Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases.


Cell culture
Immortalized mouse podocytes (provided by Dr. Shankland's group, University of Washington Seattle, Seattle, USA) were cultured according to a published procedure Griffin et al [17]. Briefly, the cells were grown in RPMI-1640 medium supplemented with 10% FBS, 2mmol/L glutamine, 10mmol/L HEPES, 1mmol/L sodium pyruvate, 100 U/ml penicillin and 0.1 mg/ml streptomycin. To induce proliferation, cells were grown on the collagen I coated plates (Becton Dickinson Labware) with the addition of 10 U/ml recombinant mouse γ-interferon to the culture medium, and incubated at 33°C (growth permissive conditions). To induce differentiation, cells were grown in the same culture medium without γ-interferon and incubated at 37°C (growth restrictive conditions).Cells cultured for 10-14 days were used for the experiments. Cortical neurons and HEK293 cells were cultured as previously described [18].

Transfection of short interfering RNA (siRNA) and infection of p35
Cdk5 siRNA (sc-29263), p35 siRNA (sc-36154), and control siRNA (sc-37007) were ordered from Santa Cruz Inc and delivered to podocytes using Pepmute siRNA transfection reagent (SignaGen Laboratories), according to the manufacturer's instructions. Briefly, podocytes were seeded in a 6-well plate and cultured for 7 days at about 50-60% confluence. For transfection, 5μl siRNA were diluted in 100μl of 1x siRNA transfection buffer (SignaGen Laboratories) in a final concentration of 50 nM siRNA. Three ul of Pepmute reagent were then mixed by pipetting up and down, incubated 15 minutes at RT, and dropped onto the cultured cells. After 72 h, cells were harvested or fixed for further experiments.
Adenovirus-p35 and empty vector (EV) were made and infected according to the methods of previous study [2,18].

Glomerular Isolation
C57BL/6J mice kidneys were drawn from the breeding adult mouse and Glomeruli were isolated by following the method of graded sieving [19].

Western blot analysis
Cells were harvested by scraping cells and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM β-glycerol phosphate, and 1 mM NaF, supplemented with a mixture of protease inhibitor cocktail and 1 mM PMSF), and incubated for 30 minutes on ice. After centrifugation for 20 minutes at 13,000× g at 4°C, the protein concentrations of the supernatants were determined using the BCA protein assay (Pierce, Rockford, Illinois). An equal amount of total protein (20 μg of protein/lane) was resolved on a 4-20%, a 15% or 8% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membrane was incubated in blocking buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% (v/v) Tween 20 (TTBS) plus 5% dry milk (w/v) for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. The membranes were then washed four times in TTBS, followed by incubation in goat anti-mouse or goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibodies (Amersham Biosciences, 1: 2500) for 2 h at room temperature. Western blots were analyzed using the Enhanced Chemiluminescence (ECL) kit (Pierce) following the manufacturer's instructions.

Cdk5 kinase assays in vitro
Kinase assays were performed as previously described [20]. Cdk5 was immunoprecipitated from supernatants of lysed cells with the polyclonal C-8 antibody overnight at 4°C and immunoglobulin was isolated using Protein A-sepharose beads for 2 h at 4°C. Immunoprecipitates were washed three times with lysis buffer and then once with 1× kinase assay buffer containing 20 mM Tris-Cl pH 7.4, 1 mM EDTA, 1 mM EGTA, 10 mM MgCl2, 10 μM sodium fluoride and 1 μM sodium orthovanadate. Kinase assays were performed in the same buffer containing 1 mM DTT, 0.1 mM ATP and 0.185 MBq [γ-32P] ATP with 20 μg of histone H1 as the substrate. Histone H1 phosphorylation was performed in a final volume of 50 μl, incubated at 30°C for 60 minutes and stopped by addition of 10% SDS sample buffer and heated at 95°C for 5 minutes. Samples were separated by SDS-PAGE, gels were stained with Coomassie, destained, dried and exposed for autoradiography.

