Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease (original) (raw)
SIRT1 is upregulated in Pkd1 mutant renal epithelial cells and tissues. To initiate our studies on the functional role of SIRT1 in ADPKD, we examined mRNA and protein levels of SIRT1 in Pkd1 mutant renal epithelial cells and kidneys. We found that mRNA and protein expression of SIRT1 was increased in _Pkd1_-null versus WT control mouse embryonic kidney (MEK) cells and in the postnatal _Pkd1_-null cell line PN24 compared with the postnatal _Pkd1_-heterozygous cell line PH2 (Figure 1, A and B). Knockdown of Pkd1 with 2 different lentivirus-mediated shRNAs in mouse inner medullary collecting duct (IMCD3) cells also resulted in upregulation of SIRT1 relative to appropriate controls (Figure 1C). SIRT1 expression was also increased in kidneys from well-characterized hypomorphic homozygous Pkd1nl/nl mice (15) compared with that in age-matched WT kidneys at P7, P14, P21, and P28 (Figure 1D). In addition, mRNA and protein expression of SIRT1 increased in P7 kidneys of Pkd1flox/flox:Ksp-Cre mice, as analyzed by quantitative RT-PCR (qRT-PCR), Western blot, and immunohistochemistry (Figure 1, E and F, and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI64401DS1). Furthermore, SIRT1 expression was upregulated in primary human ADPKD cells and ADPKD kidneys compared with primary normal human kidney (NHK) cells and normal kidneys, respectively (Figure 1G and Supplemental Figure 1B). These results suggest that the increased expression of SIRT1 in renal epithelial cells is caused by loss or mutation of Pkd1.
_Pkd1_-mutant renal epithelial cells and tissues demonstrated increased expression of SIRT1. (A) qRT-PCR analysis of relative Sirt1 mRNA expression in WT MEK (WT), _Pkd1_-null MEK (Null), PH2, and PN24 cells. (B) Top: Western blot analysis of SIRT1 and c-MYC expression from whole cell lysates. Bottom: Relative SIRT1 expression, quantified from 3 independent immunoblots and standardized to actin. (C) Top: Western blot analysis of SIRT1 expression in mouse IMCD3 cells with Pkd1 knockdown by 2 different lentivector-mediated Pkd1 shRNAs, VIRHD/P/siPkd13297 (siPKD13297) and pGIPZ-siPkd1, compared with that in the cells transduced with the respective control vectors, VIRHD/P/siLuc and pGIPZ-NS. Bottom: Relative Pkd1 knockdown efficiency, evaluated by qRT-PCR, indicated that Pkd1 expression was reduced by more than 90% and 70% in VIRHD/P/siPKD13297– and pGIPZ-siPkd1–transduced mouse IMCD3 cells, respectively, compared with that in control vector–transduced cells. (D) Top: Western blot analysis of SIRT1 expression in kidneys from WT and Pkd1nl/nl mice collected at P7, P14, P21, and P28. Bottom: Relative SIRT1 expression in the kidneys, standardized to tubulin. (E and F) qRT-PCR analysis of Sirt1 mRNA expression (E) and Western blot analysis of SIRT1 and c-MYC expression (F) in P7 kidneys from Pkd1+/+:Ksp-Cre (WT) and Pkd1flox/flox:Ksp-Cre (Flox) neonates. (G) Western blot analysis of SIRT1 expression in primary human ADPKD and NHK cells. **P < 0.01.
PC1 affects SIRT1 expression in renal epithelial cells through c-MYC. It has been reported that in ADPKD, renal c-MYC expression is elevated up to 15-fold (16). c-MYC has been shown to regulate SIRT1 expression in human cancer (HeLa) cells (17). Thus, c-MYC may regulate SIRT1 expression in renal epithelial cells. In support of this notion, we found that (a) c-MYC expression was increased in _Pkd1_-null MEK cells, PN24 cells, and kidney tissues from Pkd1flox/flox:Ksp-Cre mice (Figure 1, B and F); (b) overexpression of c-MYC increased mRNA and protein levels of SIRT1 in WT MEK cells and PH2 cells (Figure 2, A and B); (c) knockdown of c-MYC with siRNA decreased mRNA and protein levels of SIRT1 in _Pkd1_-null MEK cells and PN24 cells (Figure 2, C and D); and (d) c-MYC bound to 2 potential c-MYC–binding sites (E-boxes E1 and E2; ref. 18) of the SIRT1 promoter, as determined by ChIP assay with anti–c-MYC antibody (Figure 2E). These results suggested that loss of PC1 mechanistically altered SIRT1 expression in renal epithelial cells through c-MYC.
