The relationship among nephrin, podocin, CD2AP, and α-actinin might not be a true ‘interaction’ in podocyte

Abstract

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or α-actinin-4 mRNA by using reconstructed RNA interference vector – psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of α-actinin-4, whereas α-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them. The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or α-actinin-4 mRNA by using reconstructed RNA interference vector – psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of α-actinin-4, whereas α-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them. Podocytes are highly terminally differentiated cells with major processes and foot processes interlinked by ultrathin slit diaphragms (SD). The SD may represent a modified adhesion junction and is primarily responsible for the size selectivity of the glomerular filter.1.Reiser J. von Gersdorff G. Simons M. et al.Novel concepts in understanding and management of glomerular proteinuria.Nephrol Dial Transplant. 2002; 17: 951-955Crossref PubMed Scopus (30) Google Scholar, 2.Reiser J. Kriz W. Kretzler M. Mundel P. The glomerular slit diaphragm is a modified adherens junction.J Am Soc Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar However, the molecular components of the SD complex are still poorly understood for many years. Recently, the identification of several novel podocyte molecules and the description of their physical and functional interactions brought new insights into the components of the SD complex and the regulation of the glomerular filtration barrier. Nephrin, encoded by NPHS1, the gene mutated in congenital nephrotic syndrome (NS) of the Finnish type, has been suggested to form the SD by either homo- or heterophilic interactions.3.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome.J Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1563) Google Scholar Two other genes involved in proteinuria have also been identified. Of them, NPHS2, the causative gene for autosomal recessive steroid-resistant NS, encodes an integral membrane-associated protein, podocin, which is proposed to function as a scaffolding protein and capable of modulating nephrin-signaling activity.4.Boute N. Gribouval O. Roselli S. et al.NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephritic syndrome.Nat Genet. 2000; 24: 349-354Crossref PubMed Scopus (1198) Google Scholar, 5.Huber T.B. Köttgen M. Schilling B. et al.Interaction with podocin facilitates nephrin signaling.J Biol Chem. 2001; 276: 41543-41546Crossref PubMed Scopus (307) Google Scholar The other gene, ACTN4, has been shown to cause hereditary familial focal segmental glomerulosclerosis, and encodes an actin-filament cross-linking protein, α-actinin-4.6.Kaplan J.M. Kim S.H. North K.N. et al.Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis.Nat Genet. 2000; 24: 251-256Crossref PubMed Scopus (1040) Google Scholar In addition, the mice completely knockout of the gene CD2AP encoding CD2-assosiated protein (CD2AP), which localizes to the cytoplasmic face of the SD, develop the congenital NS and die of the massive proteinuria soon after birth.7.Shih N.Y. Li J. Karpitskii V. et al.Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar Moreover, NEPH1, another IgG superfamily protein,8.Donoviel D.B. Freed D.D. Vogel H. et al.Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.Mol Cell Biol. 2001; 21: 4829-4836Crossref PubMed Scopus (351) Google Scholar and FAT, the member of the protocadherin superfamily proteins,9.Inoue T. Yaoita E. Kurihara H. et al.FAT is a component of glomerular slit diaphragms.Kidney Int. 2001; 59: 1003-1012Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar and P-cadherin2.Reiser J. Kriz W. Kretzler M. Mundel P. The glomerular slit diaphragm is a modified adherens junction.J Am Soc Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar have been shown to localize to the SD. Targeted deletion of the FAT or NEPH1 gene in mice also leads to NS.8.Donoviel D.B. Freed D.D. Vogel H. et al.Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.Mol Cell Biol. 2001; 21: 4829-4836Crossref PubMed Scopus (351) Google Scholar, 9.Inoue T. Yaoita E. Kurihara H. et al.FAT is a component of glomerular slit diaphragms.Kidney Int. 2001; 59: 1003-1012Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar These studies suggested that these podocyte proteins play an essential role in maintaining the structural and functional integrity of the glomerular filtration barrier. A single gene mutation, such as NPHS1, NPHS2, and ACTN4, can lead to the hereditary or familial NS, implying that the abnormality of a single podocyte molecule caused by a single gene mutation, might be enough to induce NS. Further studies from acquired NS10.Guan N. Ding J. Zhang J. Yang J. Expression of nephrin, podocin, alpha-actinin, and WT1 in children with nephrotic syndrome.Pediatr Nephrol. 