i-Fect Delivers IGF1 siRNA to the Mouse Brain

Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development

Neuromics' i-Fect^TM Transfection Kit continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS in vivo (via intrathecal, epidural and intraventricular injection). Genes studied include: ABCA, ASIC, β-arrestin, CAV_1.2, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K_V1.1, K_V9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV_1.8, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. Related Publications.

It is always an honor for one our products to be referenced in one of the Nature publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii, Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. ...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Images: (a) Igf1 expression (blue) and Iba1-positive microglia (brown) in P5 brain. Scale bar represents 400 μm. (b) Magnified view of dotted square in a. Scale bar represents 50 μm. (c,d) IGF1Ra expression (red) in CTIP2-positive (c) and SATB2-positive (d) layer V neurons at P5. Scale bar represents 50 μm. (e) IGF1 protein levels in medium from cultured cortical neurons, microglia and neurons with microglia in transwell systems detected by enzyme-linked immunosorbent assay (ELISA). **P < 0.01 (neuron, microglia + neuron, n = 5; microglia, n = 6 experiments; one-way ANOVA followed by Tukey-Kramer test). (f) The number of cleaved caspase-3–positive neurons in transwell systems treated with LY294002 or H-1356 or transfected to microglia with Igf1 siRNA. *P < 0.05, **P less than  0.01 (n = 3 experiments, one-way ANOVA followed by Tukey-Kramer test). (g) Neuronal phospho-AKT expression in cultured cortical neurons and those with microglia in transwell system. (h) TUNEL-positive cells in H-1356–treated cortex (36 h after treatment). Scale bar represents 100 μm. (i) The number of TUNEL-positive apoptotic cells in each layer in vehicle- (phosphate-buffered saline, PBS) or H-1356–treated mice (36 h after injection). *P < 0.05 (n = 4 brains, one-way ANOVA followed by Tukey-Kramer test). (j) Cleaved caspase-3–labeled cells (red) expressing CTIP2 (green, arrowheads). Scale bar represents 100 μm. (k,l) TUNEL-positive cells in the cortex treated with control or Igf1 siRNA (48 h after treatment). Scale bar represents 100 μm. (m) The number of TUNEL-positive apoptotic cells in each layer in control siRNA– and Igf1 siRNA–treated mice. **P less than 0.01 (n = 5 brains, one-way ANOVA followed by Tukey-Kramer test). Error bars represent s.e.m.

I will continue to post new developments and successes.

i-Fect™ Delivers you siRNA Payload

Delivering siRNA to Dorsal Root Ganglia to Silence K_V Receptors.

Our i-Fect transfection kits continue to be used to optimize delivery in vivo and into hard to transfect cells like primary neurons. In these 2 latest expamples, researchers use i-Fect to deliver siRNA to K_V Receptors in Rat DRGs. Knocking down these receptors enable the study of their role in pain modulation: John H. Winston, Sushil K. Sarna. Developmental Origins of Functional Dyspepsia-Like Gastric Hypersensitivity in Rats. Gastroenterology. Volume 144, Issue 3, March 2013, Pages 570–579.e3. dx.doi.org/10.1053/j.gastro.2012.11.001....intrathecal treatment, 2 μg of the appropriate siRNA was mixed (1:5 vol/vol) with i-Fect transfection reagent (Neuromics, Edina, MN); rats received 2 ug siRNA/10 uL/rat/injection...

