Role of SIRT1 in HIV-associated kidney disease

Abstract

Human immunodeficiency virus (HIV) infection of kidney cells can lead to HIV-associated nephropathy (HIVAN) and aggravate the progression of other chronic kidney diseases. Thus, a better understanding of the mechanisms of HIV-induced kidney cell injury is needed for effective therapy against HIV-induced kidney disease progression. We have previously shown that the acetylation and activation of key inflammatory regulators, NF-κB p65 and STAT3, were increased in HIVAN kidneys. Here, we demonstrate the key role of sirtuin 1 (SIRT1) deacetylase in the regulation of NF-κB and STAT3 activity in HIVAN. We found that SIRT1 expression was reduced in the glomeruli of human and mouse HIVAN kidneys and that HIV-1 gene expression was associated with reduced SIRT1 expression and increased acetylation of NF-κB p65 and STAT3 in cultured podocytes. Interestingly, SIRT1 overexpression, in turn, reduced the expression of negative regulatory factor in podocytes stably expressing HIV-1 proviral genes, which was associated with inactivation of NF-κB p65 and a reduction in HIV-1 long terminal repeat promoter activity. In vivo, the administration of the small-molecule SIRT1 agonist BF175 or inducible overexpression of SIRT1 specifically in podocytes markedly attenuated albuminuria, kidney lesions, and expression of inflammatory markers in Tg26 mice. Finally, we showed that the reduction in SIRT1 expression by HIV-1 is in part mediated through miR-34a expression. Together, our data provide a new mechanism of SIRT1 regulation and its downstream effects in HIV-1-infected kidney cells and indicate that SIRT1/miR-34a are potential drug targets to treat HIV-related kidney disease.

INTRODUCTION

Following the widespread introduction of effective combination antiretroviral therapy, the natural history and epidemiology of human immunodeficiency virus (HIV) infection and HIV-associated nephropathy (HIVAN) have changed significantly. Both acquired immunodeficiency syndrome-related deaths and end-stage renal disease attributed to HIVAN have declined in populations with access to care. However, the incidence of HIVAN remains high in certain areas of Africa (19, 41), and data on the spectrum of histological findings in patients with HIV show an increase of noncollapsing focal segmental glomerulosclerosis and other forms of chronic kidney disease (CKD) (1). With increased longevity of the HIV-infected population, the prevalence of comorbid CKD risk factors is increasing (48). We and others have confirmed that HIV infection aggravates the progression of CKD such as diabetic kidney disease (DKD) (7, 30). Our recent murine study (29) has shown upregulation of local inflammation induced by both HIV infection and diabetes, indicating that both diseases share similar mechanisms. A recent study (21) has suggested that carrying any combination of two apolipoprotein L1 (APOL1) risk alleles increases the risk to develop CKD in individuals of African ancestry and that the odds ratio is highest (89-fold) in patients with HIVAN compared with HIV-positive controls. Therefore, HIVAN and other HIV-related kidney diseases remain a major public health burden, and a better understanding of its underlying mechanisms is necessary for the development of novel therapeutic approaches.

A large body of evidence points to inflammation as a key driver of the pathology of HIVAN as well as in other kidney diseases. Our previous study (20) has shown that NF-κB and STAT3 are two major transcription factors regulating proinflammatory gene expression in HIV-infected kidneys. While transcription factor activation is often regulated by phosphorylation, it is also regulated through acetylation. Sirtuin-1 (SIRT1), a NAD⁺-dependent deacetylase, represses the activity of several transcription factors through deacetylation, including STAT3 (2, 35, 45, 49). In addition, SIRT1 exerts anti-inflammatory effects through inhibition of the NF-κB pathway (6, 12, 25). Several studies have suggested a critical role of SIRT1 in kidney diseases (51). We have demonstrated that podocyte-specific knockout of Sirt1 in diabetic db/db mice led to higher levels of NF-κB and STAT3 acetylation and resulted in a greater degree of proteinuria and kidney injury than in control db/db mice (27). Recently, we further showed that SIRT1 agonist or podocyte-specific overexpression of SIRT1 was able to attenuate diabetes-induced kidney injury (17), implicating SIRT1 as a key restrainer of the inflammatory responses in DKD. Similar to DKD, HIV infection is also associated with inflammation in the kidney (34). Our recent data suggest that Sirt1 expression is significantly suppressed in HIV-infected kidney cells, and, therefore, we determined here whether SIRT1 is a key repressor of HIV-mediated CKD pathogenesis.