2003; 18: 1122-1127Crossref PubMed Scopus (37) Google Scholar, 11.Kim B.K. Hong H.K. Kim J.H. Lee H.S. Differential expression of nephrin in acquired human proteinuric diseases.Am J Kidney Dis. 2002; 40: 964-973Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 12.Koop K. Eikmans M. Baelde H.J. et al.Expression of podocyte-associated molecules in acquired human kidney diseases.J Am Soc Nephrol. 2003; 14: 2063-2071Crossref PubMed Scopus (267) Google Scholar, 13.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar, 14.Zhang S.Y. Marlier A. Gribouval O. et al.In vivo expression of podocyte slit diaphragm-associated proteins in nephrotic patients with NPHS2 mutation.Kidney Int. 2004; 66: 945-954Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and experimental NS15.Guan N. Ding J. Deng J. et al.The key molecular events occurred in puromycin aminonucleoside nephrosis rats.Pathol Int. 2004; 54: 703-711Crossref PubMed Scopus (43) Google Scholar, 16.Luimula P. Sandstrom N. Novikov D. Holthofer H. Podocyte-associated molecules in puromycin aminonucleoside nephrosis of the rat.Lab Invest. 2002; 82: 713-718Crossref PubMed Scopus (78) Google Scholar, 17.Saran A.M. Yuan H. Takeuchi E. et al.Complement mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.Kidney Int. 2003; 64: 2072-2078Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18.Yuan H. Takeuchi E. Taylor G.A. et al.Nephrin dissociates from actin, and its expression is reduced in early experimental membranous nephropathy.J Am Soc Nephrol. 2002; 13: 946-956Crossref PubMed Google Scholar suggested that many podocyte molecules were together involved in maintaining the structural and functional integrity of the SD. Although we are starting to understand the role of podocyte molecule from the view of a single molecule, we do not know much on the relationship among these molecules in maintaining the integrity of the SD and in the occurrence and development of proteinuria, and we also do not know much on the molecular effects caused by the change of each podocyte proteins on the remaining ones. So, it is necessary to explore the function of a single podocyte molecule, especially the three important proteins nephrin, podocin, and CD2AP, and the cytoskeletal protein α-actinin-4, in order to discover the potential sites or molecular target(s) for the intervention of proteinuria. In this study, we investigated the molecular behavior or response by the respective knockdown (KD) of nephrin, podocin, CD2AP, or α-actinin-4 mRNA in mouse podocyte clone 519.Mundel P. Reiser J. Zuniga Mejia-Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar with a novel gene block technique – RNA interference (RNAi), which is widely used to eliminate gene activity by degrading the specific mRNAs with complementary sequence.20.Hannon G.J. RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3515) Google Scholar In control group, the staining for podocin and nephrin distributed in perinuclear and mainly on the cell membrane surface in a filamentous pattern. CD2AP was evenly localized in the cytoplasm and on the cell membrane surface. The α-actinin staining pattern was different from that of podocin, nephrin, and CD2AP. α-Actinin mainly distributed in the cytoplasm in a filamentous pattern and also extended to the podocyte processes (Figure 1). In podocin KD group (siPod 966 and siPod 54), the intensities of podocin and nephrin were distinctly lower than those in control, and the staining for them was localized predominantly around nuclei with a loss of surface distribution. The intensity of CD2AP increased significantly, and its staining mainly distributed around nuclei with a normal membrane expression. However, the intensity and distribution of α-actinin did not change (Figure 2). In nephrin KD group (siNep 492), the changes of the intensities and distributions of nephrin and CD2AP were the same as those in podocin KD group, whereas that of podocin and α-actinin did not change (Figure 3). In siNep 1031 group, the intensities and distributions of nephrin, podocin, CD2AP, and α-actinin showed no change (data not shown). In CD2AP KD group (siCda 744 and siCda 21), the intensities of CD2AP and nephrin were obviously lower than