crosses to WT (CD1) mice, as the increasing genetic aPKC␨ using a phospho-specific antibody. A shift in the equi- heterogeneity allowed the epigenetic influence of different librium of phospho-aPKC␨(Thr410/403) expression from pre- modifying loci to emerge. Littermates and WT mice with the dominately nuclear in WT podocytes (Figure 2B, a, c, and e) to same genetic background, however, showed no functional or mainly cytoplasmic in Par4Ϫ/Ϫ (Figure 2B, b, d, and f) sup- morphologic renal phenotype (Figures 1, B, b, d, and f, and C, ported aPKC activation in Par4Ϫ/Ϫ glomeruli. a and b). These data suggested direct activation of the aPKC/ Downregulation of Wilms’ tumor 1 (WT1) and nephrin NF-␬B axis results in glomerular and renal tubular disease in was detected in Par4Ϫ/Ϫ but not WT glomeruli (Figure 3, A, vivo. B, G, and H), confirming podocyte dysregulation and loss. PAX2 expression was normal in glomeruli and tubules of Activation of RelA/NF-␬B and aPKC␨ Occurs in the Par4Ϫ/Ϫ mice (Figure 3, C through F), going against WT1- -Podocytes of Par4؊/؊ Mice mediated podocyte disease.37–39 Accordingly, through its con To confirm whether aPKC/NF-␬B pathway activation was nection with slit diaphragm signaling,26–29 we examined present in Par4Ϫ/Ϫ podocytes to contribute to glomerulosclero- whether nephrin might regulate NF-␬B activation.

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Figure 4. RelA/NF-␬B is activated in nephrin-deficient human podocytes. (A) Western blot of WT, MT, and MTϩN whole-cell protein extracts of human podocytes. All lanes were equally loaded with 20 ␮g of protein extract verified by both Ponceau S staining (data not shown) and reprobing of the membrane. Up- regulation of RelA is present in MT cells, with rescue in MTϩN. No difference was seen for p38, pJNK, BCL2, PTEN, or PDK1; data shown for BCL2. (B) Upregulation of phospho-I␬B (Ser-32/36) was Figure 3. Immunohistochemical analysis of WT and Par4Ϫ/Ϫ detected in nephrin-deficient MT cells, endorsing NF-␬B activa- glomeruli to show expression of podocyte markers WT1, PAX2, tion through release from I␬B␣, with rescue occurring on re- and nephrin. (A and B) Normal nuclear WT1 expression is seen in introduction of nephrin expression (MT-N). Total I␬B␣ expression WT podocytes (A), whereas in Par4Ϫ/Ϫ glomeruli WT1 expres- is equivalent between WT and MT, as expected. (C) Immunoflu- sion is virtually absent (B). (C and D) Nuclear PAX2 expression is orescence staining of differentiated podocytes. Expression of absent in WT and Par4Ϫ/Ϫ glomeruli. (E and F) Renal tubular RelA is cytoplasmic in WT, indicative of quiescence (a, white PAX2 expression was equivalent and is included as a control. (G arrows), whereas in mutant podocytes (MT) RelA translocates to and H) Nephrin expression is normal in WT podocytes (G) but the nucleus consistent with activation (b). Expression is rescued to significantly downregulated in Par4Ϫ/Ϫ glomeruli (H). Immuno- the cytoplasm on re-introduction of nephrin by stable transfection histochemistry was performed on paired samples under identical (c; MTϩN). Magnification, ϫ40. experimental conditions. Data are shown for 3-mo-old mice, with abnormalities detected from 2 wk of age. Par4. Upregulation of phospho-I␬B (Ser-32/36) but not total Loss of Nephrin Results in RelA/NF-␬B Activation in I␬B in MT endorsed RelA/NF-B activation through release Human Podocytes from the I␬B complex (Figure 4B). To study the mechanism of NF-␬B activation in podocytes in We used immunofluorescence confocal microscopy to more detail, we turned to an in vitro model. Cell lysates from confirm RelA/NF-␬B activation through its subcellular lo- unstimulated WT human immortalized podocytes40 were first calization in WT and MT podocytes (Figure 4C). In WT immunoblotted to identify components of the NF-␬B signal- cells, RelA/NF-␬B was sequestered in the cytoplasm, indic- ing pathway. All five isoforms RelA, RelB, c-Rel, p105/p50, and ative of quiescence. Conversely, expression was nuclear in p100/p52; I␬B␣ and I␬B␤; and the IKK␣ and IKK␤ compo- MT, indicating activation. This was specific to nephrin, be- nents of the IKK enzyme complex were detected, verifying the cause re-introduction of WT nephrin in MT podocytes presence of their orthologs in murine podocytes.41 NF-␬B iso- (MTϩN) rescued RelA/NF-␬B expression to the cytoplasm form expression was then examined in nonstimulated mutant and to WT levels on immunoblot. human podocytes (MT) with a constitutive 121delCT frame- shift mutation lacking functional nephrin.42 Interestingly, we Absence of Nephrin Relocates Phospho-aPKC␨ detected specific upregulation of the RelA/NF-␬B isoform in Expression to the Cytoplasm MT cells compared with WT (Figure 4A). Reprobing of mem- Given that our mouse studies had implicated aPKC␨ in the branes with antibodies to other proteins associated with mechanism of RelA/NF-␬B activation in Par4Ϫ/Ϫ podocytes, NF-␬B including PTEN, PDK1, pJNK, p38, Par4, and BCL2 we also compared expression levels and subcellular localiza- showed equivalent expression between WT and MT, verifying tion of aPKC␨ in WT and MT podocytes. Immunoblot of equal protein loading (data shown for BCL2). This indicated podocyte cell extracts showed upregulation of phospho- that NF-␬B activation in MT podocytes was independent of aPKC(Thr410/403) in MT podocytes, whereas total aPKC ex-