METHODS

Immunohistochemistry of human kidney biopsies.

Archival human biopsy specimens of healthy donor nephrectomies and HIVAN were collected at the Icahn School of Medicine at Mount Sinai under a protocol approved by the Institutional Review Board. Briefly, formalin-fixed and paraffin-embedded sections were heated for 20 min in a 55–60°C oven and deparaffinized, and endogenous peroxidase was inactivated with H₂O₂. Sections were then blocked in 2% goat serum in PBS for 1 h at room temperature and then incubated with rabbit anti-SIRT1 antibody (no. 110304, Abcam, Cambridge, MA) at 4°C overnight. The next day, sections were washed three times with PBS and incubated with anti-rabbit biotinylated secondary antibody at room temperature and then with the avidin-biotin-peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA).

Mice.

Animal experiments were performed under the guidelines of and approved by the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai (no. LA-10-00001). Inducible SIRT1-overexpressing transgenic mice were generated in the FVB/N background (TRE-SIRT1^OV) as previously described (17). Founder mice with germline transmission of the transgene were crossed with podocin-rtTA mice [Jackson Laboratory, FVB/N-Tg(NPHS2-rtTA2*M2)1Jbk/J] to generate inducible podocyte-specific SIRT1-overexpressing mice (Pod-SIRT1^OV). Tg26 mice on the FVB/N genetic background bearing a defective HIV-1 provirus lacking gag-pol were as described in our previous study (11). Pod-SIRT1^OV mice were crossed with Tg26 mice or their littermate controls to generate Tg26;Pod-SIRT1^OV mice or WT;Pod-SIRT1^OV mice. Age-matched Tg26 mice and normal FVB/N mice were used as nontransgenic controls. The genotyping primer sets used were as follows: SIRT1, forward 5′-AATAGGCGTATCACGAGGCCCTTTCG-3′ and reverse 5′-GGAGCCGCCGGGCTGAAG-3′; and rtTA, forward 5′-GAACAACGCCAAGTCATTCCG-3′ and reverse 5′-TACGCAGTGTAAAGTGG-3′. SIRT1 expression was induced starting at 4 wk of age by feeding doxycycline (Dox)-supplemented chow (625 mg/kg chow, Envigo). The SIRT1 agonist BF175 was as previously described (17). For in vivo treatments, mice received daily gavage of 0.4 mg/kg BF175 or vehicle (5% DMSO in saline) per day starting at 4 wk of age until 8 wk of age (n = 5 in each group). After mice had been euthanized, blood, urine, and kidney tissue were collected.

Measurement of blood urea nitrogen and urine albumin.

Blood urea nitrogen was measured by a commercially available kit (Biosystems). Urine albumin was determined using a commercial assay kit from Bethyl Laboratory (Houston, TX). Urine creatinine levels were measured in the same samples using the Creatinine Colorimetric Assay Kit (Cayman Chemical) according to the manufacturer’s instructions. The spot urine albumin excretion rate was expressed as the ratio of albumin to creatinine (in mg/mg).

Mouse kidney histology.

Kidney samples were fixed in 10% formalin, embedded in paraffin, and sectioned to 4 μm thickness. Sections were stained with periodic acid-Schiff for analysis of the glomerular area and mesangial matrix expansion. Images were taken at ×400 magnification using a Zeiss AX10 microscope (Carl Zeiss Jena, Toronto, ON, Canada). Scoring for histological changes was performed in a blinded manner as previously described for Tg26 mice (38).

Cell culture.

Conditionally immortalized human podocytes (42) obtained from Dr. Moin Saleem (University of Bristol, Bristol, UK) were cultured in RPMI-1640 medium (GIBCO, Gaithersburg, MD) containing 10% FBS, 1% insulin-transferrin-selenium-A supplement, and 0.5% penicillin and streptomycin in a 5% CO₂ humidified environment in 33°C under growth-permissive conditions and differentiated in 37°C growth-restrictive conditions for 7 days. For expression of HIV-1 genes, podocytes were stably transduced with VSV-pseudotyped pNL4–3:DeltaG/P-EGFP (HIV) lentivirus or control enhanced green fluorescent protein (EGFP) lentivirus (18). For overexpression of SIRT1 and p65 wild-type (WT) and mutant constructs, podocytes were transiently transfected using ViaFect reagent (E4981, Promega).

