Evaluation of glomerular sirtuin-1 and claudin-1 in the pathophysiology of nondiabetic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome, which is characterized by podocyte injury. Given that the pathophysiology of nondiabetic glomerulosclerosis is poorly understood and targeted therapies to prevent glomerular disease are lacking, we decided to investigate the tight junction protein claudin-1 and the histone deacetylase sirtuin-1 (SIRT1), which are known to be involved in podocyte injury. For this purpose, we first examined SIRT1, claudin-1 and podocin expression in kidney biopsies from patients diagnosed with nondiabetic FSGS and found that upregulation of glomerular claudin-1 accompanies a significant reduction in glomerular SIRT1 and podocin levels. From this, we investigated whether a small molecule activator of SIRT1, SRT1720, could delay the onset of FSGS in an animal model of adriamycin (ADR)-induced nephropathy; 14 days of treatment with SRT1720 attenuated glomerulosclerosis progression and albuminuria, prevented transcription factor Wilms tumor 1 (WT1) downregulation and increased glomerular claudin-1 in the ADR + SRT1720 group. Thus, we evaluated the effect of ADR and/or SRT1720 in cultured mouse podocytes. The results showed that ADR [1 µM] triggered an increase in claudin-1 expression after 30 min, and this effect was attenuated by pretreatment of podocytes with SRT1720 [5 µM]. ADR [1 µM] also led to changes in the localization of SIRT1 and claudin-1 in these cells, which could be associated with podocyte injury. Although the use of specific agonists such as SRT1720 presents some benefits in glomerular function, their underlying mechanisms still need to be further explored for therapeutic use. Taken together, our data indicate that SIRT1 and claudin-1 are relevant for the pathophysiology of nondiabetic FSGS.


Chronic SRT1720 administration was not effective in suppressing glomerular claudin-1 expression; however, it maintained podocyte differentiation status
For determination of the role of SIRT1 in FSGS development, BALB/c mice received a single ADR injection (10 mg/kg) and prolonged administration of SRT1720 (20 mg/kg) or vehicle (10% DMSO) for 14 days.After this period, ADR + vehicle-treated mice showed a marked increase in urinary albumin excretion, which could be significantly prevented by SRT1720 administration (Fig. 2a-c, Supplementary Fig. S1 and Table 1).Tubular protein casts and advanced glomerulosclerosis were also observed with PAS staining in the ADR + vehicle group (Fig. 2d).These results confirm the experimental model induction.In contrast, specific SIRT1 stimulation in the ADR + SRT1720 group attenuated the glomerulosclerosis index, tubular damage, and tubular protein cast formation and reduced kidney damage (Fig. 2d,e, and Table 1).Although in the ADR + vehicle group, plasma creatinine levels did not significantly increase compared to those in the CTL + vehicle group, SRT1720 administration was able to reduce this parameter (Fig. 2f and Table 1).The ADR + vehicle-treated mice also exhibited a significant drop in weight gain, which was not prevented by SRT1720 administration (Supplementary Fig. S1 and Table 1).Furthermore, no significant changes regarding water intake, food ingestion, kidney weight, or creatinine clearance were observed (Supplementary Fig. S1 and Table 1).SIRT1, claudin-1, and the transcription factor Wilms tumor 1 (WT1) were assessed by immunofluorescence.Treatment with ADR + vehicle resulted in a slight and nonsignificant decrease in SIRT1 expression compared to that in the CTL + vehicle group.However, an increased number of SIRT1-positive cell nuclei was observed in glomerular cross-sections from the ADR + SRT1720 group compared to the ADR + vehicle group (Fig. 3a,b, and Table 1).ADR + vehicle treatment induced only a slight increase in glomerular claudin-1 expression compared to that of the CTL + vehicle group, while chronic SRT1720 administration was unable to attenuate glomerular claudin-1 expression and significantly increased protein labeling in the ADR + SRT1720 