Figures. siRNA-mediated knockdown of Kv1.1 expression in thoracic DRG significantly increased gastric sensitivity in naive adult rats. (A) Western blots showed a significant decrease in Kv1.1 protein in thoracic DRG (T8–T12) after intrathecal treatment with Kv1.1 siRNA but not with control siRNA. siRNA treatment did not alter TrpV1 expression (n = 5 rats each; *P < .01 vs control siRNA). (B) Naive rats treated with Kv1.1 siRNA showed a significant increase in VMR to gastric distention (n = 5 rats each, compared with pretreatment baseline; *P < .05). (C) Treatment with control siRNA had no significant effect on gastric hypersensitivity. (D) Patch clamp recordings from freshly dissociated gastric DRG neurons from FD-like and PND 10 saline-treated littermate controls showed a significant decrease in rheobase in FD-like rats (*P < .05), and (E) a significant increase in the number of action potentials elicited by current injection at 3× the rheobase in gastric DRG neurons from FD-like rats (*P < .05). (F) Sample voltage vs time traces showing action potentials evoked at ×1, ×2, and ×3 rheobase. The patch clamp data were obtained from 16 cells from 5 PND 10 saline control rats and 19 cells from 5 FD-like rats.
Tsantoulas C, Zhu L, Shaifta Y, Grist J, Ward JP, Raouf R, Michael GJ, McMahon SB. Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury. J Neurosci. 2012 Nov 28;32(48):17502-13. doi: 10.1523/JNEUROSCI.3561-12.2012..."I'd just like to notify you about a recent paper from my team which utilised iFect for in vivo transfection of dorsal root ganglia." Dr. Christoforos Tsantoulas, University of Cambridge...i-Fect-siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors...

These add to the many publications referencing use of i-Fect to deliver siRNA. Genes studied include:  DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, Toll-Like Receptors, Kv Recptors, BDNF, Ret, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC.

BDNF and Recovery of Motor Function After Brian Injury

Researchers use i-Fect^TM to Study BDNF Silencing in Mice

Brain injury that results in an initial behavioural deficit is frequently followed by spontaneous recovery. The intrinsic mechanism of this functional recovery has never been fully understood. Here, we show that reorganization of the corticospinal tract induced by target-derived brain-derived neurotrophic factor is crucial for spontaneous recovery of motor function following brain injury:  Masaki Ueno, Yasufumi Hayano1, Hiroshi Nakagawa and Toshihide Yamashita. Intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury. Brain (2012) doi: 10.1093/brain/aws053. ... motor cortex at 14 days after the injury, using i-Fect™ transfection reagents (Neuromics) according to the manufacture's instruction .

Findings Overview: After destruction of unilateral sensorimotor cortex, intact-side corticospinal tract formed sprouting fibres into the specific lamina of the denervated side of the cervical spinal cord, and made new contact with two types of spinal interneurons—segmental and propriospinal neurons. Anatomical and electrophysiological analyses revealed that this rewired corticospinal tract functionally linked to motor neurons and forelimb muscles. This newly formed corticospinal circuit was necessary for motor recovery, because transection of the circuit led to impairment of recovering forelimb function. Knockdown of brain-derived neurotrophic factor in the spinal neurons or its receptor in the intact corticospinal neurons diminished fibre sprouting of the corticospinal tract. Our findings establish the anatomical, functional and molecular basis for the intrinsic capacity of neurons to form compensatory neural network following injury.

We will continue to post new references to our Transfection Kits.

Using i-Fect to Silence Kv9.1 in vivo

There have been multiple publications referenced here on using Neuromics' i-Fect siRNA Delivery Kit to study the effect of silencing genes known to play a role in Pain Signaling. These include: DOR,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, TRPV1 NOV, β-arrestin, TRPV1, CAV1.2 and ASIC.

I would like to highlight an exciting new study referencing how knocking down Kv9.1 Potassium Channel Subunit in vivo mediates neuropathic pain: Christoforos Tsantoulas, Lan Zhu, Yasin Shaifta, John Grist, Jeremy P. T. Ward, Ramin Raouf, Gregory J. Michael, and Stephen B. McMahon. Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury. The Journal of Neuroscience, 28 November 2012, 32(48): 17502-17513; doi: 10.1523/​JNEUROSCI.3561-12.2012.