SIRT1 expression is regulated by c-MYC and is induced by TNF-α. (A and B) Overexpression of c-MYC increased levels of (A) Sirt1 mRNA, as analyzed by qRT-PCR, and (B) SIRT1 protein, as analyzed by Western blot, in WT MEK cells and PH2 cells transfected with pcDNA3-c-MYC for 48 hours. (C and D) Knockdown of c-MYC with siRNA decreased the levels of (C) Sirt1 mRNA, as analyzed by qRT-PCR, and (D) SIRT1 protein, as analyzed by Western blot, in _Pkd1_-null MEK cells and PN24 cells transfected with c-MYC siRNA for 48 hours. (E) c-MYC bound to the promoter of SIRT1. CHIP assay was performed with anti–c-MYC antibody or normal rabbit IgG in _Pkd1_-null MEK cells. The precipitated chromatin DNA was analyzed by PCR with primers that amplified from –1,009 to –850 bp (E1) or from –2,535 to –2,385 bp (E2). The PCR amplification for distant regions (–3,178 to –3,023 bp) was used as a negative control (NC). (F and G) TNF-α (100 ng/ml) induced (F) Sirt1 mRNA, as detected by qRT-PCR, and (G) SIRT1 protein, as detected by Western blot, in _Pkd1_-null MEK cells and PN24 cells. (H) Western blot analysis of SIRT1 expression in _Pkd1_-null MEK cells and PN24 cells treated with TNF-α (100 ng/ml) and/or SN50 (50 μg/ml). *P < 0.05; **P < 0.01.
SIRT1 expression can be further induced by TNF-α in Pkd1 mutant renal epithelial cells. TNF-α, which is detected in cyst fluid and promotes cyst formation (19), has been found to induce SIRT1 expression in vascular smooth muscle cells through the NF-κB p65/RelA subunit (20). We found that TNF-α induced mRNA and protein expression of SIRT1 in _Pkd1_-null MEK cells and PN24 cells (Figure 2, F and G). TNF-α also slightly induced SIRT1 expression in WT MEK cells, but had no effect in PH2 or mouse IMCD3 cells (Supplemental Figure 2). However, the NF-κB inhibitor SN50 efficiently blocked TNF-α–induced SIRT1 upregulation in _Pkd1_-null MEK cells and PN24 cells (Figure 2H), which suggests that TNF-α induces SIRT1 expression by activating the NF-κB pathway. Although it is unclear whether the cyst fluid TNF-α is initially secreted by immune cells or by cyst lining epithelial cells, these results suggest that the presence of TNF-α in cyst fluid during cyst development may serve as a secondary stimulus to further increase expression of SIRT1 in cyst lining epithelial cells in vivo.