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pression was equivalent (Figure 5A). Immunofluorescence confocal microscopy also detected predominately nuclear phospho-aPKC␨ expression in WT, with a shift to the cytoplasm in MT podocytes (Figure 5B). Re-expression of WT nephrin in MT cells (MTϩN) rescued phospho-aPKC␨ to the cytoplasm, supporting a direct link between nephrin and aPKC␨ expression.

NF-␬B Repression in Response to Nephrin Is Specific and Independent of Podocin To substantiate a direct relationship between nephrin and NF-␬B signaling, we designed an in vitro functional assay to test NF-␬B promoter activity using a cis-acting NF-␬B en- hancer element linked to a luciferase reporter (pNF-␬B). Co- transfection of B-gal, a cytomegalovirus (CMV)-driven constitutive reporter, was used to standardize transfections. Assays were performed in cultured MDCK cells and in human podocytes. WT nephrin (pCMV-NPHS1; amino acids [AA] 1 through 1241) and two truncated nephrin mutants encompassing AA 1 through 1201 and AA 1 through 1160 were used for experiments. Equivalent expression and membrane localization of all WT and mutant nephrin proteins were verified by immunocytochemistry and immunoblot (data not shown). Truncation mutants shorter than R1160X did not demonstrate equivalent expression to WT nephrin. The R1160X mutant mimicked the most C-terminal mutation detected in human nephrotic syndrome to date,20,21 which removes a key functional region43,44

Figure 6. pNF-␬B reporter activity is repressed by nephrin. (A) pNF-␬B reporter activity was significantly repressed (P Ͻ 0.001) by co-transfection with full-length WT nephrin NPHN AA 1 through 1241. Sequential truncation to produce mutant nephrin proteins NPHN AA 1 through 1201 and NPHN AA 1 through 1160 resulted in a graded reduction in the repression of pNF-KB reporter activity. (B) Full-length podocin (POD) did not alter pNF-␬B reporter activity or its repression by WT NPHN, indicating that this Figure 5. aPKC␨/␭/␫ is activated in nephrin-deficient human observation was specific to and independent of podocin. (C) podocytes. (A) Upregulation of phospho-aPKC␨/␭/␫(Thr410/403) pNF-␬B reporter was also examined in human podocytes (MT). is seen in nephrin-deficient podocytes on Western blot. Equal Significant (P Ͻ 0.001) repression of reporter activity was again protein loading was verified by Ponceau S staining and reprobing seen with both transient and stable transfections of WT NPHN. with an antibody against total aPKC␨/␭/␫, which shows equivalent This confirmed observations made in MDCK cells. expression between WT and MT podocytes. (B) Immunofluores- cence staining of cultured human podocytes shows nuclear se- 28,29 questration of phospho-aPKC␨/␭/␫(Thr410/403) (white arrows, a) that includes three Nck SH2-binding motifs that may act 45,46 in WT podocytes and cytoplasmic expression in nephrin-deficient cooperatively to stimulate actin reorganization. ␬ podocytes (MT; b). Re-introduction of nephrin in podocytes Transfection of WT nephrin consistently repressed pNF- B (MTϩN) rescues the phenotype to predominantly nuclear expres- reporter activity to between half and two thirds of basal (P Ͻ sion (c). Magnification, ϫ40. 0.001), with the sequentially truncated mutants resulting in a