Highlights: Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.

n vivo RNA interference: Anesthetized rats were subjected to a thoracic laminectomy and a silastic tube was inserted subdurally to lie just rostral to L3 DRG and externalized to deliver bolus injections (one injection per day for 4 consecutive days). Animals were allowed to recover for 5 d before treatment commenced. On the day of injection, siRNA was mixed with i-Fect (Neuromics) to a final concentration of 0.2 μg μl−1, according to published protocols (Luo et al., 2005). For each treatment, 10–20 μl of Kv9.1 siRNA or scrambled control mixture was injected, followed by a 10 μl saline flush. Twenty-four hours after the fourth injection animals were killed and L5 DRGs fresh dissected for qRT-PCR analysis. A separate set of animals were PFA perfused and DRGs retrieved for IHC. Passenger strand sequences for Kv9.1 and scrambled control siRNAs were cuuggaaucuguaggauca and gaggcctaatcgatatgtt, respectively (Dharmacon; “in vivo processing” option).

Intrathecal Kv9.1 siRNA treatment induces pain behaviors in naive rats. A, qRT-PCR quantification of Kv9.1 mRNA in rat PASMC cultures transfected with one of three Kv9.1 siRNA sequences or control siRNA (control, n = 6; siRNA, n = 3 per group; *p < 0.05 vs control, one-way ANOVA with Tukey's). B, qRT-PCR showing Kv9.1 in vivo knock-down in L5 DRG, 4 d after intrathecal delivery of siRNA #1 compared with vehicle or matched scrambled control (vehicle, n = 4; scrambled, n = 5; Kv9.1, n = 7; *p < 0.05, t test). C, IHC for Kv9.1 in scrambled- and siRNA-treated DRG to determine protein knockdown. Graphs illustrate quantification of number of positive myelinated neurons and mean Kv9.1 signal intensity (scrambled, n = 4; siRNA, n = 6; **p < 0.01, ***p < 0.001, t test). D, Kv9.1 siRNA infusion inflicts a reduction in mechanical pain withdrawal thresholds (Kv9.1, n = 7; control, n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 vs scrambled control or baseline, two-way repeated measurements ANOVA with Tukey's). E, There was no change in heat pain thresholds after siRNA treatment. Vertical arrows on x-axis denote siRNA injections. All data represent mean ± SEM.

Kv9.1 knock-down triggers ectopic activity and a form of peripheral wind-up in response to stimulation. A, Schematic illustrating the positions of stimulating and recording electrodes. B, Example recordings from centrally disconnected L4/L5 strands demonstrating SA in Kv9.1 siRNA-treated or nerve-injured rats, but not in control (scrambled siRNA) animals. C, Frequency-dependent SEA (denoted by double arrowheads) in Kv9.1 siRNA-treated (middle) and injured (right), but not control (left) animals. This activity is not locked in time and can be seen in between stimulation events (vertical arrows on top of 5 Hz stimulation traces, only first 5 shown). Also note the prolonged after-discharge (AD) observed in siRNA-treated and injured animals. D, Percentage of units showing SA and SEA in control (n = 269), Kv9.1 siRNA-treated (n = 369) and injured (n = 176) animals (*p < 0.05, **p < 0.01, ***p < 0.001 vs control, χ2 test). E, Firing rate of SEA units at different stimulation frequencies (mean ± SEM; control, n = 4; siRNA, n = 22; injured, n = 17; *p < 0.05 vs control, two-way ANOVA with Tukey's). F, Quantification of AD rate per SEA unit (mean ± SEM; *p < 0.05 vs control, Mann–Whitney test).

Results propose that Kv9.1 downregulation after nerve injury may be the molecular switch controlling myelinated sensory neuron hyperexcitability. Intriguingly, a recent wide-genome association screen in humans identified a Kv9.1 polymorphism associated with susceptibility to develop chronic neuropathic pain after back surgery or leg amputation (Costigan et al., 2010), suggesting that the mechanisms described in our studies will be of direct clinical relevance to human pain. Future efforts to elucidate the precise pathways involved, combined with approaches aiming to compensate loss of Kv9.1 function, may create novel therapeutic opportunities for neuropathic pain management.