group compared to the ADR + vehicle group (Fig. 3c,d and Table 1).Next, we evaluated the podocyte number by glomerular WT1 labeling.Our data demonstrated a significant loss of WT1 in the ADR + vehicle-treated animals compared to the CTL + vehicle animals, which was clearly prevented by the SIRT1-specific agonist in the ADR + SRT1720 group (Fig. 3e,f and Table 1).To complement our data and to confirm the effects of SRT1720 on kidney function, we treated a group of animals with vehicle (10% DMSO).In this analysis, kidney morphology was unaffected, and there were no significant differences in creatinine or plasma urea values compared to those of the CTL group (Supplementary Fig. S2 and Supplementary Table S1).In summary, these data indicate that despite claudin-1 upregulation in the ADR + SRT1720 group, SIRT1 chronic  show the abundance of urinary albumin, which was markedly reduced by SRT1720 chronic administration.Statistical analysis was performed by two-way ANOVA.A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 5.76; DF = 1; P = 0.043; Table 1).A bovine serum albumin standard (BSA, 1 mg/ml) was loaded on the adjacent lane (left).The cropped gel is displayed, and the full-length gel is included in the supplementary files.(c) Graphic presentation of the urinary albumin-to-creatinine ratio (mg/g).Statistical analysis was performed by two-way ANOVA.A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 12.8; DF = 1; P = 0.0072; Table 1).(d) Representative PAS micrographs (4 µm thick) and graphic presentation (e) show that chronic SRT1720 administration attenuated ADR + vehicle-induced glomerular sclerotic lesions, as well as protein cast deposition inside tubular structures.The yellow arrow shows protein casts inside tubules, and black arrows indicate positive staining for segmental glomerulosclerosis.Statistical analysis was performed by two-way ANOVA.A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 5.56; DF = 1; P = 0.025; Table 1).Scale bar, 100 µm (40 ×, kidney cortex) and 10 µm (400 ×, glomerular section).(f) Plasma creatinine analysis.Statistical analysis was performed by two-way ANOVA.A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 6.93;DF = 1; P = 0.014; Table 1).Values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.CTL control mice (graph circles), ADR adriamycin-injected mice (graph squares), SRT1720 specific SIRT1 agonist, SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis, A.U arbitrary units, UACR urinary albuminto-creatinine ratio, ANOVA analysis of variance, F F-statistic, DF degrees of freedom.

Claudin-1 protein expression increases in short-term ADR-treated mouse podocytes and is counteracted by acute stimulation of SIRT1
To investigate the interplay between SIRT1 and claudin-1, we examined the ADR dose-dependent effect on podocyte apoptosis in vitro.First, podocytes were treated at concentrations of 0.25 µM, 0.50 µM, 1 µM, 2.5 µM, and 5 µM for 24 h in RPMI 1640 culture medium with 5% FBS.Then, apoptosis was analyzed by flow cytometry.
In the present study, Annexin-V-FITC and 7-AAD markers were used.Next, incubation of podocytes with ADR at concentrations of 1 µM, 2.5 µM, and 5 µM led to a significant increase in the total apoptosis rate compared to that of the CTL group (Fig. 4a,b).Therefore, the concentration used for subsequent experiments was 1 µM.Likewise, for SRT1720 standardization, cells were treated at concentrations of 1 µM, 2.5 µM, and 5 µM for 24 h in the same basal media.Subsequently, SIRT1 protein expression was evaluated by immunoblotting.SRT1720 treatment at 5 µM resulted in a significant increase in SIRT1 protein expression after 24 h compared to that of the CTL group.However, SRT1720 at 1 µM or 2.5 µM did not induce significant changes in SIRT1 expression relative to that of the CTL group (Fig. 4c,d and Supplementary Fig. S3).Therefore, the selected concentration of SRT1720 was 5 µM.Additionally, in apoptosis experiments, the results showed that prior stimulation of SIRT1 with SRT1720 was unable to attenuate ADR-induced apoptosis in podocytes in vitro (Fig. 4e,f).