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Nephrin-Mediated Repression of pNF-␬B Is Abrogated by Dominant Negative Pathway Intermediaries A number of molecules adapt between cell surface receptors and NF-␬B. We therefore used three different dominant negative pathway intermediaries linked to NF-␬B to validate findings. A key step in RelA/NF-␬B activation is the phosphorylation of the I␬B complex.4 Transfection of dominant negative I␬B␣ (pCMV-I␬M; Clontech, Mountain View, CA) squelched pNF-␬B reporter activity and blocked nephrin-mediated repression of pNF-␬B (Figure 7A), corroborating NF-␬B activation through release from cytoplasmic I␬B. In a separate experiment, we examined the effect of co- transfection of dominant negative aPKC␨. This abrogated nephrin-induced repression of pNF-␬B, substantiating a role for aPKC␨ as a pathway intermediary (Figure 7B). An interaction between aPKC␨ and cytosolic nephrin was confirmed by GST immunoprecipitation (Figure 8A) and co-localization in WT podocytes (data not shown).

Figure 7. Pathway mapping of nephrin-mediated repression of NF-␬B. (A) Repression of pNF-␬B by NPHN is abrogated by co-transfection with dominant negative I␬B␣ (I␬M) and rescued by co-transfection with WT I␬B in MDCK cells. (B) Repression of pNF-␬B by NPHN is abrogated by co-transfection with dominant negative aPKC␨ (PKC-DN) in MDCK cells. (C) Repression of pNF-␬B by WT NPHN is abrogated by increasing concentrations of dominant negative 14-3-3 (DN1 ϭ 4 ␮g/␮l; DN2 ϭ 6 ␮g/␮l) in MDCK cells.

graded reduction in pNF-␬B repression, indicating specificity to nephrin (Figure 6A), and raising the possibility of multiple Figure 8. aPKC␨ and 14-3-3␪ bind cytosolic nephrin (AA 1057 binding sites for cytoplasmic adaptor proteins linking nephrin through 1241). (A) Successful glutathione precipitation of na- to NF-␬B signaling. tive aPKC␨ from MDCK cells by cytosolic nephrin (AA 1057 AP1 was activated by nephrin, in keeping with previous through 1241). Blots were probed with aPKC␨ (rabbit poly- ␪ studies,43,46 whereas nephrin had no effect on pNFAT (data not clonal). Lanes 1 and 2, GST alone, no precipitation of 14-3-3 ␪ shown). Podocin, a key nephrin interactor47 that is known (lane 2); lanes 3 and 4, precipitation of endogenous 14-3-3 by to enhance nephrin-mediated AP1 activation in vitro44,45 the GST-cytosolic nephrin (AA 1057 through 1241) fusion protein in lane 4. (B) Successful glutathione precipitation of native had no effect on nephrin-mediated repression of pNF-␬B, in- 14-3-3␪ from MDCK cells by cytosolic nephrin (AA 1057 dicating this was independent of podocin (Figure 6B). Find- through 1241). Blots were probed with 14-3-3␪ (rabbit poly- ings in MDCK cells were mirrored by transient and stable clonal; Santa Cruz Biotechnology). Lanes 1 and 2, GST alone, transfection of WT nephrin into human podocytes, which also no precipitation of 14-3-3␪ (lane 2); lanes 3 and 4, precipitation resulted in significant repression of pNF-␬B activity (P Ͻ of endogenous 14-3-3␪ by the GST-cytosolic NPHN (AA 1057 0.001; Figure 6C). through 1241) fusion protein (lane 4).