Sirt1 and Pkd1 double conditional knockout delayed renal cyst formation. In order to explore the in vivo function of SIRT1 in a _Pkd1_-knockout mouse model, we crossed Pkd1flox/+:Sirt1flox/+:Ksp-Cre female mice with Pkd1flox/+:Sirt1flox/+:Ksp-Cre male mice, which have a kidney-specific Ksp-cadherin driving Cre expression. Cyst formation was significantly delayed in the absence of SIRT1 in Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice at P7 compared with that in age-matched Pkd1flox/flox:Sirt1+/+:Ksp-Cre and Pkd1flox/flox:Sirt1flox/+:Ksp-Cre mice (n = 10 per group; Figure 3, A–E). Kidney weight/body weight (KW/BW) ratios from Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice were dramatically reduced compared with Pkd1flox/flox:Sirt1+/+:Ksp-Cre mice (Figure 3F). In addition, blood urea nitrogen (BUN) levels were also significantly reduced in Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice compared with Pkd1flox/flox:Sirt1+/+:Ksp-Cre mice (Figure 3G), which indicates that renal function was normalized in Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice. At the same time, SIRT1 expression was not detected in cyst lining epithelial cells in kidneys from Pkd1 and Sirt1 double–conditional knockout mice, as analyzed by immunohistochemistry (Supplemental Figure 3). Proliferating cell nuclear antigen (PCNA) staining was used to determine the proliferation of cyst lining epithelial cells, which was significantly decreased in Pkd1 and Sirt1 double–conditional knockout versus Pkd1 single–conditional knockout (i.e., Sirt1+/+) renal epithelia (Figure 3H). Furthermore, we found that Pkd1 and Sirt1 double–conditional knockout mice lived to a mean age of 21.9 ± 3.6 days, while Pkd1flox/flox:Sirt1+/+:Ksp-cre mice died of polycystic kidney disease at 14.1 ± 0.9 days (P < 0.01; Figure 3I). Our in vivo data suggested that SIRT1 is involved in regulating renal cyst formation in _Pkd1_-knockout mice.
Double conditional knockout of Sirt1 and Pkd1 delayed renal cyst formation. (A–D) Histologic examination of P7 kidneys from (A) Pkd1+/+:Sirt1flox/flox:Ksp-Cre, (B) Pkd1flox/flox:Sirt1+/+:Ksp-Cre, (C) Pkd1flox/flox:Sirt1flox/+:Ksp-Cre, and (D) Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre neonates. (E) Percent cystic area relative to total kidney section area was significantly decreased in P7 kidneys from Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre (Sirt1flox/flox) versus Pkd1flox/flox:Sirt1+/+:Ksp-Cre (Sirt1+/+) neonates. Data reflect all sections quantified for each condition (n = 10 per group). (F) KW/BW ratios were dramatically reduced in P7 Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre versus Pkd1flox/flox:Sirt1+/+:Ksp-Cre neonates. (G) BUN levels of P7 Pkd1flox/flox:Sirt1+/+:Ksp-Cre, Pkd1flox/flox:Sirt1flox/+:Ksp-Cre, and Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre neonates. (H) Cell proliferation (arrows) was decreased in P7 Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre versus Pkd1flox/flox:Sirt1+/+:Ksp-Cre neonate kidneys, as detected with PCNA staining. The percentage of PCNA-positive nuclei in cystic lining epithelial cells was calculated from an average of 1,000 nuclei per mouse kidney section; only strongly stained nuclei were considered PCNA-positive. (I) Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice lived to 21.9 ± 3.6 days, whereas Pkd1flox/flox:Sirt1+/+:Ksp-Cre mice died of polycystic kidney disease at 14.1 ± 0.9 days. Scale bars: 2 mm (A–D); 30 μm (H). *P < 0.05; **P < 0.01.
A pan-sirtuin inhibitor or a specific SIRT1 inhibitor delays cyst growth in Pkd1-mutant kidneys. To test whether inhibiting the activity of SIRT1 would suppress cyst formation in Pkd1–/– embryos, we injected nicotinamide into pregnant Pkd1+/– female mice from 7.5 dpc after mating with Pkd1+/– males, and analyzed MEKs at 15.5 dpc. We found that in all E15.5 Pkd1–/– embryos from nicotinamide-injected mothers, renal cyst formation was drastically reduced compared with kidneys of Pkd1–/– embryos from control DMSO-injected mothers (n = 10 per treatment group; P < 0.01; Figure 4, A–E). Furthermore, nicotinamide induced tubular epithelial cell apoptosis in kidneys from Pkd1–/– E15.5 embryos, while apoptosis was rare and negligible in kidneys from DMSO-treated Pkd1–/– E15.5 embryos (Figure 4F). We also evaluated the effect of nicotinamide on renal cyst formation at 18.5 dpc; indeed, renal cyst growth was dramatically reduced in kidneys of E18.5 Pkd1–/– embryos from nicotinamide- versus DMSO-injected pregnant females (n = 10 per treatment group; Figure 4, G and H). Kidney weight was also significantly decreased in Pkd1–/– embryos from nicotinamide-injected pregnant females (Figure 4I). Again, tubular epithelial cell apoptosis was induced by nicotinamide in E18.5 kidneys from Pkd1–/– embryos, but was rare in E18.5 kidneys from DMSO-treated Pkd1–/– embryos (Figure 4J). Furthermore, we found that treatment with nicotinamide increased the survival of Pkd1–/– E18.5 embryos compared with those treated with DMSO (P < 0.01; Supplemental Table 1).