ABCA siRNA Transfection of Neurons & Astrocytes

Neuromics' i-Fect^TM Transfection Kit successfully meets the challenge of transfecting neurons and astrocytes in culture. The kit has been used for gene expression analysis studies for: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC. Related publication reference use of the kit for both in vitro and in vivo studies

In this study, Dr. Marina Guizzetti and his team use our i-Fect Kit and several other commercially available kits to knockdown ABCG1 and ABCG4: Jing Chen, Xiaolu Zhang, Handojo Kusumo, Lucio G. Costa, c, Marina Guizzettia. Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1831 (2), p.263-275, Feb 2013. doi:10.1016/j.bbalip.2012.09.007.
Abstract: Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.

siRNA transfection: ABCA1 SiRNA transfections: Neurons and astrocytes were transfected using the Nucleofector™ technology (Lonza/Amaxa; Walkersville, MD) as per the manufacturer's optimized protocol. In brief, primary neurons immediately after isolation, or astrocytes harvested after 7–10 days in vitro (DIV), were resuspended in Nucleofector solution. Aliquots of neurons or astrocytes were mixed with 200 pmol ABCA1 siRNA or non-targeting siRNA and were transfected using the Nucleofector programs O-007 and T-20 respectively. Exogenous cholesterol efflux was measured 96 h post transfection. ABCA1 down-regulation in ABCA1 siRNA-transfected cells was verified by Western blot. ABCG1 and ABCG4 Stealth RNAi^TM siRNA transfections: on the day of transfection primary astrocytes were switched to a medium (DMEM with 10%FBS) without antibiotics and supplemented with 50 nM ABCG1 or ABCG4 siRNA, lipofectamine RNAiMAX Transfection Reagent, and Opti-MEM I according to the manufacturer's instruction for 24 h followed by the removal of the medium containing transfection reagents. Six days after preparation, primary cortical neurons were shifted to a medium (Neurobasal/B27) without antibiotic; transfection was carried out by adding to the cultures a solution containing 12 nM ABCG1 or ABCG4 SiRNA, i-Fect siRNA Transfection Reagent, and Opti-MEM I for 24 h. Exogenous cholesterol efflux was measured 48 h after the removal of the transfection reagents. The specific silencing of ABCG1 in astrocytes and neurons was confirmed by Western blot and by qRT-PCR; silencing of ABCG4 was confirmed only by qRT-PCR because no specific antibody to ABCG4 is available.

Figures: Effect of ABCG1 and ABCG4 silencing on cholesterol efflux from neurons and astrocytes. A: Primary rat cortical neurons were transfected with a non-target (NT siRNA), an ABCG1 specific siRNA (ABCG1 siRNA) or an ABCG4 siRNA using the i-Fect™ siRNA transfection reagent. B: Primary rat cortical astrocytes were transfected with an ABCG1 siRNA or an ABCG4 siRNA using lipofectamine RNAiMAX. Twenty-four hours after transfection, cells were labeled with 1 μCi/ml [3H]cholesterol for 24 h followed by a 6 h incubation with cholesterol acceptors. [3H]Cholesterol was quantified in the medium and in the cellular lipids (n=11–12). **pb0.01; ***pb0.001 compared to acceptor-matched controls by Student's t test. C: ABCG1 (left) and ABCG4 (right) mRNA levels were quantified by qPCR in neurons transfected with NT siRNA, ABCG1 siRNA or ABCG4 siRNA (n=4). D: the levels of ABCG1, ABCG4 and ABCA1 mRNA were determined by qPCR in ABCG1 siRNA-transfected (left) and ABCG4 siRNA transfected astrocytes (n=4). Representative immunoblots of ABCG1 levels (upper blots) and β-actin levels (lower blots) in neurons transfected with NT siRNA and ABCG1 siRNA (E) and astrocytes transfected with ABCG1 siRNA (F) and the densitometric quantification of ABCG1 levels normalized to β-actin from 4 independent determination (G) are shown.