Claudin-1 protein expression was evaluated within the time-response curve of 30 min, 6 h, 12 h, 24 h, and 48 h based on 1 µM ADR treatment.Our results showed a significant increase in claudin-1 protein expression at the 30 min time point compared to that of the CTL group, as well as a significant decrease after 24 h and 48 h of treatment (Fig. 5a,b, and Supplementary Fig. S3).Given that the highest claudin-1 expression occurred 30 min after ADR incubation, we decided to explore the SIRT1/claudin-1 crosstalk at this experimental time point.For this purpose, podocytes were pretreated with 5 µM SRT1720 for 24 h and then treated with 1 µM ADR for 30 min.Our results demonstrated that early and acute SIRT1 stimulation markedly reduced ADR-induced claudin-1 expression (Fig. 5c,d, Supplementary Fig. S3, and Table 2).

Cellular distribution of SIRT1 and claudin-1 is changed in podocytes treated with ADR
To assess cellular SIRT1 and claudin-1 dynamics in mouse podocytes, we treated cells with 1 µM ADR for 30 min.Our immunofluorescence studies showed that nuclear SIRT1 expression was sharply reduced in the ADR-treated cells (Fig. 6a,b), spreading in the cytoplasm, while claudin-1 staining increased significantly, especially in the perinuclear region and plasma membrane (Fig. 6c,d).

Discussion
Interestingly, our study reveals that in nondiabetic human FSGS, reduced SIRT1 expression was associated with increased claudin-1 expression and a decrease in podocin protein expression and organization, which suggests communication between these proteins in FSGS-induced podocyte injury.
ADR-induced nephropathy results in glomerular injury in rodents, which is similar to human glomerular disease 39,40 .This model has been widely studied and allows elucidation of the mechanisms involved in CKD progression; thus, it is suitable for testing interventions that could attenuate kidney and podocyte injury 40 .For this purpose, we used SRT1720, a selective SIRT1 agonist.Regarding ADR + vehicle-treated mice, although the increase in plasma creatinine levels was not significant, strong urinary albumin excretion, low expression of WT1, Table 1.Statistical analysis of animal experimentation data.Two-way ANOVA was performed to compare data.The F, DF and exact P values considering ADR, SRT1720 and interaction are described.Significance was assumed when P < 0.05..SRT1720 administration did not reverse the body weight loss induced by ADR + vehicle.Notably, the ADR-induced nephropathy induces many metabolic alterations 43 , which could impact the ability to recover normal weight even with a SIRT1 activator.
In the glomeruli of ADR + vehicle-treated mice, claudin-1 was expressed in a manner compatible with the podocyte foot process, although the increase was not significant compared to that in the CTL + vehicle group.Claudin-1 is a tight junction protein that plays a critical role in regulating PECs and endothelial cell function 44,45 .Although the overall function of claudin-1 is conserved across species, there are differences in its expression levels and distribution pattern, as shown in humans and mice.These discrepancies could be due to evolutionary divergence or variations in tissue-specific regulation.The literature has shown that many proteins can facilitate the shifting of claudin-1 localization, such as protein kinase A (PKA) and protein kinase C (PKC) 46 , or soluble factors, such as interleukin-1 beta (IL-1β) 47,48 , transforming growth factor beta-1 (TGF-β1) 49 and tumor necrosis factor alpha (TNF-α) 50 .Using a transgenic mouse model, Gong et al. overexpressed claudin-1 specifically in  1).(e) Representative micrographs (4 µm thick) and graphic presentation (f) show that chronic SRT1720 administration attenuates WT1 depletion in ADR + SRT1720-treated mice.The white arrows indicate positive staining for WT1.Statistical analysis was performed by two-way ANOVA.A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 108; DF = 1; P < 0.0001; Table 1).Scale bar, 10 µm.Values are expressed as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.CTL control mice (graph circles), ADR adriamycin-injected mice (graph squares), SIRT1 sirtuin-1, SRT1720 specific SIRT1 agonist, WT1 Wilms tumor 1, ANOVA analysis of variance, F F-statistic, DF degrees of freedom.
podocytes.After 4 weeks, there was significant albuminuria compared to the control.The authors also observed that the increase in claudin-1 expression was compatible with podocyte ultrastructural changes, decreasing the expression of slit diaphragm components such as nephrin and podocin 51 .Taken together, our results corroborate these findings.