RNA extraction and real-time PCR.

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Total RNA (500 ng) was reverse transcribed to cDNA using the SuperScriptIII First-Strand Synthesis System (Invitrogen). Real-time PCR was performed using SYBR Green Master Mix (Applied Biosystems) with the 7600 Real-Time PCR System (Applied Biosystems, Waltham, MA). The gene level was normalized to GAPDH 18S, and the $2^{-\Delta \Delta {\text{C}}_{\text{t}}}$ method (where C_t is threshold cycle) was used for the analysis of relative gene expression.

miRNA sequencing of primary podocytes and data analysis.

Small RNA from cultured human podocytes was isolated using the mirVana miRNA isolation kit (AM1561, ThermoFisher) and sequenced based on the manufactory protocol (Illumina). After the tag sequence (5′-TCGTATGCCGTCTTCTGCTTG-3′) was clipped off, the reads with good quality were aligned to the mature miRNA database (hg19) using BWA aligner (26). The reads mapped to miRNA were counted to represent the expression level for the corresponding miRNA. To compare expression levels across samples, the read counts were log₂ transformed and normalized at an equal global median value. The paired LIMMA test (39) was performed to identify significantly dysregulated miRNAs in primary podocytes after HIV infection at an adjusted false discovery rate P value of <0.05.

miRNA-34a inhibition and quantitative PCR analysis.

Lentivectors expressing inhibitory Homo sapiens (hsa-)miR-34a (mh30567), which blocks miR-34a binding to target genes without altering its expression, and scrambled control were purchased from Applied Biological Materials (Richmond, BC, Canada). Lentiviral particles were produced in human embryonic kidney (HEK)-293T cells using pCD/NL-BH*DDD packaging plasmid (no. 17531, Addgene) and VSV-G-encoding pLTR-G plasmid (no. 17577, Addgene). miRNAs from cultured podocytes were extracted with the miRNA Extraction kit (Qiagen, Valencia, CA). Reverse transcription was conducted using the miRNA cDNA Synthesis Kit (Qiagen). Real-time PCR amplification was conducted using the miScript SYBR green PCR kit (Qiagen) in the 7600 Real-Time PCR System (Applied Biosystems) with primers of miR-34a (Qiagen). U6 small nuclear RNA was used as an internal standard for miR-34a.

Western blot analysis.

Cells were lysed in M-PER mammalian protein extraction reagent (ThermoFisher) containing protease and phosphatase inhibitor cocktail, 5 mM nicotinamide, and 1 mM trichostatin A to inhibit deacetylase activity. Protein was separated using SDS-PAGE and transferred to the PVDF membrane. Proteins were detected using the following specific antibodies: SIRT1 (ab32441, Abcam), negative regulatory factor (Nef; ab63918, Abcam), acetyl-, phospho-, and total STAT3 (nos. 2523, 9136, and 9139, Cell Signaling, Danvers, MA), acetyl-, phospho-, and total p65 (nos. 3045, 3037, and 4764, Cell Signaling), β-actin (A5316, Sigma-Aldrich), GAPDH (no. 2118, Cell Signaling), and FLAG (F1804, Sigma-Aldrich).

Statistical analyses.

Data are reported as means ± SD. An unpaired two-tailed t test was used for comparison between two groups, and one-way ANOVA followed by Tukey’s correction was used for comparison between three or more groups. GraphPad Prism software was used for statistical analyses. All experiments were repeated at least three times, and representative experiments are shown. Data were considered statistically significant when P < 0.05.

RESULTS

SIRT1 expression is suppressed in HIV-infected kidney cells.