Nicotinamide treatment delayed cyst formation in Pkd1–/– MEKs. (A–D) Histological examination of E15.5 WT and Pkd1–/– MEKs from pregnant females injected daily with nicotinamide (NIC) or DMSO vehicle control from 7.5 to 14.5 dpc. (A) Nicotinamide-treated WT. (B) DMSO-treated WT. (C) Nicotinamide-treated Pkd1–/–. (D) DMSO-treated Pkd1–/–. (E) Percent cystic area relative to total kidney section area of E15.5 kidneys from WT and Pkd1–/– embryos treated with nicotinamide or DMSO (n = 10 per treatment group). For all mice, the middle section of each kidney was quantified. (F) Nicotinamide induced cyst lining epithelial cell death (arrows) in Pkd1–/– E15.5 MEKs, while apoptosis was rare in DMSO-treated Pkd1–/– E15.5 MEKs, as detected by TUNEL assay. (G) Histological examination of E18.5 Pkd1–/– MEKs from pregnant females injected daily with DMSO or nicotinamide from 7.5 to 17.5 dpc. (H) Percent cystic area relative to total kidney section area of E18.5 kidneys from Pkd1–/– embryos treated with DMSO or nicotinamide (n = 10 per treatment group). (I) Kidney weight of Pkd1–/– E18.5 kidneys from pregnant females treated with DMSO or nicotinamide (n = 10 per treatment group). (J) Nicotinamide induced cyst lining epithelial cell death (arrows) in Pkd1–/– E18.5 kidneys, while apoptosis was rare in DMSO-treated Pkd1–/– E18.5 kidneys, as detected by TUNEL assay. Scale bars: 500 μm (A–D and G); 20 μm (F and J). **P < 0.01.
Next, we tested whether nicotinamide or EX-527, a specific SIRT1 inhibitor (21), could reduce cyst initiation or growth in Pkd1flox/flox: Ksp-Cre mice. Cyst progression is aggressive in the kidneys of Pkd1flox/flox:Ksp-Cre mice (22), which allowed us to examine the effect of nicotinamide on initiation and progressive enlargement of cyst formation. Pkd1flox/flox:Ksp-Cre pups were injected i.p. with nicotinamide (0.25 mg/g), EX-527 (2 mg/kg) or DMSO daily from P3 to P6, and kidneys were harvested and analyzed at P7. Administration of nicotinamide or EX-527 during this early phase delayed renal cyst growth (P < 0.01; Figure 5, A and B), inhibited cystic epithelial cell proliferation (PCNA staining; Figure 5E), and induced cystic epithelial cell apoptosis (TUNEL assay; Supplemental Figure 4A) in P7 kidneys from Pkd1flox/flox:Ksp-Cre mice compared with DMSO injection (n = 10 per treatment group). KW/BW ratios and BUN levels in Pkd1flox/flox:Ksp-Cre mice were dramatically reduced by nicotinamide or EX-527 treatment compared with DMSO treatment (Figure 5, C and D). Additionally, gender did not influence cyst formation and progression, as determined by comparing 5 male mice and 5 female Pkd1flox/flox:Ksp-Cre mice per treatment group with respect to cystic index and KW/BW ratios. To further confirm that nicotinamide delayed cyst formation by specifically targeting SIRT1, we treated Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice with nicotinamide daily from P3 to P6 and collected the kidneys at P7. The rationale for this experiment was that if nicotinamide delays cyst growth in Pkd1 mutant mice by targeting SIRT1, then it will not affect cyst growth in Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice, which lack SIRT1. We found that nicotinamide treatment did not further delay cyst growth in P7 kidneys of Pkd1flox/flox:Sirt1flox/flox:Ksp-Cre mice (n = 10 per treatment group; Supplemental Figure 4, B–D). These results demonstrated that nicotinamide delayed cyst growth by specifically inhibiting SIRT1 in the Pkd1flox/flox:Ksp-Cre mice.