The efficiency of transfection is higher in astrocytes than in neurons, it is possible that the lack of an effect of ABCG1 siRNA on cholesterol efflux from neurons may be due to the fact that ABCG1 is not sufficiently down-regulated in these cells. Further study on this phenomena is needed and are important for the discovery of druggable targets that could positively modulate cholesterol homeostasis.

i-Fect Assisted Transfection of ASIC-siRNAs

Dr. Eric Lingueglia and his team at LabEx Ion Channel Science and Therapeutics, 06560 Valbonne, France continue to do excellent work using our i-Fect siRNA Transfection Kit. Here they use the kit to help validate the potential of a new class of three-finger peptides from the black mamba to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons: Sylvie Diochot, Anne Baron, Miguel Salinas, Dominique Douguet, Sabine Scarzello, Anne-Sophie Dabert-Gay, Delphine Debayle, Valérie Friend, Abdelkrim Alloui, Michel Lazdunski, Eric Lingueglia. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature (2012)doi:10.1038/nature11494.

...Locally designed siRNAs targeting ASIC1 (si-ASIC1a/1b, GCCAAGAAGUUCAACAAAUdtdt), ASIC2 (si-ASIC2a/2b, UGAUCAAAGAGAAGCUAUUdtdt) and ASIC2a (si-ASIC2a, AGGCCAACUUCAAACACUAdtdt) have been validated in vitro in COS-7 cells transfected with myc-ASIC1a, ASIC1b, myc-ASIC2a or myc-ASIC2b, and the relevant siRNA or a control siRNA (si-CTR, GCUCACACUACGCAGAGAUdtdt) with TransIT-LT1 and transIT-TKO (Mirus), respectively. Cells were lysed 48 h after transfection and processed for western blot analysis to assess the amount of ASIC1a protein with the anti-Myc A14 antibody (1:500; Santa Cruz Biotechnology) or the anti-ASIC1 antibody (N271/44; 1:300; NeuroMab) and a monoclonal antibody against actin (AC-40; 1:1,000; Sigma) as a loading control. siRNAs were intrathecally injected into mice (2 µg per mouse at a ratio of 1:4 (w/v) with i-Fect (Neuromics)) twice a day for 3 days. After 3 days of treatment, the paw-flick latency was measured and the residual effect of mambalgin-1 (intrathecal or intraplantar, 34 µM) or the carrageenan (intraplantar, 2%)-induced hyperalgesia was tested. For validation of the in vivo effect of the siRNAs, lumbar DRGs or lumbar dorsal spinal cord were removed after the last siRNA injection for total RNA isolation (RNeasy kits, Qiagen) followed by cDNA synthesis (AMV First-Strand cDNA synthesis kit (Invitrogen) and High Capacity RNA-to-cDNA Kit, (Applied Biosystems)). The relative amounts of ASIC transcripts were evaluated by quantitative reverse-transcription PCR in a Light-Cycler 480 (Roche Products). Pre-designed and validated TaqMan assays for ASIC1 (ASIC1a and ASIC1b; Mm01305998_mH), ASIC1a (Mm01305996_m1), ASIC2 (ASIC2a and ASIC2b; Mm00475691_m1), ASIC3 (Mm00805460_m1) and 18S ribosomal RNA (Mm03928990_g1) were from Applied Biosystems. Each cDNA sample was run in triplicate and results were normalized against 18S and converted to fold induction relative to control siRNA treatment...

i-Fect and the Study of Glomerulosclerosis (Kidney Disease)

in vitro RANK knockdown system to determine whether RANK was necessary for podocyte survival.