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and non–immune-mediated glomerular injury implicate dysregulation of the NF-␬B pathway in its pathogenesis.30–33 In response to this, we asked whether a direct causal link between NF-␬B activation and glomerular disease exists in vivo and whether the key podocyte protein nephrin might regulate this pathway. We postulated that NF-␬B activation may result in podocyte injury. To test this, we examined the kidneys of a mouse model with genetic inactivation of Par4, which negatively regulates the RelA/NF-␬B pathway through inhibition of aPKC.12,13 Par4Ϫ/Ϫ mice exhibited hyperactivation of NF-␬B and, in keeping with our hypothesis, developed a primary podocyte defect within the first 2 wk of life. Rapid progression to severe glomerular disease combined with aPKC/RelA/ NF-␬B activation in podocytes indicated pathway control may be essential for integrity of the glomerular filtration barrier. RelA/NF-␬B activation in Par4Ϫ/Ϫ podocytes and glomerular proteinuria in Par4Ϫ/Ϫ mice connected aberrant slit diaphragm signaling with induction of NF-␬B–driven genes and podocyte injury. Constitutive activation of RelA/NF-␬Bin nephrin mutant podocytes, rescued by WT nephrin, endorsed a direct link between nephrin deficiency and RelA/NF-␬B activation in vitro. Signal transduction assays demonstrated nephrin-mediated repression of pNF-␬B, corroborating cell culture findings. Nephrin downregulation in Par4Ϫ/Ϫ podocytes might be distal to NF-␬B–induced transcription; however, identification of aPKC␨ and 14-3-3 as nephrin-binding partners illustrated the likely complexity of pathway interac- ␬ Figure 9. RelA/NF-␬B activation may lead to podocyte injury. (A) tions. Both are directly associated with NF- B activation as Opposition of aberrant slit diaphragm signaling by nephrin, no well as regulation of the actin cytoskeleton in podocytes.49–51 NF-␬B activation. (B) Absence of nephrin allows NF-␬B activation The corollary of results in the Par4Ϫ/Ϫ model and in vitro and podocyte injury. supports a hypothesis that nephrin may also exert a negative tone on NF-␬B activity in vivo. Surprising, dominant negative 14-3-3, an intracellular adapter Downregulation of developmental markers WT1 and neph- that modulates RelA/NF-␬B signaling through an independent rin in Par4Ϫ/Ϫ glomeruli was consistent with podocyte injury mechanism,48 also blocked nephrin-induced repression of and loss. Although Par4 co-represses transcription with pNF-␬B (Figure 7C). The significance is unclear, although yeast WT152, absence of other WT1-linked developmental abnor- two-hybrid screening revealed that 14-3-3 interacts with nephrin malities and normal expression of WT1 targets PAX2 and (data not shown), confirmed by GST immunoprecipitation (Fig- BCL2 in Par4Ϫ/Ϫ podocytes precluded WT1 as a direct cause. ure 8B) and co-localization (data not shown). Notably, the cystic tubular phenotype supported a direct Par4 effect, because this is a crucial factor in the regulation of renal tubular cell death and survival, although renal carcinoma was DISCUSSION not detected.53 Par4 deficiency also resulted in mild T cell proliferation,54 although after the onset of kidney disease. The lack NF-␬B deviates from the classical receptor signal paradigm of an overt immunologic phenotype or inflammatory cell in- through an ability to respond to cellular stress and microbial filtrate in Par4Ϫ/Ϫ glomeruli also indicated systemic immu- pathogens, both implicated in the pathogenesis of glomerular nologic derangement was an unlikely primary cause. disease. Its stimuli and target genes are extraordinarily diverse, The shift in aPKC␨ expression may be associated with its differing greatly depending on cell and stimulus context. Con- ability to act as a molecular switch,55 playing a beneficial sequently, tight regulation of NF-␬B activity is a prerequisite role in the nucleus through combinatorial transcriptional for ensuring that coordinated processing and integration of co-repression or co-activation10 but moving to the cyto- stimuli occur. plasm to phosphorylate I␬B56 and release RelA/NF-␬Bas Although the significance of NF-␬B activation in glomeru- occurs in renal tubular cell injury.57 aPKC␨ and NF-␬B di- lar disease is incompletely understood, a series of correlations rectly regulate cytoskeletal stability,58–60 which may aug- between the activation of NF-␬B and onset of both immune- ment podocyte damage. Upregulation of RelA/NF-␬Bin