Treatment with nicotinamide or EX-527 delayed cyst growth in Pkd1flox/flox:Ksp-Cre neonates. (A) Histologic examination of P7 kidneys from Pkd1flox/flox:Ksp-Cre neonates treated with DMSO, nicotinamide, or EX-527 (n = 10 per treatment group). (B) Percent cystic area relative to total kidney section area of P7 kidneys from Pkd1flox/flox:Ksp-Cre neonates treated as in A. Data reflect all sections quantified for each condition. (C and D) KW/BW ratios (C) and BUN levels (D) were decreased in Pkd1flox/flox:Ksp-Cre P7 neonates treated with nicotinamide or EX-527 compared with DMSO treatment. (E) Nicotinamide and EX-527 treatment reduced cyst lining epithelial cell proliferation (arrows) in Pkd1flox/flox:Ksp-Cre P7 kidneys, as detected by PCNA staining. Scale bars: 2 mm (A); 20 μm (E). **P < 0.01.
Finally, we examined whether nicotinamide or EX-527 could delay cyst growth in the progressive hypomorphic Pkd1nl/nl mouse model (15). Pkd1nl/nl pups were injected i.p. with nicotinamide (0.25 mg/g), EX-527 (2 mg/kg), or DMSO daily from P5 to P27, and kidneys were harvested and analyzed at P28. Administration of nicotinamide or EX-527 delayed cyst progression (Figure 6, A and B), inhibited cystic epithelial cell proliferation (Figure 6E), and induced cystic epithelial cell apoptosis (Supplemental Figure 5) in P28 Pkd1nl/nl kidneys compared with kidneys of age-matched DMSO-injected Pkd1nl/nl mice (n = 10 per treatment group). Nicotinamide or EX-527 treatment also significantly decreased KW/BW ratios and BUN levels in Pkd1nl/nl mice compared with DMSO injection (Figure 6, C and D). We also found that gender did not affect cyst formation and progression in Pkd1nl/nl mice by comparing 5 male mice and 5 female mice per group. These results further supported the notion that targeting SIRT1 with pharmacological inhibitors may delay cyst growth in ADPKD patients.
Treatment with nicotinamide or EX-527 delayed cyst growth in Pkd1nl/nl mice. (A) Histologic examination of P28 kidneys from Pkd1nl/nl mice treated with DMSO, nicotinamide, or EX-527 (n = 10 per treatment group). (B) Percent cystic area relative to total kidney section area of P28 kidney sections from Pkd1nl/nl mice treated as in A. Data reflect all sections quantified for each condition. (C and D) KW/BW ratios (C) and BUN levels (D) were decreased in P28 Pkd1nl/nl mice treated with nicotinamide or EX-527 compared with DMSO treatment. (E) Nicotinamide and EX-527 treatment reduced cyst lining epithelial cell proliferation (arrows) in P28 kidneys from Pkd1nl/nl mice, as detected by PCNA staining. Scale bars: 2 mm (A); 20 μm (E). **P < 0.01.
Silence or inhibition of SIRT1 decreases renal epithelial cell growth, but increases apoptosis. Our findings that genetic deletion of Sirt1 or inhibition of SIRT1 with nicotinamide or EX-527 in _Pkd1_-mutant background mice not only delayed cyst formation, but also decreased cystic epithelial cell proliferation and increased cystic epithelial cell apoptosis, suggested that SIRT1-mediated downstream pathways are involved in this process. To support this notion, we examined the effect of SIRT1 overexpression and SIRT1 depletion or inhibition on cell proliferation with a BrdU proliferation assay in mouse IMCD3 cells and _Pkd1_-null renal epithelial cells, respectively. We found that overexpressing HA-tagged WT SIRT1, but not the deacetylase catalytically inactive mutant SIRT1-H355A (23), increased BrdU incorporation in mouse IMCD3 cells (Figure 7A). In contrast, knockdown of SIRT1 with siRNA decreased BrdU incorporation in _Pkd1_-null MEK cells and PN24 cells (Figure 7, B and C). In addition, treatment with different concentrations of nicotinamide resulted in a dose-dependent decrease in BrdU incorporation in _Pkd1_-null MEK cells and PN24 cells (Figure 7, D and E). These results suggest that upregulation of SIRT1 increases S-phase entry in _Pkd1_-mutant renal epithelial cells.