Curiously, in ADR + SRT1720-treated mice, SRT1720 administration increased glomerular claudin-1 labeling.The interplay between SIRT1 and claudin-1 was elegantly described by Hasegawa et al. using diabetic mouse models 24 .Under diabetic conditions, the decreased levels of proximal tubular nicotinamide mononucleotide and podocyte SIRT1 indicate H3 and H4 histone acetylation and diminished H3K9 methylation.These histone modifications decrease the activity of DNA (cytosine-5)-methyltransferase 1 (Dnmt1), which in turn causes hypomethylation of the claudin-1 gene, leading to increased podocyte expression of claudin-1 25,26,28 .Indeed, in several experimental models, including diabetic nephropathy and renal fibrosis induced by unilateral ureteral obstruction (UUO), SIRT1 is described as a therapeutic target, reducing endoplasmic reticulum stress and fibrosis 52 .In contrast, Ponnusamy et al. observed that in a UUO model, the SIRT1 agonist SRT1720 accelerated the deposition of collagen fibers and increased the expression of fibroblast activation markers such as alpha smooth muscle actin (α-SMA), collagen I and fibronectin 53 .The fibrogenic effect of SRT1720 was associated with connections between SIRT1 and other proteins, such as increased phosphorylation of epidermal growth factor receptor (EGFR Tyr1068) and platelet-derived growth factor β (PDGFRβ Tyr751).SRT1720 also increased the phosphorylation of STAT3 (Tyr705) and AKT (Ser473) in renal fibroblasts 53 and PKA (Thr197) in vascular smooth muscle cells 54 .Additionally, PKA phosphorylation is known to regulate the subcellular expression of claudin-1 in myeloma cells 46 .In this sense, we strongly believe that in our experimental model, PKA was targeted by SRT1720, resulting in increased claudin-1 expression.In this sense, despite their short-term benefits for kidney function, caution should be taken when using SIRT1 activators to avoid late podocyte injury.Table 2. Statistical analysis of cell culture data.Two-way ANOVA was performed to compare data.The F, DF and exact P values considering ADR, SRT1720 and interaction are described.Significance was assumed when P < 0.05.ADR adriamycin, SRT1720 specific SIRT1 agonist, ANOVA analysis of variance, F F-statistic, DF degrees of freedom.Given that ADR induces podocyte injury, we investigated the effect of ADR on cell apoptosis.First, we standardized ADR concentrations to validate podocyte injury.Our results showed that apoptosis was dependent on the ADR dose, as observed by Yi et al. 55 .In the context of podocytes, apoptosis is essential for glomerulosclerosis progression 56 .The ADR effects on podocytes have already been described by previous studies: generation of reactive oxygen species (ROS), increased apoptosis rate, expression of cleaved caspase-3 and BAX, and reduced Bcl-2 57 .A recent study reported that an ADR concentration of 1 µM on podocytes for 48 h promoted chromatin condensation and nuclear fragmentation 58 .It has been observed that a concentration of 2 µg/ml (equivalent to 3.44 µM) induces epithelial-to-mesenchymal transition (EMT), mainly by increasing integrin-linked kinase activity 59 .Furthermore, a concentration of 3 µg/ml (equivalent to 5.17 µM) for 72 h was associated with podocyte advanced glycation end products and ROS synthesis 60 .In contrast, in our experimental model, concentrations above 5 µM would be impractical, as the rate of apoptosis would be up to 80%, characterizing nonviable cells.