We first determined the expression level of SIRT1 in immortalized human podocytes transduced with HIV-1 pseudovirus lacking gag and pol (pNL4-3:Δgag-pol-EGFP or HIVΔgp-EGFP) under the endogenous viral long terminal repeat (LTR) promoter (see Supplemental Fig. S1 in the Supplemental Material, available online at https://doi.org/10.6084/m9.figshare.12098616.v2) or control pseudovirus expressing EGFP alone. Podocytes stably expressing HIVΔgp-EGFP showed reduced SIRT1 mRNA and protein levels compared with control podocytes (Fig. 1, A and B). SIRT1 expression was similarly reduced in isolated glomeruli of HIV-1 transgenic mice (Tg26) compared with their WT littermates (Fig. 1, C and D). The immunohistochemical analysis further confirmed the reduction in SIRT1 protein in the glomeruli of human HIVAN kidneys compared with healthy controls (Fig. 1E).

Fig. 1.

Sirtuin-1 (SIRT1) expression is reduced in human immunodeficiency virus (HIV)-infected kidney cells. A: quantitative PCR analysis of SIRT1 mRNA in immortalized human podocytes stably transduced with a control virus expressing (Ctrl)) or HIVΔgp-EGFP (HIV). B, left: Western blot analysis of SIRT1 and negative regulatory factor (Nef) proteins in Ctrl or HIV podocytes (n = 3 experiments). Representative blots of three independent experiments are shown. Densitometric analysis is shown on the right. C: quantitative PCR analysis of Sirt1 mRNA in isolated glomeruli of Tg26 mice and wild-type (WT) control mice. D, left: Western blot analysis of Sirt1 expression in isolated glomeruli of Tg26 mice and WT mice (n = 3 mice). Densitometric analysis is shown on the right. E: representative images of SIRT1 immunostaining in a kidney biopsy sample of an indvidual with HIV-associated nephropathy (HIVAN) or a control nephrectomy sample (n = 3). Scale bar = 50 μm. **P < 0.01 and ***P < 0.001 between samples by a two-tailed unpaired t test. EGFP, enhanced green fluorescent protein.

Suppression of SIRT1 leads to increased acetylation of NF-κB p65 and STAT3.

As to be expected, the reduction of glomerular SIRT1 expression in Tg26 mice was associated with increased acetylation p65 (Lys³¹⁰) and STAT3 (Lys⁶⁸⁵) compared with WT mice (Fig. 2A). These specific acetylation sites of p65 and STAT3 are required for their full activation and respective downstream gene transcription (10, 22, 50). Therefore, the increased acetylation of p65 and STAT3 was also associated with their increased phosphorylation (Fig. 2A). In cultured podocytes, overexpression of SIRT1 (SIRT1^OV) led to decreased acetylation of p65 and STAT3 (Fig. 2B), whereas knockdown of SIRT1 (SIRT1^KD) conversely elevated their acetylation (Fig. 2C).

Fig. 2.

Sirtuin-1 (SIRT1) regulates the acetylation and activation of NF-κB p65 and STAT3 in kidney cells. A: Western blot analysis of SIRT1, acetylated p65 (ac-p65), phosphorylated p65 (p-p65), and total p65 (t-p65) as well as acetylated STAT3 (ac-STAT3), phosphorylated STAT3 (p-STAT3), and total STAT3 (t-STAT3) in isolated glomeruli of Tg26 mice and wild-type (WT) mice. B: Western blot analysis of SIRT1, ac-STAT3, and ac-p65 in immortalized human podocytes stably expressing FLAG-tagged SIRT1 (SIRT1^OV) or control vector (Ctrl). C: Western blot analysis of SIRT1, ac-STAT3, and ac-p65 in immortalized human podocytes stably expressing shRNA specific for SIRT1 (SIRT1^KD) or scrambled control (Ctrl^KD). Representative blots of three independent experiments are shown.

SIRT1 suppresses HIV-1 gene expression through NF-κB p65 deacetylation.

Interestingly, SIRT1^OV led to a significant reduction of Nef in HIV-infected podocytes (Fig. 3A), whereas overexpression of mutant SIRT1 lacking the deacetylase activity (4) (SIRT1^H363Y) had little effect (Fig. 3B). Moreover, SIRT1^OV, but not mutant SIRT1^H363Y, enhanced acetylation of NF-κB p65 (Fig. 3C). Binding of NF-κB p65 to the LTR promoter was shown to be one of the strongest triggers for HIV-1 transcription (5, 32). Therefore, we next determined whether overexpression of NF-κB affects HIV gene expression in podocytes. Indeed, overexpression of WT NF-κB p65 enhanced Nef expression in podocytes, whereas mutant NF-κB with a defective acetylation site (K319R) did not (Fig. 3D). Therefore, we conclude that SIRT1 suppresses HIV-1 gene expression in podocytes through inhibition of NF-κB-mediated transactivation of HIV LTR.