those in control, and the staining for them predominantly distributed around nuclei with a loss of surface expression. Compared with control, the intensity of podocin was distinctly higher, and its staining was mainly localized around nuclei with the normal expression of the cytoplasm and the membrane. No change was observed for α-actinin (Figure 4). In α-actinin-4 KD group (siAct 1790 and siAct 319), the intensity of α-actinin was distinctly lower with normal distribution. All other proteins changed with distribution and expression alteration, that is, nephrin decreased with a loss of surface expression, whereas the perinuclear staining for podocin and CD2AP increased evidently with a normal expression of the membrane surface (Figure 5). The specific DNA bands of podocin, nephrin, CD2AP, α-actinin-4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were observed with the size of 193, 211, 191, 277, and 201 bp, respectively, and the quantitative results in relation to GAPDH at mRNA level were shown in Table 1 and Figure 6.Table 1The mRNA quantification results of podocin, nephrin, α-actinin-4, and CD2AP in relation to GAPDHsiPod 966siPod 54siNep 1031siNep 492siAct 319siAct 1790siCda 744siCda 21ControlPodocin0.16±0.09*0.20±0.10*0.67±0.040.68±0.140.86±0.161.12±0.23*1.00±0.16*0.70±0.080.66±0.08Nephrin0.14±0.06*0.18±0.05*1.03±0.290.14±0.07*0.48±0.11*0.33±0.08*0.52±0.16*0.59±0.13*1.03±0.15α-Actinin-41.10±0.221.09±0.151.09±0.191.12±0.250.41±0.13*0.31±0.16*1.12±0.181.10±0.171.06±0.34CD2AP1.24±0.16*1.21±0.15*0.84±0.101.11±0.15*1.03±0.281.23±0.20*0.15±0.05*0.24±0.13*0.78±0.04The quantification of mRNA was carried out by detecting the band intensity of PCR products revealed by agarose gel electrophoresis. The data in the table are the means±s.d. of the ratios between podocin, nephrin, CD2AP, α-actinin-4, and the corresponding GAPDH from four independent RNA interference experiments. *Statistical significance relative to control, P<0.05. Open table in a new tab The quantification of mRNA was carried out by detecting the band intensity of PCR products revealed by agarose gel electrophoresis. The data in the table are the means±s.d. of the ratios between podocin, nephrin, CD2AP, α-actinin-4, and the corresponding GAPDH from four independent RNA interference experiments. *Statistical significance relative to control, P<0.05. The specific protein bands of podocin, nephrin, CD2AP, α-actinin, and GAPDH were detected with the size of 44, 180, 80, 100, and 36 kDa, respectively, and the quantitative results at protein level in relation to GAPDH were shown in Table 2 and Figure 7.Table 2The protein quantification results of nephrin, podocin, CD2AP, and α-actinin in relation to GAPDHsiPod 966siPod 54siNep 1031siNep 492siAct 319siAct 1790siCda 744siCda 21ControlPodocin0.05±0.03*0.10±0.05*0.52±0.160.47±0.120.53±0.070.70±0.08*0.66±0.07*0.52±0.060.44±0.07Nephrin0.20±0.05*0.33±0.11*1.15±0.170.05±0.03*0.38±0.10*0.29±0.10*0.28±0.07*0.59±0.35*1.12±0.10α-Actinin-41.45±0.291.55±0.231.48±0.171.47±0.070.63±0.12*0.28±0.10*1.44±0.081.50±0.101.50±0.12CD2AP0.93±0.27*0.91±0.20*0.53±0.130.75±0.12*0.69±0.081.03±0.20*0.05±0.01*0.12±0.04*0.58±0.08The quantification of protein expression was carried out by detecting the intensity of the Western blotting bands. The data in the table are the means±s.d. of the ratios between podocin, nephrin, CD2AP, α-actinin-4, and the corresponding GAPDH from four independent RNA interference experiments.*Statistical significance relative to control, P<0.05. Open table in a new tab The quantification of protein expression was carried out by detecting the intensity of the Western blotting bands. The data in the table are the means±s.d. of the ratios between podocin, nephrin, CD2AP, α-actinin-4, and the corresponding GAPDH from four independent RNA interference experiments. *Statistical significance relative to control, P<0.05. The massive proteinuria is the most important clinical manifestation of NS. The SD plays a crucial role in preventing proteins and other larger molecules from leaking to urine.1.Reiser J. von Gersdorff G. Simons M. et al.Novel concepts in understanding and management of glomerular proteinuria.Nephrol Dial Transplant. 2002; 17: 951-955Crossref PubMed Scopus (30) Google Scholar, 2.Reiser J. Kriz W. Kretzler M. Mundel P. The glomerular slit diaphragm is a modified adherens junction.J Am Soc Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar Recently, exciting studies demonstrated that the SD complex contains at least three important proteins: nephrin, podocin, and CD2AP, the genes mutated leading to congenital or familial NS.3.