Immunocytochemistry
Podocytes were cultured and infected on glass cover slips. They were washed twice in PBS, fixed for 30 minutes at room temperature in 4% paraformaldehyde in PBS, and permeabilized with a buffer (25 mM Tris, pH7.4, 150 mM NaCl, and 0.1% Triton X-100) for 15 min. The coverslips were incubated overnight at 4°C with primary antibodies. All antibodies were diluted in PBS with 1% Triton-X-100. After three washes in PBS, coverslips were incubated with fluorescein goat anti-mouse IgG, or Texas Red goat anti-rabbit IgG for 1 h at room temperature, followed by three washes with PBS. Fluorescent images were obtained with a Zeiss LSM-510 laser-scanning confocal microscope and images were managed with Adobe Photoshop.
In situ cell death detection (TUNEL and cleaved caspase-3 staining assays) Podocytes were cultured and tranfected with siRNAs using Pepmute siRNA trasfection reagent (SignaGen Laboratory). TUNEL staining was performed according to the manufacturer's instructions using the in situ cell death detection kit TMR red (Roche). TUNEL staining and cleaved caspase-3 fluorescent images were captured with a Zeiss LSM-510 laser-scanning confocal microscope and images were managed with Adobe Photoshop. Cell counts were performed to measure cell apoptosis.

Viability of cells assay
Podocytes were cultured and tranfected with siRNAs using Pepmute siRNA trasfection reagent. Viable cells was counted using cell counting kit-8 (CCK-8) according to the manufacturer's instructions.

Statistical analysis
Analysis of variance (ANOVA) with Tukey-Kramer adjustment for multiple comparisons was applied. A p-value below 0.05 was considered significant.

Results
The expression of Cdk5, p35, and Cdk5 activity in immortalized mouse podocytes and glomeruli Mouse cortical neuronal cells expressing Cdk5 and p35, were used as positive control [1]. The expression of Cdk5 was approximately equal in mouse cortical neurons (CN), cultured immortalized mouse podocytes (P), mouse glomeruli (G), and HEK293 cells (Fig 1A. lower panel, lane 1, 2, 3 and 4, respectively). p35, however, was positively expressed in mouse podocytes and glomeruli ( Fig 1A. upper panel, lane 2&3), and at higher levels in the cortical neurons ( Fig 1A. upper panel, lane 1), but absent in HEK293 cells (Fig 1A. upper panel, lane 4). We further confirmed the expression of Cdk5 and p35 in immortalized cultured podocytes by using immunofluorescent analysis (Fig 1Ba and 1Bb, respectively); Cdk5 and p35 localization overlapped in the podocytes (Fig 1Bd). Cdk5/p35 activity assays in podocytes and glomeruli also showed activities (Fig 1C & 1D. lane2&3), that were much lower than in cortical neurons (compared lane 2&3 with lane 1). There was no Cdk5 activity in the HEK293 cells (Fig 1C & 1D. lane4). These results indicate that p35 is present in podocytes, activates Cdk5 activity and may play an important role in podocyte regulation.

Time course expression of Cdk5 and p35 in podocyte cultures and cortexes from developing mouse kidneys
To detect the expression levels of Cdk5 and p35 during podocyte maturation in culture and during kidney development we used Western blots to determine the expression of Cdk5 and p35 in cultured podocytes and cortexes from mouse kidneys at different times (Fig 2A & 2C). The expression of Cdk5 tended to increase over time in cultured podocytes (