Fig. 3.

Sirtuin-1 (SIRT1) suppresses human immunodeficiency virus (HIV)-1 gene expression through modulation of NF-κB p65. A: Western blot analysis of SIRT1 and negative regulatory factor (Nef) expression in HIVΔgp-EGFP podocytes that were transiently transfected with control (Ctrl) or SIRT1 overexpression vector (SIRT1^OV). B: Western blot analysis of SIRT1 and Nef expression in HIVΔgp-EGFP podocytes that were transiently transfected with control (Ctrl), SIRT1 (SIRT1^OV), or SIRT1 H363Y mutant overexpression vector (SIRT1^H363Y). C: Western blot analysis of SIRT1 and p65 in podocytes as described above. D: Western blot analysis of SIRT1 and p65 in HIVΔgp-EGFP podocytes that were transiently transfected with control (Ctrl), p65 overexpression (p65^WT), or p65 K310R mutant overexpression vector (p65^K310R). EGFP, enhanced green fluorescent protein; ac-p65, acetylated p65; t-p65, total p65.

The SIRT1 agonist BF175 attenuates NF-κB and STAT3 acetylation.

We have recently described a novel SIRT1 agonist, BF175, that was more potent than resveratrol (17). Using BF175, we also examined acetylation of NF-κB and STAT3. In cultured podocytes stably transduced with lentivirus expressing HIV proviral genes under the cytomegalovirus (CMV) promoter instead of its LTR promoter, BF175 reduced the acetylation of p65 and STAT3 (Fig. 4A). We confirmed here that CMV-driven HIV viral gene expression was not affected by BF175. However, BF175 suppressed HIV Nef expression in podocytes with LTR-driven HIV-1 gene expression (Fig. 4B). These data support our conclusion that SIRT1 regulates HIV gene expression through inactivation of NF-κB-mediated LTR regulation.

Fig. 4.

The SIRT1 agonist BF175 reduces the acetylation of NF-κB p65 and STAT3 in human immunodeficiency virus (HIV)-infected podocytes. Immortalized podocytes stably transduced with lentivirus expressing HIV-1 proviral genes under the cytomegalovirus promoter (CMV-HIV) or control enhanced green fluorescent protein (EGFP) were treated with vehicle or BF175 (10 μg/mL) for 24 h. A: Western blot analysis of total p65 (t-p65), acetylated p65 (ac-p65), total STAT3 (t-STAT3), acetylated STAT3 (ac-STAT3), and negative regulatory factor (Nef) expression. B: Western blot analysis of Nef expression in long tandem repeat-HIV podocytes after treatment with 0, 5, or 10 μg/mL BF175 for 24 h. Representative blots of three independent experiments are shown.

BF175 attenuates kidney injury in Tg26 mice.

To confirm the above findings in vivo, 4-wk-old Tg26 mice and their WT littermates received either BF175 (0.4 mg/kg body wt) or vehicle (5% DMSO in saline) daily by oral gavage for 4 wk. BF175 treatment attenuated albuminuria and renal injuries in Tg26 mice compared with Tg26 mice treated with vehicle (Fig. 5, A–C), whereas no difference was observed in WT mice treated with or without BF175. We confirmed the reduction in HIV Nef protein in the kidneys of Tg26 mice treated with BF175 compared with Tg26 mice treated with vehicle (Fig. 5D). Finally, expression of inflammatory markers was also suppressed in the kidneys of Tg26 mice by treatment of BF175 (Fig. 5, E and F).

Fig. 5.