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome.J Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1563) Google Scholar, 5.Huber T.B. Köttgen M. Schilling B. et al.Interaction with podocin facilitates nephrin signaling.J Biol Chem. 2001; 276: 41543-41546Crossref PubMed Scopus (307) Google Scholar, 7.Shih N.Y. Li J. Karpitskii V. et al.Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar Moreover, further in vitro studies suggested that there might be some interactions among these podocyte molecules. Schwarz et al.21.Schwarz K. Simons M. Reiser J. et al.Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.J Clin Invest. 2001; 108: 1621-1629Crossref PubMed Scopus (514) Google Scholar revealed that podocin might recruit nephrin and connect via its C-terminal domain with CD2AP. Studies from Huber's group showed that nephrin-induced signaling could be greatly enhanced by podocin in HEK 293 cells.5.Huber T.B. Köttgen M. Schilling B. et al.Interaction with podocin facilitates nephrin signaling.J Biol Chem. 2001; 276: 41543-41546Crossref PubMed Scopus (307) Google Scholar In addition, Saleem et al.22.Saleem M.A. Ni L. Witherden I. et al.Co-localization of nephrin, podocin and the actin cytoskeleton.Am J Pathol. 2002; 161: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar found that nephrin and podocin might partially colocalize with actin filaments, and might be dependent on integral actin polymers for their intracellular distribution in podocyte. Simultaneously, studies from some congenital/hereditary and acquired NS implied that the expression or distribution abnormality of a single or many podocyte molecule(s) can lead to the abnormalities of the SD structure and proteinuria. For example, nephrin and podocin decreased evidently at protein level in some NS patients with minimal change nephropathy, focal segmental glomerulosclerosis, lupus nephritis, or diabetic nephropathy,12.Koop K. Eikmans M. Baelde H.J. et al.Expression of podocyte-associated molecules in acquired human kidney diseases.J Am Soc Nephrol. 2003; 14: 2063-2071Crossref PubMed Scopus (267) Google Scholar and the remarkable redistributions of nephrin, podocin, CD2AP, and α-actinin-4 were revealed in the NS patients with NPHS2 mutations,14.Zhang S.Y. Marlier A. Gribouval O. et al.In vivo expression of podocyte slit diaphragm-associated proteins in nephrotic patients with NPHS2 mutation.Kidney Int. 2004; 66: 945-954Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and in puromycin aminonucleoside nephrosis rat, the expressions of nephrin and podocin decreased along with an obvious redistribution of podocin before the occurrence of proteinuria.15.Guan N. Ding J. Deng J. et al.The key molecular events occurred in puromycin aminonucleoside nephrosis rats.Pathol Int. 2004; 54: 703-711Crossref PubMed Scopus (43) Google Scholar These data highlighted the critical role of the SD complex and the actin cytoskeleton in the maintenance of the glomerular filtration barrier, and implied that these molecules were together involved in the occurrence and development of proteinuria. However, the molecular behavior or response induced by the change of a single podocyte molecule has not been completely elucidated. Therefore, it is necessary to clarify the molecular interaction(s) among the podocyte molecules, which is crucial to understand the molecular mechanism of proteinuria and further discover the potential molecular target(s) for the intervention of proteinuria. RNAi can rapidly and specifically degrade the target mRNA by generating an active RNA-induced silencing complex, and is also termed as gene KD compared with gene knockout (KO).20.Hannon G.J. RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3515) Google Scholar, 23.Agrawal N. Dasaradhi P.V. Mohmmed A. et al.RNA interference: biology, mechanism, and applications.Microbiol Mol Biol Rev. 2003; 67: 657-685Crossref PubMed Scopus (785) Google Scholar, 24.Caplen N.J. A new approach to the inhibition of gene expression.Trends Biotechnol. 2002; 20: 49-51Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Recently, studies have shown that 21-nucleotide hairpin siRNA duplexes can efficiently and specifically inhibit the expressions of cognate genes in mammalian cells, and that RNAi technique can be widely used to study the gene function and to identify interacting pairs of genes in a rapid and an inexpensive way.25.Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar, 26.Elbashir S.M. Harborth J. Lendeckel W. et al.Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8144) Google Scholar, 27.Paddison P.J. Caudy A.A. Bernstein E. et al.Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.Genes Dev. 2002; 16: 948-958Crossref PubMed Scopus (1320) Google Scholar So, we designed and reconstructed two recombinant RNAi vectors targeted to the 5′ and 3′-terminal of the coding sequences of nephrin, podocin, CD2AP, or α-actinin-4 mRNA, respectively, to study the effect of the KD molecule on the remaining non-KD ones and the molecular relationship among these podocyte molecules. Our results showed that all interference target sites except the siNep 1031 significantly led to the corresponding gene silencing, but the efficacy of individual RNAi was different. The RNAi efficacy of siPod 966, siAct 1790, and siCda 744 was obviously higher than that of siPod 54, siAct 319, and siCda 21. This phenomenon may be related to the location and secondary structure of the interference target sites. Moreover, the different degree changes of podocin and/or CD2AP were induced by the different RNAi efficacy of the two target sites for α-actinin-4 or CD2AP. That is, the expression increments of podocin and CD2AP were much more obvious in siAct 1790 group than in siAct 319 group because the RNAi efficacy in siAct 1790 group was higher than that in siAct 319 group. Similarly, the expression increment of podocin was much more significant in siCda 744 group than in siCda 21 group because the RNAi efficacy in siCda 744 group was higher than that in siCda 21 group. With the nephrin KD, only the quantitative expression of CD2AP increased, whereas podocin and α-actinin-4 showed no change. Contrarily, the quantitative expressions of nephrin all significantly decreased and its distributions changed evidently with the podocin, CD2AP, or α-actinin-4 KD. Previously, we also observed the reduction of nephrin with the podocin KD.28.Fan Q. Ding J. Zhang J. et al.Effect of the knockdown of podocin mRNA on nephrin and alpha-actinin in mouse podocyte.Exp Biol Med. 2004; 229: 964-970Google Scholar Data from Hamano et al.29.Hamano Y. Grunkemeyer J.A. Sudhakar A. et al.Determinants of vascular permeability in the kidney glomerulus.J Biol Chem. 2002; 277: 31154-31162Crossref PubMed Scopus (106) Google Scholar showed that the expressions and distributions of podocin, CD2AP, and α-actinin-4 did not change in NPHS1 KO mice with the obvious absence of the SD and podocyte effacement. In other words, nephrin deficiency did not cause alterations in the expression of podocin, CD2AP, and α-actinin-4, and only nephrin abnormality might be enough to induce the change of the SD structure and proteinuria. So, the role of nephrin might be more important than podocin, CD2AP, and α-actinin-4 on maintaining the normal structure and function of the SD and in turn the proper function of the glomerular filter compared to other podocyte molecules. With the podocin, nephrin, CD2AP, or α-actinin-4 KD, not only did the quantity of the affected non-KD molecules change, but also their redistributions were observed from the membrane surface to the predominant perinuclear. For example, no matter the expressions decreased (because of KD) or increased (caused by α-actinin-4 KD), the stainings for podocin and CD2AP all disappeared in the membrane surface along with the loss of nephrin in the cell surface. Therefore, we postulate that the existence of podocin or CD2AP might be very important and necessary for anchoring nephrin to the membrane surface, and the mechanisms underlying this should be further explored. Interestingly, such reduction of nephrin and increment of CD2AP and their redistributions were reported in the podocin-deficient mice,30.Roselli S. Heidet L. Sich M. et al.Early glomerular filtration defect and severe renal disease in podocin-deficient mice.Mol Cell Biol. 2004; 24: 550-560Crossref PubMed Scopus (224) Google Scholar and the changes in the distribution of nephrin, CD2AP, and α-actinin-4 were observed in the NS patients with NPHS2 mutations, which resulted in profound alteration of podocin distribution restricted to the podocyte body,14.Zhang S.Y. Marlier A. Gribouval O. et al.In vivo expression of podocyte slit diaphragm-associated proteins in nephrotic patients with NPHS2 mutation.Kidney Int. 2004; 66: 945-954Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and a small fraction of glomeruli had lost the reactivity with the nephrin antibody in the CD2AP KO mice.31.Li C. Ruotsalainen V. Tryggvason K. et al.CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere.Am J Physiol Renal Physiol. 