Over expression of p35 promotes podocyte differentiation
To investigate the relation between Cdk5/p35 and podocyte differentiation, we over expressed p35 gene in podocytes and observed cell differentiation. Podocytes were infected with adenovirus-p35 and EV on the day1of differentiation phase culture (37°C without γ-IFN). After 48h, cells were fixed or harvested for ICC staining or WB analysis. The results showed that p35 infected cells exhibit obviously spike like differentiation processes (Fig 3Ae-3Ah, arrows). However, there are negative spike like processes in EV infection cells (Fig 3Aa-3Ad). The expression of synaptopodin, a podocyte differentiation marker, was increased in p35 infected cells (Fig 3f vs 3b). This result indicated that p35 may promote the podocyte differentiation. To confirm the result, we detected the expression of synaptopodin with WB using the same cells as above. The result showed that synaptopodin expression in the p35 infected cells was remarkably increased (Fig 3B,

Knockdown of Cdk5 expression induced apoptosis, decreased the expression of WT1 and viable cell number in immortalized mouse podocytes
It should be noted that Wilms' Tumor 1(WT1), a transcription factor, is required for podocyte maturation and is often used as a molecular marker for differentiated podocytes [21][22]. To further confirm the effect of Cdk5 in podocytes, we deleted Cdk5 from cultured mouse podocytes by using Cdk5 siRNA transfection technology. The Cdk5-siRNA transfected into cultured podocytes (day 7). After 72h incubation, cells were harvested for western blotting, kinase assay and viable cell counting. Cdk5 expression in the Cdk5 siRNA cells is significantly eliminated (Fig 4A.     vs bar 1, p<0.01). The roscovitine was used as positive control (Fig 4A. lane3 and 4B, bar 3). To test if the down regulation of Cdk5 affected podocyte survival, apoptosis analysis was carried out by western blotting using cleaved caspase3 antibodies, a cell apoptosis marker. The results showed that the cleaved caspase3 expression was significantly upregulated in the Cdk5 siRNA treated cells (Fig 4C. lane 2 and Fig 4D, bar 2), compared to the control siRNA cells (p<0.01). This was confirmed by a reduction in WT1, a podocyte-specific marker (Fig 4E. lane 2) with decreased cell number (Fig 4F, bar 2), compared to the control siRNA cells (p<0.05). As a positive control, roscovitine-treated cells showed a significantly increasing level of cleaved caspase 3 (Fig 4C. lane 3), reduction in WT1 expression (Fig 4E. lane 3) and viable cell number (Fig 4F, bar 3). These results show that knockdown of the Cdk5 expression and activity caused cell apoptosis and decreased podocyte number.
Knockdown of p35 decreased the expression of WT1 and viable cell number without affecting cleaved caspase 3 apoptosis marker as detected by western blotting P35 is the main activator of Cdk5. We now ask whether knockdown of p35 with p35 siRNA results in decreased cell number and elevated cleaved caspase 3 (Fig 5). The results showed a down regulation of p35 expression in Fig 5A, lane 2. There is a significant difference compared to the control siRNA treated cells (Fig 5B. bar graph, bar 2 vs bar 1, P<0.01). There is also a significant decrease in Cdk5 activity in p35 siRNA and roscovitine treated podocytes (Fig 5C. bar  2 and 3 vs bar 1, p<0.01 and <0.001, respectively). Cyclin I expression, however, was not changed in the three groups of cells (Fig 5A, panel 3). Moreover, WT1 expression and viable cell number decreased in p35 siRNA treated cells (Fig 5D. upper panel,