BF175 treatment attenuates kidney injury in Tg26 mice. Six-week-old Tg26 or wild-type (WT) littermates were administered 0.4 mg/kg BF175 or vehicle daily for 4 wk (n = 5–6 mice/group). A: urinary albumin-to-creatinine ratio (UACR). *P < 0.05, **P < 0.01, and ***P < 0.001 compared between Tg26 mice and BF175-treated Tg26 mice. B: representative periodic acid-Schiff-stained images of mouse kidneys. Scale bar = 50 μm. Arrows show sclerotic glomeruli in Tg26 kidneys. C: average glomerulosclerosis (GS) score per mouse kidney (45–75 glomeruli were scored per mouse kidney). D: Western blot analysis of negative regulatory factor (Nef) in mouse kidneys. E: quantitative PCR analysis of chemokine (C-C motif) ligand 2 (Ccl2) and chemokine (C-X-C motif) ligand 11 (Cxcl11) mRNA in kidney cortexes of mice. **P < 0.01 and ****P < 0.0001 vs. vehicle-treated control mice; #P < 0.5 and ###P < 0.001 vs. vehicle-treated Tg26 mice.

Podocyte-specific SIRT1 overexpression attenuates kidney injury in Tg26 mice.

Pod-SIRT1^OV mice were generated as described in our previous study (17) and crossed with either Tg26 mice or their WT littermates on the same FVB background. WT, Pod-SIRT1^OV, Tg26, and Tg26;Pod-SIRT1^OV mice were given Dox-supplemented chow starting at 4 wk of age for the duration of 6 wk. Urine albumin was monitored weekly until the mice were euthanized. We found that SIRT1 overexpression in podocytes attenuated albuminuria (Fig. 6A) and reduced glomerulosclerosis in Tg26 mice (Fig. 6, B and C). We also confirmed that expression of the HIV Nef gene was also suppressed in isolated glomeruli in Tg26;Pod-SIRT1^OV mice compared with Tg26 mice (Fig. 6D). Expression of inflammatory markers was also reduced in Tg26;Pod-SIRT1^OV mice compared with Tg26 mice (Fig. 6, E and F).

Fig. 6.

Podocyte-specific sirtuin-1 (SIRT1) overexpression attenuates kidney injury in Tg26 mice. Doxycycline-supplemented chow was administered starting at 4 wk of age, and all mice were euthanized at 10 wk of age. A: urinary albumin-to-creatinine ratio (UACR). *P < 0.05 and **P < 0.01 compared between Tg26 and Tg26;Pod-Sirt1^OV mice. B: representative periodic acid-Schiff-stained images of mouse kidneys. Scale bar = 50 μm. C: average glomerulosclerosis (GS) score per mouse kidney (45–75 glomeruli were scored per mouse kidney). D: quantitative PCR analysis of negative regulatory factor (Nef) in isolated glomeruli in Tg26 mice. E: quantitative PCR analysis of chemokine (C-C motif) ligand 2 (Ccl2) and chemokine (C-X-C motif) ligand 11 (Cxcl11) mRNA in kidney cortexes of mice. *P < 0.5, **P < 0.01, and ****P < 0.0001 vs. wild-type (WT) control mice without Pod-SIRT1^OV expression; #P < 0.5 and ####P < 0.0001 vs. Tg26 mice.

miR-34a mediates the regulation of SIRT1 by HIV infection.

We next determined the potential mechanism involved in suppression of SIRT1 expression by HIV-1 in podocytes. In endothelial cells, SIRT1 expression was shown to be downregulated by several SIRT1-targeting miRNAs, such as miR-34a (52). Recent studies have also shown that miR-34a-mediated SIRT1 repression increases Tat-mediated HIV transcription (53, 55). Therefore, we performed miRNA sequencing in podocytes stably transduced with either HIV-1 or EGFP lentivirus. Table 1 shows the series of miRNAs that were upregulated and downregulated by HIV infection compared with controls. Interestingly, miR-34a was one of the miRNAs that were significantly upregulated by HIV in podocytes. We confirmed that miR-34a was upregulated by HIV infection in podocytes by quantitative PCR analysis (Fig. 7, A and B), which was associated with a reduction in Sirt1 mRNA. In addition to HIV, overexpression of NF-κB was also sufficient to increase expression of miR-34a (Fig. 7C). Indeed, miR-34a inhibitor diminished the suppression of SIRT1 by HIV (Fig. 7D). Together, the data indicate that the suppression of SIRT1 by HIV is mediated in part through expression likely through NF-κB-mediated miR-34a expression.

Table 1.