To date, few studies have investigated SRT1720 in podocytes in vitro.Wang et al. evaluated the inhibitory effect of astragaloside IV on podocyte EMT induced by high glucose concentrations 61 .In this case, podocytes were treated with SRT1720 at a concentration of 10 µM for 48 h to verify the role of SIRT1.To enhance proximal tubular SIRT1, studies have shown that the SRT1720 concentration varies from 2.5 to 10 µM [62][63][64] .Thus, we decided to standardize SRT1720 concentration in podocytes to evaluate SIRT1 protein expression.According to our data, SRT1720 at a concentration of 5 µM increased SIRT1 protein expression in podocytes after a 24 h treatment period.Based on the ADR and SRT1720 concentrations in in vitro studies, we observed a substantial increase in claudin-1 expression after 30 min of podocyte treatment with 1 µM ADR.The initial increase in claudin-1 expression at 30 min may be a response to the cellular damage caused by ADR.In this case, podocytes could try to restructure the cytoskeleton and their tight junctions by increasing claudin-1.After an initial response, cells enter a more severe state of stress or injury, which can result in a rapid decrease in claudin-1 expression.This process can be related to cellular mechanisms involved in apoptosis.
Since chronic and concomitant administration of SRT1720 did not attenuate claudin-1 expression in the glomeruli of ADR + SRT1720-treated mice, we decided to examine whether prior stimulation of SIRT1 with SRT1720 could affect claudin-1 expression in podocyte culture.Our results showed that prior stimulation of SIRT1 in podocytes prevented ADR-induced claudin-1 protein expression.Our study reveals that podocytes produce their own SIRT1, which enhances their local regulatory responses regardless of proximal tubule/podocyte crosstalk, as observed in diabetic models.Next, we observed that the treatment of podocytes with 1 µM ADR for 30 min resulted in SIRT1 translocation from the nucleus to the cytoplasm, as well as recruitment of claudin-1 to the perinuclear/plasma membrane.These modifications in the expression pattern of SIRT1 and claudin-1 could be linked to cellular adaptation and actin cytoskeleton changes, respectively, which were induced by treatment with ADR 65,66 .
Taken together, our data suggest that the lack of SIRT1 is associated with increased glomerular claudin-1 and podocyte injury in human FSGS.In an animal model of ADR-induced nephropathy, the chronic administration of SRT1720 prevented renal function changes induced by ADR.In immortalized mouse podocytes, although SRT1720 did not attenuate ADR-induced apoptosis, the agonist decreased claudin-1 protein expression.This finding suggests that in all three circumstances, SIRT1 can regulate the activity and expression of podocyte claudin-1.On the other hand, attention should be given when using SIRT1 agonists as therapeutic targets for glomerular diseases since they can stimulate renal fibrogenesis.

Human biopsies
All methods were performed in accordance with the relevant guidelines, regulations, and the Declaration of Helsinki.Kidney biopsy samples were obtained from Hospital das Clínicas FMUSP, University of Sao Paulo, with written informed consent.Twelve control patients (CTL; autopsied or healthy kidney parenchyma removed due to renal carcinoma) and eight patients diagnosed with FSGS were selected.Patients diagnosed with diabetic glomerulosclerosis were excluded from the study.The baseline characteristics of the FSGS patients are described in Supplementary Table S2.The protocol was approved by the Research Ethics Committee (CEP/ICB-USP), Institute of Biomedical Sciences, University of Sao Paulo, Brazil, protocol no.3.812.162.