J Am Soc Nephrol 20: 1733–1743, 2009 Nephrin Regulates NF-␬B 1739 BASIC RESEARCH www.jasn.org nephrin-deficient podocytes indicated abnormal NF-␬B Verification of cDNA constructs was by ABI automated sequencing turnover, usually under tight dynamic control.61 Total I␬B (Applied Biosystems, Warrington, UK). expression was equivalent, indicating NF-␬B degradation was intact; however, loss of pathway modulation by nephrin Antibodies would lead to upregulation, supporting its key role in the Anti-Ha tag (mouse IgG) and rabbit polyclonals anti-14-3-3␪, anti- regulation of the process. PKC-␨, anti-RelA, anti-WT1 (C-19), p-JNK, Par4, p38, and Bcl2 were Although our data indicated that RelA/NF-␬B activation from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GFP (rabbit causes podocyte damage, the identity of inducing signals re- polyclonal) was from Abcam (Cambridge, UK). Anti-acetylated tu- mains unknown. The association between NF-␬B and stress bulin (mouse) was from Sigma-Aldrich (Dorset, UK). I␬B-␣ and and innate immunity provides a link between podocyte injury phospho-PKC␨/␭(Thr410/403) rabbit polyclonals were from Cell and NF-␬B activation stimuli potentially originating from cy- Signaling Technology (Danvers, MA). Anti–Alexa Fluor 488 antibody tokines or microbial pathogens.62,63 LPS-induced proteinuria (rabbit) and anti–Alexa Fluor 496 (mouse) secondaries were from is also associated with NF-␬B activation in podocytes.64,65 Be- Invitrogen (Paisley, UK). cause receptors linked to innate immunity, including Toll-like receptor 4, are constitutively expressed on podocytes,66,67,68 it Generation of Par4؊/؊ Mice is tempting to speculate that these might also participate in Mice genetically deficient for Par4 were produced by homologous NF-␬B activation in podocytes outside the LPS model. RelA/ recombination on a CD1 outbred background as described by Garcia- NF-␬B activation may represent an end point for a number of Cao et al.36 convergent pathways in glomerular damage, with regulation by nephrin constituting an essential cellular survival mecha- Histologic Analysis of Murine Kidney Sections nism. Further support for this concept comes from clinical The kidneys of killed mice were fixed in 10% neutral buffered forma- observations that glucocorticoids and angiotensin inhibitors lin, dehydrated, and subsequently wax embedded in paraffin. Sections commonly used to limit proteinuria downregulate NF-␬B and were cut at 4-␮m thickness and stained with hematoxylin and eosin upregulate nephrin.69,70 using a standard method. In summary, we propose that nephrin maintains a negative tone on NF-␬B activation signals, triggering its nuclear trans- Immunohistochemistry location in podocytes. As illustrated in Figure 9, either primary Sections were dewaxed in Histoclear (National Diagnostics, Atlanta, or secondary nephrin deficiency could remove this brake, al- GA) and rehydrated through graded ethanols. Antigen retrieval was lowing inappropriate induction of NF-␬B transcriptional tar- performed by boiling in citrate buffer (10 mM citric acid and 0.05% gets that are able to cause cellular injury directly or indirectly. Tween 20 [pH 6.0]) for 10 min. Nonspecific binding was blocked by Our data verify NF-␬B activation as an important mediator of incubation in blocking medium. Two-hour incubations with primary aberrant slit diaphragm signaling and provide a novel insight antibody at dilutions between 1:50 and 1:200 and secondary antibody into the regulation of this ancient pathway in nonimmune at a dilution of 1:200 were performed in blocking medium at room cells. temperature. For double labeling, cells were additionally incubated with acetylated-tubulin antibody (1:50) and an anti–Alexa Fluor 496 (red) secondary. Sections were mounted under coverslips in Citifluor CONCISE METHODS (London, UK). Fluorescence was visualized on a confocal laser scan- ning microscope (Leica Aristoplan, Heidelberg, Germany). Plasmids and Generation of cDNA Constructs Full-length pcDNA3-NPHS1 (gift from Prof. K. Trygvasson, Karolin- Electron Microscopy ska Institute, Stockholm, Sweden) was used to generate constructs. Kidney tissue was fixed in 3% gluteraldehyde, 0.1 M sodium cacody- Bsu36I and Xba1 digestion excised cDNA corresponding to the cyto- late, and 5 mM calcium chloride (pH 7.2) at room temperature. Post- solic tail encoding AA 1142 through 1241. cDNA encoding AA 1142 fixation was carried out with 1% osmium tetroxide for 1 h, followed through 1160 and AA 1142 through 1201 was amplified by Pfu PCR by dehydration of tissue in graded ethanols and infiltration with 1:1 using gene-specific primers and subcloned in frame to encode mutant propylene oxide/epoxy resin for3hatroom temperature. Samples nephrin proteins truncated at R1160 and W1201. Full-length NPHS2 were re-infiltrated with pure epoxy resin overnight at 4°C, embedded, cDNA was amplified from QUICK-Clone kidney cDNA (Clontech, and polymerized at 60°C for 48 to 72 h. Ultrathin sections (60 nm) Mountain View, CA) by Pfu PCR and subcloned as an EcoRI/EcoRI were collected onto copper grids. Sections were stained with 25% fragment into pcDNA-HA (in-house). Dominant negative 14-3-3 is uranyl acetate, washed in methanol, and contrasted with lead citrate. described by Thorson et al.71 Dominant negative pcDNA-HA-PKC␨ Electron micrographs were taken with a Zeiss 910 Advanced Trans- is described by Diaz-Meco et al.72 pcDNA-GST-NPHS1 was generated mission Electron Microscope (Zeiss, Oberkochen, Germany). from pcDNA-FLAG-NPHS1 by EcoRI excision of cDNA corresponding to AA 1057 through 1241, blunt ending, Xho1 digestion, and liga- Cell Culture tion into pcDNA-GST (gift from Dr. S. Parkinson, Cancer Research MDCK cells were cultured in DMEM (Glutamax I) and 10% FBS (Life UK, London, UK). GFP-PKC␨ was also a gift from Dr. Parkinson. Technologies, Grand Island, NY) using standard techniques. Condi-