Knockdown of p35 resulted in defective podocyte morphology and apoptosis
Brinkkoetter et al had shown that deletion of p35 in podocyte or p35 KO in mice kidneys didn't directly induce podocyte apoptosis, but did so indirectly [5]. Our results confirmed their data although, we showed that podocyte number decreased in the p35 siRNA treated cells. To further explore whether depletion of p35 affects podocyte survival, we examined podocyte morphology by immunocytochemical staining with β-actin antibody. The results showed that podocyte structure was abnormal in p35 siRNA transfected cells (Fig 6Ab) similar to Cdk5 siRNA transfected cells (Fig 6Ac), which showed the cell morphological changes and actin rearrangement with a loss of the typcal transverse stress fibers; whereas roscovitine destroyed podocyte structure (Fig 6Ad). The results suggest that p35 knockdown may indeed cause podocyte apoptosis. Accordingly using tunel analysis we could demonstrate significant apoptosis in the p35 siRNA treated cells (Fig 6Bb and 6C. TUNEL -TMR, bar 2 vs bar 1 p = 0.041,). To further confirm this result, the same experiment was carried out using immunofluorecence staining with cleaved caspase 3 antibody. A similar result was obtained (Fig 6Bf and 6C. cleaved caspase 3, bar 2 vs bar 1, p = 0.036). The positive control cells which were treated with Cdk5 siRNA and roscovitine showed remarkable apoptosis in both staining analysis (Fig 6Bc, 6Bd,  6Bg and 6Bh). The significant differences were shown in Fig 6C (both TUNEL -TMR and cleaved caspase 3, bar3 and 4 vs bar 1, P<0.0001). Evidently, TUNEL and immuflouresence staining are more sensitive than Western blotting as a measure of apoptosis. All these findings indicated that Cdk5/p35 expression and activities play a cytoprotective role in maintaining podocyte function and survival.