Silence or inhibition of SIRT1 decreased renal epithelial cell proliferation, but increased apoptosis. (A) Overexpressing WT SIRT1, but not deacetylase catalytically inactive mutant SIRT1 H355A, increased BrdU incorporation in mouse IMCD3 cells compared with cells transfected with vector alone. (B–E) Silencing SIRT1 with siRNA (B and C) or inhibiting SIRT1 with the indicated concentrations of nicotinamide (D and E) decreased BrdU incorporation in (B and D) _Pkd1_-null MEK and (C and E) PN24 cells. BrdU incorporation index in the control cells was assigned as 100%. An average of 300 cells was counted for each experiment. n = 3. (F and G) Flow cytometry analysis indicated that apoptosis was induced in (F) _Pkd1_-null MEK cells treated with 10 mM nicotinamide for 24 hours and (G) PN24 cells treated with 40 mM nicotinamide for 48 hours. Nicotinamide-treated cells were labeled with annexin V and PI and analyzed by flow cytometry. Early and late apoptotic cells were represented by annexin V+PI– and annexin V+PI+ cells, respectively. n = 3. (H) Caspase-3 activation was examined from whole cell lysates of WT MEK, _Pkd1_-null MEK, PH2, and PN24 cells treated or not with 10 mM nicotinamide for 24 hours. Nicotinamide treatment increased caspase-3 activation and caused the appearance of cleaved PARP (a substrate of caspase-3) in _Pkd1_-null MEK and PN24 cells. *P < 0.05; **P < 0.01.
Next, we examined whether nicotinamide had a proapoptotic effect on WT MEK, _Pkd1_-null MEK, PH2, and PN24 cells by TUNEL assay. Nicotinamide induced apoptosis in _Pkd1_-null MEK cells and PN24 cells, but not in WT MEK cells or PH2 cells (Supplemental Figure 6, A and B). Flow cytometry analysis demonstrated that apoptosis was significantly increased in _Pkd1_-null MEK cells and PN24 cells treated with nicotinamide compared with vehicle (Figure 7, F and G). We further found that treatment with nicotinamide markedly increased the level of active caspase-3 in _Pkd1_-null MEK cells and PN24 cells, but not that in WT MEK cells or PH2 cells (Figure 7H). Caspase-3 activation was confirmed by the appearance of cleaved poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3 (Figure 7H), which suggests that caspase-3 is the downstream executioner of nicotinamide-induced apoptosis in _Pkd1_-mutant renal epithelial cells.
SIRT1 regulates cystic epithelial cell proliferation by altering Rb acetylation and phosphorylation. Previous studies demonstrated that acetylation of Rb inhibits its phosphorylation by cyclin-dependent kinases and that SIRT1-mediated deacetylation of Rb increases its phosphorylation in vitro (12, 24). However, whether endogenous SIRT1 regulates Rb activity through this process is unknown. We demonstrated that knockdown of Pkd1 in mouse IMCD3 cells with 2 different lentiviruses expressing shRNAs increased not only SIRT1 expression (Figure 1C), but also Rb phosphorylation (Figure 8A), compared with control mouse IMCD3 cells transduced with the respective control siLuc or pGIPZ-NS lentivectors. Phospho-Rb was also increased in _Pkd1_-null MEK cells and PN24 cells, as well as in kidney tissues from Pkd1nl/nl mice, compared with that seen in the respective WT MEK cells, PH2 cells, and control kidney tissues (Figure 8, B and C).
