Mesangial cells (MCs) in the glomeruli of the kidney are essential in maintaining the structural integrity of the glomerular capillary network [1
]. In healthy adult kidneys, MCs remain in a quiescent state with a cell renewal rate of < 1% per day, as shown by autoradiography [2
]. With various stress stimuli, including increases in blood pressure and blood sugar, MCs show aberrant proliferation. The uncontrolled MC proliferation can induce extracellular matrix (ECM) accumulation in the glomeruli, leading to glomerulosclerosis [3
]. These features are recognised as a critical element in the pathogenesis of chronic kidney disease (CKD) secondary to many glomerular diseases, including diabetic nephropathy and IgA nephropathy.
The proliferation of MC is tightly regulated by various mitogenic stimuli including platelet derived growth factor (PDGF), PI3/AKT and tyrosine kinases [6
]. In addition to this, the influx of Ca2+
via calcium channels appears to be involved in the regulation of MC proliferation. Alongside the different types of high voltage-gated calcium channels including P/Q type, N-type, R-type, and L-type channels are the low voltage-gated T-type calcium channels (TTCC) [10
]. It has been recognised for decades that calcium channel blockers can impact MC proliferation through mechanisms beyond L-type inhibition [11
]. The specific target of these actions is uncertain, though they are unlikely to be P/Q-type, N-type or R-type calcium channels as there is no evidence of their expression in MC. TTCC on the other hand, are expressed in human and rat MC and are sensitive to inhibition by the TTCC blocker TH1177 in vitro and in vivo in the Thy1 nephritis rat model [12
]. Also, the knockdown of TTCC in human and rat MC has been shown to inhibit proliferation [12
]. TTCC exhibit a similar structure to that of LTCC but differ in kinetics and activation threshold [10
]. The blockade of TTCC also exhibits anti-proliferative properties in vascular and pulmonary arterial smooth muscle cells [15
] and human cancer cells [17
]. In aortic smooth muscle cells, T-type calcium currents are predominantly present in the G1 phase and the synthesis phase of the cell cycle [15
]. There are three isoforms of TTCC including Cav3.1(α1G
) and CaV
]. To date, the role of specific TTCC isoforms in MC proliferation is not well studied. Additionally, there are no known reports available that show the presence of TTCC in mouse MC.
As no selective and specific small molecule inhibitors of TTCC isoforms exist, we aimed to investigate the expressions of TTCC isoforms in mouse MC in vitro and the impact of deletion of specific TTCC isoforms on cell proliferation and phosphorylation of ERK1/2, a key process in the Ras-MAPK pathway controlling cell-cycle progression [18
]. We have, therefore, developed single and double knockouts (SKO and DKO) of CaV
3.1 and CaV
3.2 by using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) gene editing system. We have also examined the stimulated phosphorylation of ERK1/2 in CaV
3.1 and CaV
3.2 SKO MC clones as compared to WT cells and evaluated proliferation of CaV
3.1 and CaV
3.2 SKO MC with and without non-selective TTCC blockers (mibefradil and TH1177).
Mouse mesangial cells (SV40 MES13) purchased from ATCC (Virginia, United States) were maintained in a 3:1 solution of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Paisley, UK) and F-12 (Invitrogen). The medium was supplemented with 5% fetal bovine serum (FBS), 14 mM HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin (Invitrogen). Cultured cells were maintained at standard cell culture condition at 37 °C with 95% O2 and 5% CO2.
Calcium channel inhibitors
Mibefradil (Sigma Aldrich, Dorset, UK) and verapamil (Sigma Aldrich) were made from 10 mM stock solutions in distilled water and stored at 4 °C, and every two months, fresh stock solutions were prepared. TH1177 was a kind gift from Dr Lloyd Gray, University of Virginia, Charlottesville, Va., USA. TH1177 was prepared from a 10 mM stock solution in 100% ethanol and stored at -20 °C.
Serum, PDGF and TGF-β1 stimulation
MES13 cells were seeded at 3 × 105 cells in 35 mm dishes with 1% FBS for 48 h. At 48 h, cells were cultured in varied conditions: 0% FBS, 20% FBS, or 20 ng/ml PDGF-BB and 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN) and vehicle control [TGF- β1 buffer-4 mM HCl plus 0.1% BSA, pH3] for 24, 48 and 72 h time points. Cells were harvested for RNA extraction at the end of each time point. The medium was removed and washed with PBS and 350 µl of RLT buffer with beta-mercaptoethanol was used to lyse the cells. The RNA extraction was carried out using the RNeasy Mini Kit (Qiagen Ltd, Crawley, UK) by following the manufacturer’s instructions.