2000; 279: F785-F792PubMed Google Scholar So, the normal localization of podocyte molecules might depend on their normal expression quantity and the normal molecular reactions among these molecules, which is very critical for maintaining the normal podocyte biology behaviors. On the other hand, the behavior of α-actinin-4 showed no change with the podocin, nephrin, or CD2AP KD, that is to say, the KD of any SD complex molecule did not induce the abnormality of α-actinin-4. Contrarily, the α-actinin-4 KD evidently changed the expressions and distributions of nephrin, podocin, and CD2AP. Namely, nephrin decreased with the α-actinin-4 KD, whereas podocin and CD2AP increased. Similarly, Michaud et al.13.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar detected the reduction of nephrin mRNA in the ACTN4 mutant mice. Our results imply that there might be a chain reaction between nephrin, podocin, CD2AP, and α-actinin-4, and that α-actinin-4 might directly or indirectly interact with nephrin, podocin, or CD2AP. So, α-actinin-4 might also play an important role in maintaining the structural and functional integrity of the SD, and its abnormality might indirectly affect the expression of the SD complex critical protein nephrin via podocin or CD2AP. However, the role and significance of the increment of podocin or CD2AP with the α-actinin-4 KD is still unclear, and a compensative increment should be considered for the reduction of nephrin in the membrane surface. It is also worth noticing that some results from gene KO were contrary to those from gene KD. Kos et al.32.Kos C.H. Le T.C. Sinha S. et al.Mice deficient in alpha-actinin-4 have severe glomerular disease.J Clin Invest. 2003; 111: 1683-1690Crossref PubMed Scopus (212) Google Scholar found that the expressions and distributions of nephrin and podocin did not change in the ACTN4 KO mice, and Hamano et al.29.Hamano Y. Grunkemeyer J.A. Sudhakar A. et al.Determinants of vascular permeability in the kidney glomerulus.J Biol Chem. 2002; 277: 31154-31162Crossref PubMed Scopus (106) Google Scholar and Rantanen et al.33.Rantanen M. Palmen T. Patari A. et al.Nephrin TRAP mice lack slit diaphragms and show fibrotic glomeruli and cystic tubular lesions.J Am Soc Nephrol. 2002; 13: 1586-1594Crossref PubMed Scopus (101) Google Scholar observed that the expression and distribution of CD2AP showed no change in the NPHS1 KO mice. The phenomena may be induced by the different biology effect between the complete gene deficiency caused by the gene KO and the gene expression reduction caused by the gene KD. In many diseases, the gene expression is often reduction but not complete deficiency. So, the change of molecular expression, distribution and reaction induced by the gene KD compared with the gene KO, seems to be much closer to the real in vivo situations. Additionally, our KD experiments indicate that the expression of the KD molecule decreased both at mRNA and protein level, which suggests that RNAi is a post-transcriptional regulation that specifically degrades the target mRNA. Furthermore, the change of the remaining affected non-KD molecules at mRNA level is consistent with that at protein level. For example, the nephrin KD increased the expression of CD2AP not only at protein level but also at mRNA level. This phenomenon suggests that there might be an underlying mechanism by which the reduction of the KD molecule at mRNA level causes the mRNA and protein change of the affected non-KD molecules. In addition, there is another very interesting finding in the podocyte molecular KD experiments. The relationship among these podocyte molecules might not be a true ‘interaction.’ Our study shows that the nephrin or podocin KD could increase CD2AP expression, in turn, the CD2AP KD could decrease nephrin and increase podocin expression. Nevertheless, the nephrin KD did not change the expression of podocin, whereas the podocin KD decreased the expression of nephrin. A similar phenomenon was also found between nephrin, podocin, or CD2AP and α-actinin-4. Namely, there was no change on the α-actinin-4 expression after the nephrin, podocin, or CD2AP KD, whereas the α-actinin-4 KD begetted the reduction of nephrin, and the increment of podocin and CD2AP, respectively. All of these imply that the response between the four podocyte molecules is very complicated and evidently different. Further understanding the relationship and interaction of these podocyte molecules will help us to identify the critical molecular target(s) in intervening the proteinuria, and some experiments about the