Adriamycin nephropathy
All methods were performed in accordance with the relevant guidelines, regulations and with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.Male BALB/c mice aged 4 weeks were acquired from the animal care facility of the Medicine School, University of Sao Paulo, and kept in the experimental animal care facility of the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Brazil.All animals were maintained under standard conditions (22 °C, 12/12-h light/dark cycles, 60% relative humidity, standard mice chow-fed, and ad libitum water) for 1 week.Experiments began when the animals reached 5 weeks of age.Adriamycin hydrochloride and SRT1720 were purchased from Cayman Chemicals (Cayman Chemicals, Ann Arbor, MI, USA).ADR-induced nephropathy was induced by a single slow intravenous administration of ADR (10 mg/kg body weight, diluted in 0.9% NaCl) into the tail vein.SRT1720 was first dissolved in 100% DMSO and then diluted in 10% DMSO with 0.9% NaCl.SRT1720 was administered intraperitoneally daily for 14 days (20 mg/kg body weight).Mice were randomly distributed into four groups: control + vehicle group (CTL + vehicle), ADR + vehicle nephropathy group, CTL + SRT1720 group, and ADR + SRT1720 group.The groups without ADR received the same dose of 0.9% NaCl via the tail vein.The groups without SRT1720 received the same dose of vehicle (10% DMSO) intraperitoneally.After this, mice were individually placed inside metabolic cages (Tecniplast, Buguggiate, VA, Italy) for 24 h urine collection.The animals were kept under inhalation anesthesia on the last day using 5% isoflurane.After the complete loss of pain reflexes, an abdominal incision was made using a scalpel (DL Micof, Sao Paulo, SP, BR).Blood samples were collected from the heart, and the kidneys were removed.Euthanasia was performed by exsanguination.All animal procedures were approved by the Ethics Committee on Animal Use of the Institute of Biomedical Sciences, University of Sao Paulo, Brazil (CEUA-ICB/USP), protocol no.3425170518.

Kidney function analysis
Plasma and urine creatinine levels were assessed in a Cobas c111 biochemical analyzer (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions.As previously described 67 , urinary albumin was analyzed by SDS-PAGE after urinary creatinine normalization.Next, gels were stained with a SilverQuest Silver Staining Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions.ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for quantification.In addition, urine albumin was quantified using the Albuwell M 1011 kit (Ethos Bioscience, Logan Township, NJ, USA) according to the manufacturer's instructions and is presented as the urinary albumin-to-creatinine ratio (UACR).

Cell culture
As first described by Dr. Peter Mundel 70 , a conditionally immortalized mouse podocyte cell line was certified by Dr. Karlhans Endlich (University of Heidelberg, Germany) and generously provided by Dr. Niels Olsen Saraiva Câmara (Institute of Biomedical Sciences, University of Sao Paulo).Cells were cultured as previously described and summarized here 16,71 .For induction of proliferation, mouse podocytes were grown in 75 cm 2 flasks coated with type-I collagen and maintained in RPMI 1640 medium (Invitrogen) with 30 IU/ml recombinant mouse interferon γ (BioLegend, San Diego, CA, USA) and 10% fetal bovine serum (FBS, Invitrogen) at a permissive temperature (33 °C).For induction of differentiation, the cells were shifted to 37 °C in RPMI 1640 medium with 5% FBS and without interferon γ for 15 days.

Flow cytometry
As previously described 16,71 , control and ADR-treated podocytes were trypsinized, and cell staining buffer (BioLegend) was added to the cell samples.The cell suspensions were centrifuged at 2500 rpm for 5 min.This procedure was repeated once more, followed by suspension of the cells in Annexin V binding buffer (BioLegend).A total of 2 × 10 5 cells were transferred to cytometry sample tubes, and FITC-Annexin V (BioLegend) was incubated for 30 min.After this, 7-AAD (Invitrogen) was added.Cells were subsequently analyzed using a BD FACSCanto II Flow Cytometer/BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, US), calibrated to detect 10,000 or 200,000 events.Cells positive for FITC-Annexin V and 7-AAD were considered apoptotic.The values are shown as percentages (%).