SIRT1 interacts with Rb. (A and B) Western blot analysis of Rb and phospho-Rb expression (A) in mouse IMCD3 cells with Pkd1 knockdown by 2 different lentivirus-mediated Pkd1 shRNAs (as in Figure 1C) and (B) from whole cell lysates of WT MEK, _Pkd1_-null MEK, PH2, and PN24 cells. In B, relative phospho-Rb level (standardized to actin) from 3 independent immunoblots is also shown. (C) Western blot analysis of phospho-Rb expression in kidneys from WT and Pkd1nl/nl mice collected at P7, P14, P21, and P28. (D) Interaction between SIRT1 and Rb and levels of acetyl-Rb in WT MEK and _Pkd1_-null MEK cells, examined by IP with anti-Rb antibody and then blotting with SIRT1 antibody and anti–acetyl-α-lysine antibody. IgG was used as a negative control. **P < 0.01.
To support the functional relationship between SIRT1 and Rb in renal epithelial cells, we found that SIRT1 interacted with Rb by demonstrating that anti-Rb antibody could pull down SIRT1 (Figure 8D). Due to the lack of antibodies for acetyl-Rb, we used anti-Rb antibody to pull down Rb and subsequently used an anti–acetyl-α-lysine antibody to evaluate the acetylation of Rb, as performed by other laboratories (24, 25). We found that acetylated Rb was decreased in SIRT1 upregulating _Pkd1_-null MEK versus WT MEK cells (Figure 8D). In addition, we found that (a) overexpressing WT SIRT1, but not SIRT1-H355A, decreased the acetylation level of Rb and increased the level of phospho-Rb in mouse IMCD3 cells (Figure 9, A and B); (b) silencing SIRT1 with siRNA or inhibiting SIRT1 activity with nicotinamide increased the acetylation level of Rb in _Pkd1_-null MEK cells compared with appropriate controls (Figure 9, C and D); and (c) silencing SIRT1 with siRNA or inhibiting SIRT1 activity with nicotinamide decreased phospho-Rb levels in _Pkd1_-null MEK cells and PN24 cells compared with untreated control cells (Figure 9, E and F). Rb regulates the cell cycle through its interaction with the E2F family of transcription factors, in that Rb dephosphorylation increases Rb-E2F1 complex formation, and Rb phosphorylation releases E2F1 from Rb–E2F complexes, enabling E2F-dependent transcription of genes that mediate S-phase entry (26, 27). We found that the expression of E2F1 downstream targets DHFR, cyclin D3, and cyclin E, which are involved in cell cycle regulation, was upregulated in PN24 cells compared with PH2 cells, while levels of these proteins decreased in nicotinamide-treated versus untreated PN24 cells (Supplemental Figure 7). These results suggested that SIRT1 regulates renal cystic epithelial cell proliferation through Rb-E2F1 signaling.
Interaction of SIRT1 with Rb mediates Rb deacetylation and phosphorylation. (A and B) Overexpression of WT SIRT1, but not deacetylase catalytically inactive mutant SIRT1 H355A, (A) decreased the level of acetylated Rb, as examined with anti–acetyl-α-lysine antibody after Rb was pulled down with anti-Rb antibody, and (B) increased the level of phospho-Rb, as examined by Western blot, in mouse IMCD3 cells transfected with WT SIRT1, mutant SIRT1 H355A, or empty vector for 48 hours. (C and D) Knockdown of SIRT1 with siRNA or inhibition of SIRT1 with nicotinamide increased the acetylation level of Rb in _Pkd1_-null MEK cells that were (C) transfected with SIRT1 siRNA for 48 hours or (D) treated with 10 mM nicotinamide for 24 hours. Rb acetylation was analyzed as above. (E and F) Knockdown of SIRT1 with siRNA or inhibition of SIRT1 with nicotinamide decreased Rb phosphorylation, but did not affect Rb expression, in WT MEK, _Pkd1_-null MEK, PH2, and PN24 cells that were (E) transfected with SIRT1 siRNA for 48 hours or (F) treated with 10 mM nicotinamide for 24 hours. *P 0.05; **P 0.01.