co-immunoprecipitation and signal transduction pathway should be done in vivo in the future to elucidate the response of these important molecules in the occurrence and development of proteinuria. Taken together, we successfully silenced the expressions of nephrin, podocin, CD2AP, and α-actinin-4 with RNAi technique, respectively. Our data suggest that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. Moreover, the normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reaction between them. The mRNA sequences of the mouse NPHS1, NPHS2, CD2AP and ACTN4 gene were obtained from the GenBank (accession number AF191090, NM130456, NM009847, and NM021895, respectively). The coding sequences of mRNA were searched for the interference target sites. The potential targets were the oligonucleotides starting with A or G as the transcription initiation point of hH1 promoter followed by 20 nucleotides, in which a stretch of more than four Ts as the terminal signal for RNA polIII, or a set of six G and C or T (e.g., GCCGGC or GGTGTT) should be excluded, and the sequences containing a palindrome should also be omitted to avoid unwanted hairpin structures. By BLAST search, we chose two potential interference targets for every target mRNA, which did not have homology to other mouse mRNA sequences. Two cohesive cloning sites of BbsI were designed at the ends of the forward and reverse insert template with a loop sequence (TCAAGAG) for cloning into the RNAi vector—psiRNA-hH1GFPzeo (Invivogen, San Diego, CA, USA). Table 3 shows the location and sequence of every potential interference target, and the name of every recombinant RNAi vector.Table 3Location and sequence of the potential interference target site, and the recombinant vector nameTarget geneLocation of target siteaAccording to the number from the first base of the start codon of the target mRNA.Sequence of target siteRecombinant vector nameNPHS254–745′-GTCCTCCTCTAGGGATGACAA-3′siPod 54966–9865′-GCTTCGATACTTGCACACTCT-3′siPod 966NPHS1492–5125′-GGTAGCTGGACAGGAATATGT-3′siNep 4921031–10515′-GAAGCATCACTCTGCAGGTCA-3′siNep 1031ACTN4319–3395′-GTGCACAAGATCAACAATGTA-3′siAct 3191790–18105′-GGATCGCTGAGAGCAATCACA-3′siAct 1790CD2AP21–415′-GTATGACTATGATGCTGTACA-3′siCda 21744–7645′-GCCTCTGGGATCTAGAACTCA-3′siCda 744a According to the number from the first base of the start codon of the target mRNA. Open table in a new tab The forward and reverse oligonucleotides were annealed, and the resulted double strand DNA was ligated into the psiRNA-hH1GFPzeo digested with BbsI (Invivogen). Then, Escherichia coli GT116 competent cells (Invivogen) were transformed with the above ligation products, and spread on E. coli FastMedia™ Zeo Xgal (Invivogen) plate containing 25 μg/ml zeocin (Invivogen) to take advantage of the white/blue selection. After confirmed the presence of the siRNA insert by digesting with SpeI (Invivogen), and further verified by sequencing with primer OL381 and OL178 (Invivogen), the recombinant plasmids were prepared with Wizard® PureFection Plasmid DNA Purification System (Promega, Madison, USA), and quantitated with UV spectrophotomer (UV 2100, Shimadzu, Japan). Figure 8 shows the schematic drawing of the recombinant siPod 54 vector. Conditionally immortalized mouse podocyte clone was a kindly gift from Dr Mundel,19.Mundel P. Reiser J. Zuniga Mejia-Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar and was cultured at 33°C in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco), 100 U/ml of penicillin–streptomycin and 10 U/ml of mouse γ-interferon (PEPRO Tech, London, UK). To induce differentiation, podocytes were reseeded and cultured at 37°C in six-well plates with glass coverslips and 25 cm2-flasks coated with 10 μg/ml of type-I collagen (Sigma, St Louis, MO, USA) by removal of γ-interferon. When they grew to about 60% confluence, podocytes were transfected with the above-verified recombinant plasmid DNA and LyoVec™ (Invivogen) according to the manufacturer's instruction. Cells were harvested about 48 h after transfection to assay the activities of target genes. Expression of green flurescence protein enables the detection of cells transfected with the recombinant RNAi plasmid. The vector – psiRNA-hH1GFPzeo was used as the control plasmid, which cannot produce any hairpin siRNA, then was expected to have no any interference effect on mouse genes. The following primary antibodies were used: rabbit polyclonal antibody against mouse nephrin (a kindly gift from Professor Karl Tryggvason); rabbit polyclonal antibody against the C-terminal part of human podocin (a generous gift