Reverse transcriptase- polymerase chain reaction (RT-PCR)
cDNA was synthesised from 2 µg of RNA using high capacity RNA to cDNA synthesis kit (Applied biosystems, Massachusetts, USA) in the 20 µl reaction mix. RT-PCR amplification was carried out from 5 µl of cDNA using Dream Taq polymerase (Fischer Scientific, Loughborough, UK) using a thermocycler. The PCR amplification was carried out with an initial denaturation of 95 °C for 3 min, 35 cycles of denaturation 95 °C for 30 s, annealing Tm-5 for 30 s, extension 72 °C for 1 min and final extension 72 °C for 5 min. The RT-PCR primers are listed in Suppl. Table S1
plenti-CRISPR-cas9-gRNA for CaV
3.2 was purchased from GenScirpt, New Jersey, USA. The plasmid contained a plentiCRISPRv2.0 backbone cloned with cas9, ampicillin, bleomycin, puromycin selection markers, and 20 nucleotides of the gRNA sequence complementary to exon 4 of Cacna1G and exon 6 of Cacna1H gene under the control of U6 promoter (Suppl. Fig. S2
a, c and b).
Transfection, antibiotic and clonal selection
plenti-CRISPR-cas9-gRNA for CaV3.1/CaV3.2 plasmids were used to generate stable knockouts which were transfected into MES13 cells using lipofectamine 3000 (Thermo fisher Scientific, Massachusetts, USA). Briefly, 6 × 105 cells were seeded in 6 well plates overnight in the antibiotic-free growth medium. The following day, for SKO generation, 5 µg of plenti-CRISPR-cas9-gRNA for CaV3.1 or CaV3.2 plasmids and for DKO generation, 2.5ug each of plenti-CRISPR-cas9-gRNA for CaV3.1 and CaV3.2 were transfected by lipofection, and the medium was changed after 6 h. Post 72 h of transfection, complete growth medium with 2 µg/ml of puromycin was added for antibiotic selection. The cells resistant to puromycin post 72 h antibiotic selection were trypsinised and sub-cultured to the new six-well plate and the second round of antibiotic selection was carried out using puromycin to minimise the contamination of untransfected cells. Although the cells were sorted with a selection marker, there remained a possibility of a mixed population of cells with a variable length of base-pair deletion of ~ 1–19 nucleotides. Hence, the knockout cells were subjected to single-cell clonal selection by serial dilution starting with 500 cells in 96-well plates. The SKO clones were picked, expanded and confirmed at the genomic level by Sanger sequencing and confirmed at the translational level by Western blot. This second round of puromycin selection resulted in too few cells to achieve single cell clonal selection for the DKO clones and expansion for Western blotting, hence DKO cells from the first round of antibiotic selection went through clonal selection in the conditional medium. The survived clones were sent for Sanger sequencing alone.
MES13 control cells and CaV
3.2 SKO and DKO clones were subjected to genomic DNA extraction by using the QIAamp DNA extraction kit (Qiagen Ltd, Crawley, UK), following the manufacturer’s instructions. The genomic DNA was quantified in a Nanodrop, and 100 ng of DNA was amplified by using Dream Taq polymerase and primers spanning the gRNA sequence region in 20 µl reaction mix. The primer list is detailed in Suppl. Table S1
. The PCR products were cleaned using the ExoSAP-IT Express PCR product clean-up kit (Thermo Fisher Scientific). The PCR product was preceded to Sanger sequencing by GATC Biotech, Ebersberg, Germany.
MES13 cells and SKO clones of CaV3.1 and CaV3.2 treated with or without calcium channel blockers (mibefradil and TH1177) were subjected to a microculture tetrazolium (MTS) assay (Promega, Southampton, UK) to measure the cell number. Briefly, MES13 cells, CaV3.1 and CaV3.2 SKO clones were subjected to serum deprivation in 1% FBS for 48 h. Cells were seeded into 96-well plates at a density of 5000 cells per well and incubated with varied concentration of mibefradil and TH1177 (0, 5, 10 and 20 µM) in 5%FBS. Absorbance at 492 nm was measured at 48, and 72 h in a microplate reader.
Signal transduction evaluation
MES13 and SKO clones of CaV3.1 and CaV3.2 were seeded at 6X105 cells per well in 6 well plates and left overnight. The cells were serum deprived to 1% FBS for 48 h. At 48 h, the cells were stimulated for 30 min with either: 5% FBS, 20 ng/ml PDGF + 1% FBS or 10 ng/ml TGF-β1 + 1% FBS.