Our i-Fect^TM Transfection Kit is widely used and frequently published as a potent tool for gene expression analysis. In this study i-Fect was used to consistently achieved close to 71.6% siRNA transfection efficiency in podocytes: Shuangxin Liu, Wei Shi, Houqin Xiao, Xinling Liang, Chunyu Deng, Zhiming Ye, Ping Mei, Suxia Wang, Xiaoying Liu, Zhixin Shan, Yongzheng Liang, Bin Zhang, Wenjian Wang, Yanhui Liu, Lixia Xu, Yunfeng Xia, Jianchao Ma, Zhilian Li. Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury. PLoS ONE: Research Article, published 25 Jul 2012 10.1371/journal.pone.0041331......RANK small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled, functionally validated Cy3–labeled RANK siRNA (Invitrogen) [16]. Podocytes that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above, and transfected using the RANK siRNA transfection reagent (Neuromics). For determination of the transfection efficiency, a Cy3–labeled RANK siRNA was analyzed by flow cytometry. Western blot analysis for RANK was performed with samples from cells 24 to 96 h after the transfection. Several concentrations of RANK siRNA (20, 40, and 60 nM) were tested to determine optimal knockdown conditions...
Data Highlights: To better understand RANK function in immortalized mouse podocytes, we used an in vitro RANK knockdown system to determine whether RANK was necessary for podocyte survival [33]. We consistently achieved close to 71.6% siRNA transfection efficiency in podocytes, as visualized by transfecting a fluorescently tagged Cy3-RANK siRNA. Flow cytometry fluorescence of podocytes not transfected with siRNA-Cy3 was measured as negative control; the value in podocytes was 4.1% (Figure 5A,5B and 5C). Knockdown of RANK was determined to be maximal between days 3 and 4 after transfection (Figure 5D). We did not observe any morphologic changes between cells with or without RANK siRNA knockdown during the 4 d after transfection. To test whether RANK was involved in the apoptosis of podocytes in vitro, we studied podocytes apoptosis before and after stable knockdown of RANK with siRNA. The knockdown of RANK alone did not induce podocytes apoptosis, but increased mildly the apoptosis of podocytes exposed to PA. However, RANKL reduced apoptosis of podocytes transfected with RANK siRNA exposed to PA compared with control siRNA (RANK siRNA 16.5±1.5% versus control siRNA 24.0±1.8%, p<0.01, Figure 5E).

Images: RANKL and RANK Protects Mouse Podocytes from Apoptosis.(A) Mouse podocytes were transfected with RANK siRNA at a concentration of 100 nM. (B) Flow cytometry fluorescence of podocytes not transfected with siRNA-Cy3 was measured as negative control; the value in podocytes was 4.1%. (C) Transfection efficiency was measured by counting fluorescence-positive cells by flow cytometry; the value in podocytes was 71.6%. (D) RANK immunoblotting of podocytes revealed that RANK was downregulated with RANK siRNA. RANK protein was low abundance in the podocytes 3 d after transfection (lane 1 and lane 2). (E) The percentage of apoptotic cells was measured by flow cytometry. In cells transfected with RANK siRNA, RANK knockdown was associated with mild increase in apoptosis compared with control siRNA (24.0±1.8% versus 22.8±1.1%; p>0.05) after podocytes were exposed to PA. However, RANKL (40 ng/ml) decreased PA-induced apoptosis of podocytes with RANK siRNA (RANKL 16.5±1.5% versus control 22.8±1.1%). (F) Quantification of apoptosis of podocytes with PA and RANKL. Apoptosis was measured by flow cytometry in control podocytes without PA induction (G) (8.7±0.97%) and podocytes that were exposed to RANKL (40 ng/ml) without PA after 48 h (H) (5.7±0.81%). (I) PA increased the apoptosis of podocytes. Apoptosis was measured by flow cytometry of podocytes exposed to PA (25 µg/ml) for 48 h (26.3±3.6%). (J) Exogenous RANKL protected podocytes from PA induction apoptosis. RANKL (40 ng/ml) decreased PA-induced apoptosis (15.5±2.2%). All the experiments were conducted in three times. **Compared with control, p<0.01; *Compared with control, p<0.05.

The upregulation of RANKL and RANK, in combination with the significant protective effects of RANKL, indicates that RANK is part of an adaptive, recovery response to podocyte injury. This is the first observation that RANKL, acting through RANK, functions in an injury paradigm in the kidney. These data raise the exciting therapeutic possibility of giving exogenous RANKL to patients with glomerular disease that is characterized by a loss of podocytes, such as membranous nephropathy and focal segmental glomerulosclerosis.