Role of nephrin phosphorylation inducted by dexamethasone and angiotensin II in podocytes

The phosphorylation of nephrin plays an important role in maintaining the normal structure and function in podocytes. Dexamethasone (Dex) is usually used to treat glomerular diseases with proteinuria. In this study, we observated the effect of Dex and angiotensin II (AngII) on the change of nephrin phosphorylation in cultured podocytes. In vitro, cultured podocytes were exposed to AngII (10^?6 mol/L) pretreated with or without Dex (100 nM) for different time periods. Nck or Fyn were silenced by small interfering RNA (siRNA), nephrin and its phosphorylation expression were analyzed by Western blotting. In vitro, the phosphorylation of nephrin was significantly reduced after AngII stimulation (P < 0.05). Dex significantly resisted podocyte injury inducted by AngII via increasing the phosphorylation of nephrin (P < 0.05), siRNA silencing Nck can partially inhibited nephrin phosphorylation, siRNA silencing Fyn can completely inhibited nephrin phosphorylation. Phosphorylation of nephrin is important for the survival status of podocytes. Glucocorticoid treatment for human glomerulonephritis may exert its function by regulating Nck and Fyn complex to promote phosphorylation of nephrin. These results elucidate a novel mechanism of glucocorticoid treatment for glomerulonephritis.     

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.     

Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr¹¹²⁷ and Tyr¹¹⁵² are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2^+/C96Y) compared with control littermate mice (Ins2^+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.     

Downregulation of CD2-associated protein impaired the physiological functions of podocytes

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.     

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.     

Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6-PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type-specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.     

CD2相关蛋白在足细胞分化中的作用

本文旨在研究肾脏足细胞的分化特点及CD2相关蛋白(CD2-associated protein,CD2AP)在足细胞分化过程中的作用.用RPMI 1640培养基在33.C许可条件下培养永生化小鼠足细胞系(未分化组),转染针对CD2AP的小分子干扰RNA(smallinterfering RNA,siRNA)后置于37.C非许可条件下培养(转染组),并将非许可条件下未转染组作为对照组.用MTT法检测足细胞的生长速度;用RT-PCR方法检测CD2AP、WTI、synaptopodin和nephrin mRNA表达;用Western blot检测CD2AP、wTl和nephrin蛋白表达;用免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、F-actin和tubulin在分化及未分化足细胞中的分布及其共定位情况.结果显示,CD2AP、WTl和nephrin在分化及未分化足细胞中均可稳定表达,而synaptopodin仅表达于已分化足细胞,在未分化足细胞无表达.在足细胞分化过程中,CD2AP和nephrin的表达上调(P<0.05);CD2AP、tubulin和F-actin在细胞内的分布发生改变,CD2AP与nephrin及F-actin在未分化足细胞中存在共定位关系.转染特异性siRNA下调CD2AP表达,细胞生长速度明显减慢,synaptopodin mRNA表达下调(P<0.05),细胞分化迟滞.结果表明,足细胞分化过程中伴随细胞骨架的重新分布和细胞形态的改变;CD2AP可能作为足细胞裂孔隔膜分子与细胞骨架的连接蛋白,在足细胞分化过程中发挥重要作用.     

Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D₃ through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25‐dihydroxyvitamin D₃ analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)‐diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose‐mediated down‐regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose‐induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co‐localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down‐regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ‐diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

Establishment of Protein Delivery Systems Targeting Podocytes

Background

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.

Methodology/Principal Findings

Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as β-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95–100%. The delivered β-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.

Conclusions

Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.     

Innovative Delivery of siRNA to Solid Tumors by Super Carbonate Apatite

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.     

Angiotensin II induces nephrin dephosphorylation and podocyte injury: role of caveolin-1

Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10⁻⁶ M) pretreated with or without losartan (10⁻⁵ M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.     

Structural modification of siRNA for efficient gene silencing

Small interfering RNA (siRNA) holds a great promise for the future of genomic medicine because of its highly sequence-specific gene silencing and universality in therapeutic target. The medical use of siRNA, however, has been severely hampered by the inherent physico-chemical properties of siRNA itself, such as low charge density, high structural stiffness and rapid enzymatic degradation; therefore, the establishment of efficient and safe siRNA delivery methodology is an essential prerequisite, particularly for systemic administration. For an efficient systemic siRNA delivery, it is a critical issue to obtain small and compact siRNA polyplexes with cationic condensing reagents including cationic polymers, because the size and surface properties of the polyplexes are major determinants for achieving desirable in vivo fate. Unfortunately, synthetic siRNA is not easily condensed with cationic polymers due to its intrinsic rigid structure and low spatial charge density. Accordingly, the loose siRNA polyplexes inevitably expose siRNA to the extracellular environment during systemic circulation, resulting in low therapeutic efficiency and poor biodistribution. In this review, we highlight the innovative approaches to increase the size of siRNA via structural modification of the siRNA itself. The attempts include several methodologies such as hybridization, chemical polymerization, and micro- and nano-structurization of siRNA. Due to its increased charge density and flexibility, the structured siRNA can produce highly condensed and homogenous polyplexes compared to the classical monomeric siRNA. As a result, stable and compact siRNA polyplexes can enhance serum stability and target delivery efficiency in vivo with desirable biodistribution. The review specifically aims to provide the recent progress of structural modification of siRNA. In addition, the article also briefly and concisely explains the improved physico-chemical properties of structured siRNA with respect to stability, condensation ability and gene silencing efficiency.     

Mammalian Target of Rapamycin Complex 2 Signaling Pathway Regulates Transient Receptor Potential Cation Channel 6 in Podocytes

Transient receptor potential cation channel 6 (TRPC6) is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR) complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2]) signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.