1740 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1733–1743, 2009 www.jasn.org BASIC RESEARCH

tionally immortalized WT and mutant human podocytes were cul- NETN 48 h after transfection. A total of 400 ␮g of cell lysate and 50 ␮l tured according to methods described by Saleem et al.40 of beads were used per precipitation at 4°C for 3 h. Pelleted glutathione precipitates were washed with NETN and resuspended in 2ϫ Protein Extraction from Cultured Cells Laemmli Buffer (20 ␮l). Samples were boiled for 5 min before SDS- Ice-cold NETN buffer (20 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM PAGE. Precipitated native 14-3-3␪ was detected using a rabbit poly- EDTA, 0.5% NP40, and protease inhibitor cocktail [Boehringer clonal 14-3-3␪ antibody (Santa Cruz Biotechnology). Mannheim, Manheim, Germany]) was used for protein extraction for Western blotting and glutathione precipitation. Phospho-proteins were extracted using ice-cold HEPES buffer (50 mM HEPES [pH 7.5], ACKNOWLEDGMENTS 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 150 mM NaCl, 1 mM EGTA, and 1 mM PMSF, with protease inhibitors and phosphatase This work was funded by the Wellcome Trust (A.K. and S.H.); the Sir inhibitor cocktail [Roche Applied Science, Burgess Hill, UK]). Lysates Jules Thorn Trust (A.K. and L.R.); and a Charlotte Eyke & Elizabeth were centrifuged at 4°C, and supernatants were stored at Ϫ70°C. Wherry Award, British Medical Association (A.K.). We thank Prof. K Trygvasson and Dr. S. Parker for gifts of anti- Immunocytochemistry bodies and plasmids. MDCK cells and human podocytes were propagated to 70% confluence on glass microscope slides using the Lab-Tek Chamber Slide system (Nunc, Fisher Scientific Loughborough, UK). Cells were fixed DISCLOSURES Ϫ with either methanol at 20°C or 2% paraformaldehyde for 10 min. None. Permeabilization with 0.3% Triton X was used for WT1, nephrin, and Par4. Cells were blocked for1hin10%FCSinPBSand1%BSAand incubated with primary and anti–Alexa Fluor 488 (green, rabbit) sec- REFERENCES ondary antibody for 1 h, respectively, at room temperature. Cells were

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