Discussion
Podocytes are terminally differentiated glomerular epithelial cells which adhere to the outer flank of the glomerular basement membrane, that play an essential role in the prevention of proteinuria. Similar to other terminally differentiated cells podocytes have a limited capacity to proliferate. Accordingly, when they undergo apoptosis, overall cell number tends to decrease. Loss of podocytes is involved in the onset and progression of many primary or secondary kidney diseases [23][24][25].Dysfunction and loss of podocytes has become a critical indicator of the severity of glomerular damage and sclerosis. Cdk5 is activated by p35, p39, and cyclin I, and plays multiple roles in the growth and function of such terminally differentiated cells as neurons, pancreatic β cells and podocytes [17][18][26][27]. A recent study showing a role for Cdk5/ p35 in the pathogenesis of high glucose induced podocyte injury [28].
Griffin et al showed that Cdk5 protein increased during differentiation of cultured podocytes, and that p35 is also expressed in adult mouse glomeruli and cultured podocytes [5,17]. Here, we first confirmed the expression of Cdk5 and p35 in immortalized cultured mouse podocytes and glomeruli, and also showed that expression of both proteins increased over time in culture and during glomerular development respectively. This is the first demonstration that cdk5 and p35 are expressed in differentiating embryonal glomeruli, and persist in mature adult glomeruli. Similarly, in cultured podocytes, both Cdk5 and p35 expressions increased during the switch from a proliferating phenotype to a differentiated and quiescent one. These results suggest that Cdk5/p35 expression is important in podocyte differentiation. These results were confirmed by over-expressing p35 in podocytes which showed a spike like differentiation processes and increased expression of synaptopodin, a podocyte differentiation marker, in the beginning of differentiation culture condition.
Podocytes and neurons share many similarities, both cells are specialized, terminally differentiated cells, with a complicated cytoarchitecture and wide, highly organized cell processes [29]. It was originally thought that p35, an activator of Cdk5, is largely expressed in the neurons responsible for high levels of Cdk5 activity [30]. The activity is essential for neuronal migration and ordered cortical lamination during development of the mammalian brain [31]. Cdk5 knockout (KO) mice exhibit perinatal lethality and defective positioning of several types of neurons [15], p35 KO mice, however, show a milder phenotype of abnormal neuronal cytoarchitecture [15][16]. We ask whether a Cdk5 KO and /or a p35 KO express an abnormal Fig 6. p35 siRNA directly induced podocyte apoptosis and abnormal morphology as detected by TUNEL analysis and immunofluorescence staining respectively. After p35 siRNA transfection 72 hs, cells were fixed and submitted to TUNEL analysis and immufluorescence staining using cleaved caspase 3 and β-actin antibodies. A. podocyte morphological analysis (a: control siRNA; b: p35 siRNA; c: Cdk5 siRNA; d: roscovitine); B. podocytes apoptosis analysis: upper panel: tunel analysis (a: control siRNA; b: p35 siRNA; c: Cdk5 siRNA; d: roscovitine); lower panel: cleaved caspase 3 staining (e: control siRNA; f: p35 siRNA; g: Cdk5 siRNA; h: roscovitine). C. TUNEL staining and cleaved caspase-3 fluorescent images semi-quantity anlysis. The images were captured with a Zeiss LSM-510 laser-scanning confocal microscope and were managed with Adobe Photoshop. Cell counts were performed as follows: 10 independent fields were analyzed with a total of 500 cells where TUNEL and cleaved caspase-3 could be counted. DAPI staining gave the total number of podocyte cells. The bar graph shows the quantity of podocyte apoptosis expressed as mean from three separate transfections. podocyte or glomerular phenotype. Our current study showed that a Cdk5 KO (siRNAinduced) resulted in podocyte apoptosis, decreased WT1 and diminished podocyte number. These results, consistent with Taniguchi's reports [32] indicate a crucial role of Cdk5 in podocyte survival.
On the other hand, deletion of p35 in podocytes cultures or in p35 KO mouse kidneys did not induce podocyte apoptosis and loss directly, but affected them indirectly [5,21]. We obtained a similar result; the cleaved caspase 3 marker of apoptosis showed no effect in the p35 KO (siRNA-induced) podocytes but WT1 and viable cell number decreased indicating loss of podocyte number. Because of this finding, we further applied TUNEL and immufluorescence staining for apoptosis analysis in the p35 knockdown cells. Here, knockdown of p35 evoked abnormal podocyte morphology due to actin rearrangement (Fig 6Ab), and podocyte apoptosis (Fig 6Bb & 6Bf). This finding indicates that p35 is also involved in maintaining podocyte normal morphology and function. It also indicates that the TUNEL and immunofluorescence staining maybe more sensitive than WB for cell apoptosis analysis. The mechanism of p35 KO induction of podocyte apoptosis is not well understood. Apoptosis is differentiated from necrosis by morphologic and functional features and by the requirement for energy and intracellular proapoptotic proteins [33]. It is likely that decreased Cdk5 activity-dependent apoptosis results from deregulation of podocyte proliferation and differentiation. Cdk5 is critical for the cell development and differentiation in various cell types, such as myoblasts [34], lens epithelial cells [35], and human hematopoietic cells [17,36]. Maintaining podocyte differentiation is a prerequisite for normal podocyte cytoskeletal structure and survival. Since, cycline I is another activator of cdk5 in podocytes [5,17], it is likely that decreased p35 causes cell apoptosis indirectly. Our study showed that knockdown either Cdk5 or p35 does not affect cycline I expression. This result indicates that knockdown p35 caused podocytes apoptosis is independent from cycline I. A recent study examined the relationship between subcellular distribution of Cdk5 with its activator cyclin I and p35 in podocytes and found that the predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These results strongly indicate that cyclin I and p35 do not compensate for one another and that both are needed to maximize cell survival [27]. The presence of p39 in the podocyte has not been determined.
An alternative mechanism for the role of p35 in survival is seen in a study of a mutant p35 in Drosophila [37]. The mutation causes an increase in apoptotic and necrotic cell death, axonal fragmentation, accumulation of autophagosomes packed with crystalline-like depositions, and finally leads to neurodegeneration. The study revealed aggregated depositions of endogenous material as well as enhanced accumulation of autophagic organelles from ultrastructural analysis of the p35 mutant, which may contribute to neuronal death [37]. Whether it occurs in a podocyte p35KO and initiates apoptosis will be an important subject for future investigation.
In summary, in this study we show that both Cdk5 and p35 are expressed in cultured immortalized mouse podocytes and glomeruli and increase in expression over time. Knockdown of Cdk5 or p35 caused podocyte apoptosis and morphological abnormalities. Taken together, these data demonstrate that Cdk5/p35 expression and activity are essential for podocyte survival.