Retinoic Acid Inhibits HIV-1–Induced Podocyte Proliferation through the cAMP Pathway
John Cijiang He*,,,
Ting-Chi Lu*,
Margaret Fleet,
Masaaki Sunamoto*,
Mohammad Husain*,
Wei Fang*,
Susana Neves,
Yibang Chen,
Stuart Shankland,
Ravi Iyengar and
Paul E. Klotman*
Departments of * Medicine and Pharmacology and Biochemistry, Mount Sinai School of Medicine, New York, and Nephrology/Medicine, James J. Peters VA Medical Center, Bronx, New York; and Department of Medicine, University of Washington, Seattle, Washington
Address correspondence to: Dr. John Cijiang He, Box 1243, Division of Nephrology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: 212-241-8004; Fax: 212-987-0389; E-mail: cijiang.he{at}mssm.edu
Received for publication July 12, 2006.
Accepted for publication October 23, 2006.
HIV-associated nephropathy is characterized by renal podocyteproliferation and dedifferentiation. This study found that all-transretinoic acid (atRA) reverses the effects of HIV-1 infectionin podocytes. Treatment with atRA reduced cell proliferationrate by causing G1 arrest and restored the expression of thedifferentiation markers (synaptopodin, nephrin, podocin, andWT-1) in HIV-1–infected podocytes. It is interesting thatboth atRA and 9-cis RA increased intracellular cAMP levels inpodocytes. Podocytes expressed most isoforms of retinoic acidreceptors (RAR) and retinoid X receptors (RXR) with the exceptionof RXR. RAR antagonists blocked atRA-induced cAMP productionand its antiproliferative and prodifferentiation effects onpodocytes, suggesting that RAR is required. For determinationof the effect of increased intracellular cAMP on HIV-infectedpodocytes, cells were stimulated with either forskolin or 8-bromo-cAMP.Both compounds inhibited cell proliferation significantly andrestored synaptopodin expression in HIV-infected podocytes.The effects of atRA were abolished by Rp-cAMP, an inhibitorof the cAMP/protein kinase A pathway and were enhanced by rolipram,an inhibitor of phosphodiesterase 4, suggesting that the antiproliferativeand prodifferentiation effects of atRA on HIV-infected podocytesare cAMP dependent. Furthermore, both atRA and forskolin suppressedHIV-induced mitogen-activated protein kinase 1 and 2 and Stat3phosphorylation. In vivo, atRA reduced proteinuria, cell proliferation,and glomerulosclerosis in HIV-1–transgenic mice. Thesefindings suggest that atRA reverses the abnormal phenotype inHIV-1–infected podocytes by stimulating RAR-mediated intracellularcAMP production. These results demonstrate the mechanism bywhich atRA reverses the proliferation of podocytes that is inducedby HIV-1.
HIV-associated nephropathy (HIVAN) is the most common causeof chronic renal failure in HIV-1–seropositive patients(1). The key histologic features of HIVAN are collapsing FSGSand microcystic tubular dilation (2). A unique characteristicof HIVAN is that podocytes, which normally are quiescent andhighly differentiated cells, undergo marked proliferation anddedifferentiation (3,4). Expression of HIV-1 viral proteinsis critical for pathogenesis as shown in HIV-1–transgenicmice (Tg26) as well as humans (5,6). Podocytes from HIV-1–transgenicmice exhibit increased growth rate and loss of contact inhibitionwhen compared with those of control littermates (7). In vitro,infection of podocytes with HIV-1 also causes proliferationand dedifferentiation (8). We have shown that the HIV-1 nefgene is the major determinant of podocyte proliferation anddedifferentiation (8,9) by inducing Src-dependent mitogen-activatedprotein kinase (MAPK) 1 and 2/Stat3 activation (10).
Retinoids are derivatives of vitamin A and have multiple cellularfunctions, including inhibition of proliferation, inductionof cell differentiation, regulation of apoptosis, and inhibitionof inflammation (11). During kidney development, retinoic acid(RA) affects tubulogenesis and nephron number (12). In additionto their established benefits in treating some malignancies,retinoids have been found to provide protection in several experimentalmodels of kidney disease (13–16). In rat models of acuteand chronic mesangioproliferative glomerulonephritis, retinoidspreserve renal function, decrease albuminuria, and reduce glomerularand tubular damage (13,14). In the rat model of puromycin aminonucleoside–inducednephrosis, retinoids prevent proteinuria from developing byprotecting podocytes from injury (15,16).