Immunofluorescence
Kidney sections (4 µm-thick) were deparaffinized in xylene and rehydrated in a graded ethanol series, ending in running tap water.We performed heat-induced epitope recovery for 5 min using an electric pressure cooker and sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) or Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0).After that, the sections were blocked with 1 × TBS plus 1% bovine serum albumin (BSA, VWR, Radnor, PA, USA) for 2 h at room temperature.The blocking solution was removed, and then, the primary antibody was added.The incubation was overnight at 4 °C.The next day, the sections were washed, and Fab fragment secondary antibodies were applied to the sections for 1 h at room temperature.Sections were covered with an aqueous mounting medium (Sigma-Aldrich) and coverslips.For cell immunofluorescence, differentiated podocytes were cultured on glass coverslips (Knittel, Braunschweig, NI, DE).Control or treated cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in 1X PBS (phosphate buffered saline, pH 7.4) for 4 min, permeabilized with 0.1% Triton X-100 in 1 × PBS for 5 min and blocked with 2.5% horse serum solution (Vector Laboratories, Burlingame, CA, USA) for 30 min.Primary antibodies were incubated overnight.Next, the glass coverslips were washed thrice with 1X PBS, followed by secondary antibody incubation for 90 min.F-Actin was labeled with rhodamine phalloidin (Invitrogen).Coverslips were sealed with Vectashield Hardset plus DAPI (Vector Laboratories) for nuclear staining.The fluorescence signal was analyzed with an Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with a 20 ×/40 × plane objective using excitation wavelengths of 488 nm (green) and 543 nm (red) and NIS-Elements Basic Research software (Nikon).ImageJ software (National Institutes of Health) was used to open files and fluorescence quantification, as described in the Supplementary Methods.The primary antibodies used were as follows: anti-claudin-1 (1:100, #51-9000, Invitrogen), anti-SIRT1 (1:100, #9475, Cell Signaling Technology, Inc.), and anti-WT1 (1:200, ab89901, Abcam

Immunohistochemistry
Kidney sections (4 µm-thick) were deparaffinized in xylene and rehydrated in a graded ethanol series, ending in running tap water.We performed heat-induced epitope recovery for 5 min using an electric pressure cooker and sodium citrate buffer.After that, endogenous peroxidase was quenched with 3% hydrogen peroxide in 1X PBS for 10 min.Next, the sections were incubated with serum-free protein blocks (Agilent Technologies, Santa Clara, CA, USA) for 2 h at room temperature.The blocking solution was removed, and the primary antibody was applied.The incubation was overnight at 4 °C.The next day, the sections were washed, and a polymer horseradish peroxidase antibody (Agilent Technologies) was applied to the sections for 2 h at room temperature.Subsequent

Figure 4 .
Figure 4. Prior stimulation of sirtuin-1 with SRT1720 does not attenuate podocyte adriamycin-induced apoptosis in vitro.(a) Representative flow cytometry analysis and graphic presentation (b) show that ADR increases podocyte apoptosis in a dose-dependent manner.Cells were treated with various concentrations of ADR for 24 h, as indicated.Q1, cells in necrosis; Q2, cells in late apoptosis; Q3, cells in early apoptosis; and Q4, healthy cells.Total apoptosis means the sum of early and late apoptosis.Statistical analysis was performed by one-way ANOVA.(c) Representative immunoblots (10% SDS-PAGE) and graphic presentation (d) show SIRT1 protein abundance in SRT1720-treated cells, as indicated.The cropped blot is displayed, and the full-length gel is included in the supplementary files.(e) Representative flow cytometry analysis and graphic presentation (f) show that SRT1720 did not attenuate ADR-induced podocyte apoptosis.ADR cells were treated with 1 µM ADR for 24 h.ADR + SRT1720 cells were pretreated with SRT1720 [5 µM] for 24 h and then treated with ADR [1 µM] for 24 h.Total apoptosis means the sum of early and late apoptosis.Statistical analysis was performed by one-way ANOVA.Values are expressed as the mean ± SEM. *P < 0.05, ****P < 0.0001.CTL control cells, ADR adriamycin-treated cells, SIRT1 sirtuin-1, SRT1720 specific SIRT1 agonist, SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis, ANOVA analysis of variance.
value P value F value DF value P value F value DF value P value Claudin-1/β-actin, fold change 15