Differential expression of miRNAs in podocytes infected with human immunodeficiency virus

miRNA	Log2 Ratio
Decreased genes
hsa-miR-15a	−1.96863
hsa-miR-1291	−1.948
hsa-miR-629	−1.52797
hsa-miR-339-3p	−1.50051
hsa-miR-193b	−1.47787
hsa-miR-1308	−1.37005
hsa-miR-138	−1.09022
hsa-miR-15b	−1.00575
hsa-miR-93	−1.00279
hsa-miR-503	−0.80514
hsa-miR-27a	−0.7995
hsa-miR-92a	−0.75521
hsa-miR-378	−0.73409
hsa-miR-148b	−0.71922
hsa-miR-222	−0.63522
hsa-miR-151-5p	−0.62644
hsa-miR-1290	−0.59861
hsa-miR-1245	−0.59831
hsa-miR-22	−0.58485
hsa-miR-340	−0.58074
Increased genes
hsa-miR-193a-5p	0.591773
hsa-miR-30e	0.728323
hsa-miR-206	0.734568
hsa-miR-744	0.785838
hsa-miR-34a	0.881661
hsa-miR-100-5p	0.918304
hsa-miR-423-3p	0.956719
hsa-miR-1825	0.96257
hsa-miR-1826	0.976436
hsa-miR-1268	0.988855
hsa-miR-615-5p	0.994253
hsa-miR-217	1.087617
hsa-miR-1975	1.118701
hsa-miR-1974	1.602858

hsa, Homo sapiens.

Fig. 7.

miR-34a mediates the repression of sirtuin-1 (SIRT1) by human immunodeficiency virus (HIV). A: Western blot analysis of negative regulatory factor (Nef) expression in immortalized human podocytes infected with enhanced green fluorescent protein (EGFP) control or HIVΔgp-EGFP (HIV) lentivirus. B: quantitative PCR analysis of miR-34a and SIRT1 mRNA expression in EGFP control or HIVΔgp-EGFP podocytes. C: quantitative PCR analysis of miR-34a expression in podocytes with EGFP control or NF-κB p65 (p65) overexpression. D: quantitative PCR analysis of SIRT1 mRNA expression in control or HIVΔgp-EGFP podocytes expressing scrambled or miR-34a inhibitor. *P < 0.05, **P < 0.01, and ***P < 0.001 compared between the indicated groups.

DISCUSSION

With new combination antiretroviral therapy, the incidence of HIVAN has dramatically decreased and the spectrum of HIV-related kidney disease has been changed to more classic focal segmental glomerulosclerosis, acute interstitial nephritis, and drug-induced nephrotoxicity (1, 46). HIV infection has been shown to aggravate the progression of other CKDs, such as diabetic and hypertensive kidney disease (7, 29−31). In addition, it has been shown that HIV infection could aggravate the progression of other CKDs, such as diabetic and hypertensive kidney disease, likely through an enhanced inflammatory response. Therefore, it is still important for us to understand how HIV infection contributes to the development and progression of CKD. We have previously shown that NF-κB p65 and STAT3 are two major transcription factors regulating proinflammatory gene expression in HIV-infected kidneys (20). Systems biology analysis also supports JAK-STAT and NF-κB as key pathways activated in DKD (3, 47). HIV kidney disease is also a great model to study APOL1 kidney disease (18). Therefore, the findings from HIV kidney disease could be extrapolated to other kidney diseases and the drugs identified in HIV kidney disease could be also applied to other kidney diseases.

SIRT1 has been studied extensively in kidney injury and disease, including acute kidney injury, DKD, aging kidney disease, and others (13–16, 23). At the cellular level, SIRT1 is involved in a variety of processes that include autophagy (43), energetic homeostasis (40), mitochondrial biogenesis (44), and apoptosis (28). These biological effects of SIRT1 are thought to be mediated through transcription repression by deacetylation of histones and multiple transcription factors, including p53, FOXO, NF-κB p65, STAT3, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor (33). We have previously demonstrated a clear role of SIRT1 in protecting podocytes from aging and diabetes-induced injury (8, 27). In the present study, we confirmed that SIRT1 expression in renal cells is significantly suppressed by HIV infection in both mouse and human kidneys, which leads to enhanced acetylation and activation of transcription factors NF-κB p65 and STAT3.