Similarly, MES13 cells alone were seeded at 6X105 cells per well in 6 well plates and left overnight. The cells were serum deprived to 1% FBS for 48 h. At 44 h, the cells were treated with 5 µM mibefradil + 1% FBS and at 48 h, the cells were stimulated with 5% FBS, 20 ng/ml PDGF + 1%FBS and 10 ng/ml of TGF- β1 + 1%FBS for 30 min.
The medium was removed immediately after 30 min stimulation, washed with PBS, and the cells were lysed with RIPA buffer with protease and phosphatase inhibitor (Sigma). The lysate was scraped off from the plates and incubated on ice for 30 min with intermittent vortexing. The lysates were centrifuged for 14,000 rpm at 4 °C for 20 min. The supernatant from the lysate was transferred to new labelled tubes and stored at -80 °C. The experiments were repeated three times.
The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20–40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for CaV3.1 and CaV3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: CaV3.1 (1:1000) (Alomone, Jerusalem, Israel) and CaV3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the blots were washed thrice with 1 × TBS + 0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1 × TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.
All data were tested for normality and no difference in the variances between groups was detected using the Shapiro–Wilk test. Parametric variables were analysed using a One-Way Analysis of Variance (for multiple group comparisons) with a post hoc Bonferroni test or Two-Way Analysis of Variance with post hoc Tukey test p < 0.05 is considered as statistical significance. Statistical analyses were performed using Prism software version 8.0 (Graph-Pad Software, San Diego, CA).
In this study, we have demonstrated that i) mouse MC express TTCC and are sensitive to two TTCC inhibitors: mibefradil and TH1177. However, MC are insensitive to the LTCC inhibitor, verapamil; ii) The TTCC isoforms CaV3.1 and CaV3.2 were successfully knocked out in mouse MC by CRISPR-cas9 gene editing, creating viable single-cell TTCC isoform SKO clones; iii) CaV3.1 SKO, but not CaV3.2 SKO inhibits the serum, PDGF and TGF- β1 stimulated phosphorylation of ERK1/2; iv) The effect of CaV3.1 SKO on pERK1/2 is similar to the actions of TTCC blockers (mibefradil and TH1177) in WT cells; v) Knock out of either CaV3.1 or CaV3.2 alone does not alter the anti-proliferative effects of mibefradil or TH1177 and has minimal effects on stimulated cell proliferation, whereas knock out of both isoforms completely inhibits proliferation.
TTCC and their role in MC proliferation have been reported in a few studies. Findings in rat and human MC have shown that only CaV
3.1 and CaV
3.2 were detected in rat MC and CaV
3.2 is expressed in human MC [12
]. However, in our study, we have shown that all three TTCC isoforms (CaV
3.2 and CaV
3.3) were expressed in mouse MC. Various studies have used mibefradil and TH1177 as an inhibitor of TTCC in proximal tubular epithelial cells, MC and other cell types. Mibefradil has a 10- fold selectivity to TTCC than LTCC [21
], whereas TH1177 may inhibit other ion channels. In prostate cancer cells, TH1177 inhibits proliferation by impeding the entry of Ca2+
, but it does not affect calcium release from internal stores [23
]. TH1177 is reported to affect a range of cation channels including TRPV5 channels [24
]. Studies in rat and human MC show that the MC were sensitive to the TTCC inhibitors mibefradil and TH1177 but there was no effect seen by the LTCC inhibitor (verapamil) [12
]. Similar sensitivity was shown in our mouse MC to mibefradil and TH1177 with no effect of verapamil. As TTCC blockers can be non-selective, to accurately understand the role of TTCC, it is important to look at the effect of the different isoforms in signaling and proliferation.