from Professor Corinne Antignac); rabbit polyclonal antibody against the C-terminus of CD2AP of human origin (Santa Cruz, CA, USA); and rabbit polyclonal antibody against the C-terminus of α-actinin-1 of human origin, which can react with mouse, rat as well as human α-actinin-1, -2, -3 and -4 (Santa Cruz). Tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit IgG (Santa Cruz) was used as the secondary antibody for immunolabeling. The dye hoechst (Sigma) was used for nuclei staining. The immunolabeling was carried out as previously described.28.Fan Q. Ding J. Zhang J. et al.Effect of the knockdown of podocin mRNA on nephrin and alpha-actinin in mouse podocyte.Exp Biol Med. 2004; 229: 964-970Google Scholar, 34.Saleem M.A. O’Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Crossref PubMed Google Scholar Briefly, cells were fixed with ice-cold acetone, then permeabilized and blocked with 0.3% Triton X-100 and 5% bovine serum albumin (Sigma) in phosphate-buffered saline. The primary antibodies, the secondary antibody and hoechst were applied at appropriate dilutions, and the coverslips were mounted on glass slides with 15% Mowiol. Images were obtained by confocal laser-scanning microscopy using a Bio-Rad Radiance 2100 TM confocal system, and processed with Adobe Photoshop 7.0 software. Total RNA extracted with Trizol (Gibco) was reversely transcribed as previously described.28.Fan Q. Ding J. Zhang J. et al.Effect of the knockdown of podocin mRNA on nephrin and alpha-actinin in mouse podocyte.Exp Biol Med. 2004; 229: 964-970Google Scholar The sequence-specific primers for CD2AP with a size of 191 bp were a forward primer 5′-AGCGAATCAGCACTTATGGA-3′ and a reverse primer 5′-CCACCAGCCTTCTTCTACCT-3′, and the PCR conditions were as follows: 5 min at 94°C for the initial denaturation, followed by 1 min at 94°C, 45 s at 58°C and 1 min at 72°C for amplification, and a final extension at 72°C for 5 min. Sequence-specific primers for nephrin, podocin, α-actinin-4 and GAPDH, and the PCR conditions were previously described.7.Shih N.Y. Li J. Karpitskii V. et al.Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (696) Google Scholar PCR amplification was performed with 32, 30, and 28 cycles, respectively. The PCR products were separated on agarose gel. For the quantification, an image of the gel stained with ethidium bromide was captured, and the intensity of the bands was quantitated using AlphaImager gel analysis system (Alpha Innotech, San Leandro, CA, USA). In this study, the semiquantification analysis was performed using the PCR products of 30 cycles, which existed distinct difference between KD group and control group. Podocyte cells were lysed in a lysis buffer containing 1% Tritonx-100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin (Sigma) on ice.28.Fan Q. Ding J. Zhang J. et al.Effect of the knockdown of podocin mRNA on nephrin and alpha-actinin in mouse podocyte.Exp Biol Med. 2004; 229: 964-970Google Scholar, 34.Saleem M.A. O’Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Crossref PubMed Google Scholar An 8% sodium dodecyl sulfate-polyacrylamide gel was run, loading 75 μg of total protein in each lane. The gel was set up for transfer to nitrocellulose membranes (Amersham Life Science, Piscataway, NJ, USA). Then, the membranes were rinsed in a Tris-buffered saline with 0.02% Tween-20, followed by immersing in 5% low-fat milk powder. Subsequently, the membranes were incubated with antinephrin, podocin, CD2AP, α-actinin, or GAPDH antibodies at appropriate dilutions. Mouse monoclonal antibody against GAPDH (Chemicon, Temecula, CA, USA) was used in this study. After rinsing three times, the membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Santa Cruz). After final washing, the membranes were developed using ECL chemiluminescence reagent (Santa Cruz) and the specific protein bands were scanned and quantitated in relation to the GAPDH (36 kDa). In this study, all independent experiments were performed four times and the values reported are the means±s.d. Statistical comparisons were performed by using the Mann–Whitney test. P<0.05 were considered statistically significant. We thank Professor Peter Mundel (America) for the podocyte cell line, Professor Karl Tryggvason (Sweden), and Professor Corinne Antignac (France) for the gifts of nephrin and podocin antinbodies, respectively. We thank Professor Dingfang Bu (China) for the excellent technical assistance. This study was supported by the National Nature Science Foundation of China (30170992) and the fund from Ministry of Education (2003-14).