Nicotinamide induces cystic epithelial cell death through p53-mediated cell death pathway. Treatment with nicotinamide increased cystic epithelial cell death in _Pkd1_-knockout renal tissues. Previous studies demonstrated that SIRT1 protects cells from p53-mediated apoptosis through a deacetylation-dependent mechanism (10, 28, 29). Thus, we examined whether SIRT1-mediated p53 deacetylation was involved in nicotinamide-induced cystic epithelial cell death. We found that (a) SIRT1 interacted with p53 by demonstrating that anti-p53 antibody could pull down endogenous SIRT1 and that anti-SIRT1 antibody could pull down endogenous p53 in WT MEK cells and _Pkd1_-null MEK cells (Figure 10A); (b) p53 acetylation was decreased in _Pkd1_-null MEK cells versus WT MEK cells, while p53 expression exhibited no difference between these cells (Figure 10B); (c) overexpressing HA-tagged WT SIRT1, but not SIRT1-H355A, in mouse IMCD3 cells decreased p53 acetylation, but had no effect on p53 expression (Supplemental Figure 8); (d) silencing SIRT1 with siRNA or inhibiting SIRT1 activity with its inhibitor, nicotinamide, increased the level of acetyl-p53 but had no effect on p53 expression in _Pkd1_-null MEK cells and PN24 cells compared with untreated control cells (Figure 10, C and D); (e) knockdown of p53 with siRNA prevented nicotinamide-induced caspase-3 activation and cystic epithelial cell death in _Pkd1_-null MEK cells and PN24 cells (Figure 11, A and B, and Supplemental Figure 9, A and B); and (f) overexpression of WT p53, but not mutant p53-8KR (which is mutated at 8 acetylation sites; ref. 30), increased apoptosis in _Pkd1_-null MEK cells treated with nicotinamide (Figure 11C). These results suggested that SIRT1 mediates p53 deacetylation involved in nicotinamide-induced cell death in _Pkd1_-mutant epithelia.
SIRT1 interacts and deacetylates p53 in renal epithelial cells. (A) The interaction between SIRT1 and p53 in WT and _Pkd1_-null MEK cells was examined by co-IP using either anti-SIRT1 or anti-p53 antibody. IgG was used as a negative control. (B) Western blot analysis of p53 and acetyl-p53 expression from whole cell lysates of WT MEK, _Pkd1_-null MEK, PH2, and PN24 cells. Acetyl-p53 expression was decreased in _Pkd1_-null MEK versus WT MEK cells, whereas p53 expression was almost at same levels in these cells. (C and D) Knockdown of SIRT1 with siRNA or inhibition of SIRT1 with nicotinamide increased p53 acetylation, but did not affect p53 expression, in _Pkd1_-null MEK cells and PN24 cells that were (C) transfected with SIRT1 siRNA for 48 hours or (D) treated with 10 mM nicotinamide for 24 hours. *P 0.05, **P 0.01.
Nicotinamide induces cystic epithelial cell death through p53. (A) Western blot analysis of p53 and active caspase-3 expression in _Pkd1_-null MEK cells transfected or not with p53 siRNA for 24 hours and then treated or not with 10 mM nicotinamide for another 24 hours. (B) Knockdown of p53 with siRNA prevented nicotinamide-induced apoptosis, as detected by TUNEL assay, in _Pkd1_-null MEK cells that were transfected or not with p53 siRNA for 24 hours and then treated or not with 10 mM nicotinamide for 24 hours. (C) Overexpression of WT p53, but not mutant p53-8KR (which is mutated at 8 acetylation sites), increased apoptosis in _Pkd1_-null MEK cells treated with nicotinamide. _Pkd1_-null MEK cells were transfected with WT p53, mutant p53-8KR, or empty vector together with or without nicotinamide for 24 hours, then analyzed by TUNEL assay. (D) SIRT1-mediated pathways in _Pkd1_-mutant renal epithelial cells. Pkd1 knockout or mutation upregulates SIRT1 through c-MYC. Upregulated SIRT1 in _Pkd1_-mutant renal epithelial cells (i) is a target of nicotinamide, which decreases proliferation and induces apoptosis of cystic epithelial cells to delay cyst growth in _Pkd1_-null mouse kidneys; (ii) regulates the acetylation and phosphorylation of Rb and further affects Rb-E2F–mediated S-phase entry; (iii) regulates the p53 acetylation and p53-dependent apoptosis in response to nicotinamide; and (iv) can be regulated by c-MYC and induced by TNF-α. Scale bars: 50 μm. **P 0.01.