The role of CaV3.1 in cell proliferation has been described in heart, lung and cancer cells [15
]. Human pulmonary artery myocytes express CaV3.1 and silencing of CaV3.1 inhibits serum-induced proliferation [25
]. This is consistent with studies in preadipocytes in primary culture. The level of CaV3.1 in preadipocytes is high and is downregulated during differentiation. Knockdown of CaV3.1 or use of mibefradil inhibits preadipocyte proliferation [29
]. CaV3.1 also plays a different role in the cancer cells. Knockdown of CaV3.1 indeed induces cell proliferation and reduces apoptosis in MCF-7 breast cancer cells. The effect was reversed by overexpression of CaV3.1. The effect of CaV3.1 knockdown is similar to the TTCC blocker (tarantula toxin ProTx-1). In MCF-7 cells, knockdown of CaV3.2 did not affect proliferation or apoptosis [28
]. All these studies used siRNA to transiently silence the CaV3.1 or CaV3.2 isoforms and evaluate their roles in proliferation. There have been no studies to date demonstrating the specific role of TTCC isoform on the MC proliferation in the kidney. TTCC inhibition experiments in rat and human MC also did not delineate the specific role of TTCC isoforms, though we have previously shown that although Cav3.2 is upregulated in the cortex of kidneys with Thy1 nephritis, this is not in the glomerular compartment. Cav3.1 is predominantly expressed in glomeruli, though this does not increase with disease [12
]. We have therefore addressed the specific mesangial roles of Cav3.1 and Cav3.2 in this study using CRISPR-cas9 gene editing. In our study, mouse MCs lacking either CaV
3.1 or CaV
3.2 survived and formed single-cell clones whereas CaV
3.1 and CaV
3.2 DKO clones didn’t survive to carry out the western blot. This suggests that expression of either one of the TTCC isoforms is sufficient to support cell proliferation and cell survival.
Given the wealth of evidence suggesting a role for T-type Ca channels in cell proliferation, it is surprising that knocking out each isoform individually has virtually no impact on cell proliferation. However, since the relationship between T-type Ca current and proliferation is extremely steep (see Fig. 4
]), with only a small residual current being permissive for proliferation, it seems that the presence of either isoform is sufficient to allow effectively normal proliferation. The absence of both isoforms however is lethal—presumably by completely preventing cell cycle progression. On the other hand, pharmacological inhibition, with drugs showing little of no isoform selectivity, allows a graded reduction in Ca influx and hence a graded anti-proliferative effect.
MC proliferation is controlled by various regulators of the cell cycle, including PI3/AKT, Ras/MAPK, and calmodulin-dependent kinase signaling [8
]. These are likely to be involved in the changes seen in MC proliferation in TTCC KO clones. MAPK plays an important role in mesangioproliferative disease and inhibition of Ras/ERK1/2 by a pharmacological inhibitor or Ras antagonist reduces glomerular cell proliferation [19
]. It has been shown that in rat pulmonary artery smooth muscle cells (PASMCs) stimulation with insulin growth factor -1 (IGF-1) induces CaV
3.1 expression, and the level was decreased when treated with both PI3K and MEK inhibitor [15
]. PASMCs treated with constitutively active AKT increase basal CaV
3.1 expression more than with constitutively active MEK treatment, suggesting upregulation of CaV
3.1 is via AKT signaling [15
]. Similarly, CaV
3.1 is highly expressed in prostate cancer tissue. Knockdown of CaV
3.1 in PC-3 and Dol45 cells suppressed cell proliferation by inhibiting the transition of cells to G1/S phase. The CaV
3.1 knockdown in the cancer cells decreases phosphorylated AKT (pAKT) protein which was recovered by ectopic AKT expression [27
]. Furthermore, idiopathic pulmonary arterial hypertension cells (iPAH) are more proliferative than cells from control pulmonary arteries. TTCC blocker TTA-A2 reduced the proliferation of iPAH cells by delaying the S/G2 transition. TTA-A2 induces pAKT but not pERK1/2 or phosphorylated P38, suggesting the proliferation of iPAH is diverted to AKT signaling [26
]. All these studies suggest that the CaV
3.1-dependent regulation of proliferation is mostly via AKT signalling.
The present results in mouse MC reveal a significant role for CaV
3.1 in the stimulated phosphorylation of ERK1/2 with no such role for CaV
3.2. The effects of both Cav3.1 and 3.2 SKO on MC proliferation were much more subtle. CaV
3.1 SKO cells without pERK1/2 responses were slightly but not profoundly hypoproliferative. This may be explained by compensatory changes in the SKO cells to amplify pERK1/2-independent proliferative signaling. Similarly, TTCC inhibitors were effective in reducing proliferation in CaV
3.1 SKO cells that already had impaired pERK1/2 responses. This implies that the pERK1/2 pathway actions of these agents may not be the mechanism of their anti-proliferative actions. TTCC activity may be coupled to proliferation by multiple pathways. Moreover, these small molecule TTCC inhibitors likely have a range of actions on cation channels, including inhibition of TRP channels [24
]. These off-target effects are another possible explanation for their anti-proliferative actions.
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