Recently published results have indicated epigenetic silencing of SIRT1 expression via SIRT1-targeting miRNAs (52). We performed miRNA sequencing in human podocytes infected with or without HIV-1 infection and found that miR-34a was upregulated. We confirmed that miR-34a mediates inhibitory effects of HIV-1 infection on SIRT1 expression in podocytes. Consistent with our findings, recent studies (53, 55) have suggested that downregulation of SIRT1 by miR-34a increases Tat-mediated HIV transcription. Interestingly, we found that SIRT1 reciprocally can suppress HIV-1 nef expression in kidney cells in part through inhibition of NF-kB-mediated LTR promoter activity. These data suggest that SIRT1 could have dual beneficial effects in HIV kidney disease: inhibition of the inflammatory response and suppression of viral gene expression. SIRT1 has been previously shown to directly regulate HIV transcription via deacetylation of tat protein (37) and indirectly through the inhibition of tat-induced HIV transcription (54). Interestingly, in T cells, one study has shown that tat can directly interact with the deacetylase domain of SIRT1 and block its ability to deacetylate NF-κB p65 at Lys³¹⁰ (24), a key acetylation site leading to its activation (22), suggesting a reciprocal negative regulation between tat and SIRT1. Together, these and our studies suggest a complex regulatory mechanism between SIRT1, NF-κB, and HIV viral gene expression.

Our study not only demonstrates the mechanisms of SIRT1 in HIV-infected kidney cells but also provides evidence that SIRT1 agonists could be potentially developed as a novel therapy for HIV kidney disease. A recent study (36) has suggested that resveratrol and other purported SIRT1 agonists, SRT1720, SRT2183, and SRT1460, are also not director activators of SIRT1. Furthermore, resveratrol has been associated with renal toxicity in rodents (9). Therefore, we developed a new SIRT1 agonist that is more specific and has less toxicity (17). We reported that BF175 directly binds to SIRT1 and increases SIRT1 activity more significantly than other known SIRT1 agonists . Our previous and present studies showed that in vitro BF175 inhibits acetylation of peroxisome proliferator-activated receptor-γ coactivator-1α, NF-κB, and STAT3 in podocytes and in vivo BF175 significantly attenuated proteinuria and kidney injury in both diabetic and HIV-infected mice with kidney disease. We did not find any obvious toxicity of this drug in our previous studies in diabetic mice and the present study in Tg26 mice. BF175-treated mice exhibited normal behavior and physical activity and maintained normal growth without weight loss compared with mice treated with vehicle. In summary (Fig. 8), we demonstrated that HIV infection suppresses SIRT1 expression through induction of miR-34a expression in podocytes. Increased activation or expression of SIRT1 can attenuate HIV-induced podocyte injury in vitro and in vivo likely though deacetylation of NF-kB/Stat3, which leads to inhibition of the proinflammatory response and HIV viral gene expression. Our data support a critical role of SIRT1 in HIV kidney disease and suggest that SIRT1 agonists could be potentially developed to treat HIV-related kidney disease.

Fig. 8.

Schematics of suppression of sirtuin-1 (SIRT1) by human immunodeficiency virus (HIV). HIV infection suppresses Sirt1 expression through induction of miR-34a. Increased activation or expression of SIRT1 can thereby attenuate HIV-induced podocyte injury through reduced acetylation of NF-κB/Stat3, which leads to inhibition of the proinflammatory response and HIV viral gene expression. Nef, negative regulatory factor; CKD, chronic kidney disease.

GRANTS

J. C. He is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P01DK56492, 1R01DK078897, and 1R01DK088541 and by Veterans Affairs Merit Award IBX000345C. K. Lee is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 1R01DK117913.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.L., D.P.H., W.Y., P.E.K., and J.C.H. conceived and designed research; X.W., R.L., G.C.D., B.D., Z.L., and A.W. performed experiments; X.W., R.L., W.Z., Z.L., and K.L. analyzed data; X.W., K.L., and J.C.H. interpreted results of experiments; K.L. prepared figures; X.W., K.L., and J.C.H. drafted manuscript; D.P.H., K.L., and J.C.H. edited and revised manuscript;X.W., R.L., W.Z., K.L., and J.C.H. approved final version of manuscript.