Retinoids exert their effects by binding two families of nuclearreceptors, retinoic acid receptors (RAR) and retinoid X receptors(RXR). All-trans-RA (atRA) binds and activates the RAR (RAR, , and ), whereas 9-cis RA binds and activates both the RARand the RXR (RXR , , and ) (17). RAR are expressed in many tissues,including the kidney (18). They affect gene transcription eitherdirectly by binding to the RA-response elements of a promoterregion (18,19) or indirectly by modulating transcription factorssuch as activator-protein 1 (20,21) or NF-B (22). Activationof cytosolic signaling molecules by retinoids also has beenreported as an important pathway to induce leukemia cell differentiation(23). Retinoids directly alter the activity of various proteinkinases, including MAPK and protein kinase C isoforms (24,25).A recent study reported that atRA induced rapid cAMP productionand increases protein kinase A (PKA) activity in acute myeloblasticleukemia cells, leading to cell differentiation (26). A synergisticeffect between atRA and inhibitors of phosphodiesterase alsowas observed for myeloid differentiation (27).
On the basis of the antiproliferative nature of atRA and thepathogenesis of HIVAN (podocyte proliferation and dedifferentiation),we hypothesized that retinoids may exert a beneficial effecton podocytes that are infected with HIV-1. In this study, weshow that atRA can inhibit HIV-1–induced podocyte proliferationand dedifferentiation through activation of an RAR-mediatedcAMP/PKA pathway.
Infection of Conditionally Immortalized Murine Podocytes with HIV-1 Expressing or Control Vector
Conditionally immortalized murine podocytes were isolated asdescribed previously (28). These cells proliferate under permissiveconditions (IFN- at 33°C) but differentiate under nonpermissiveconditions (37°C). The HIV-1 constructs were described previously(8). Briefly, the HIV-1 gag/pol deleted construct pNL4–3:d1443was derived from the provirus pNL4–3. A fragment thatcontained the EGFP gene (from pEGFP-C1; Clontech, Palo Alto,CA) was inserted at the SphI/MscI gag/pol deletion site. Theexpression of HIV-1 genes was confirmed by Western blot analysis.The HIV-1 gag/pol genes and VSV.G envelope glycoprotein wereprovided in trans using pCMV R8.91 and pMD.G plasmids, respectively(gifts of Dr. Didier Trono, Salk Institute, La Jolla, CA). Asa negative control, virus also was produced from pHR-CMV-IRES2-GFP-B,which contains the HIV-1 long-term repeat and EGFP. As the pilotstudy, we performed experiments in both freshly infected podocytesand a subset of established podocytes. We obtained similar results;therefore, we used here the established HIV-infected podocytes.In all experiments, cells were grown at 37°C on type 1 collagen–coateddishes for 10 d to inactivate the temperature-sensitive T antigenand to allow for differentiation. By Western blot, we confirmedthat T antigen was absent in these cells.
Cell Growth Assay
Control and HIV-1–infected podocytes were plated on collagen-coated24-well plates at a density of 20,000 cells/well. Podocyteswere cultured further for 3 to 5 d with atRA or 9-cis RA (0.1to 10 µM; Sigma, St. Louis, MO), RAR agonists (Am580 andRo-23-4217), RAR antagonist, and RXR agonist Ro-25-7386, and4-hydroxyphenylretinamide, a poor activator of RAR, which servedas a negative control (Roche, Basel, Switzerland) (29). Fortesting of PKA pathway involvement, podocytes were culturedwith H89 (10 µM; Calbiochem, San Diego, CA), rolipram(10 µM; Calbiochem), Rp-cAMP (100µM; Sigma), Forskolin(Sigma), or various combinations as indicated in the figures.Vehicle alone served as a control. After intervention, cellswere trypsinized and counted. For studying contact inhibition,podocytes were cultured for 10 d under nonpermissive conditionsand were plated at 80% confluence on collagen-coated dishes.Podocytes were cultured further with atRA (10 µM) or vehicle(DMSO), and photographs were taken on day 7. 3-(4,5-dimethylthiazol)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay for cell proliferation also was performed after3 d using a protocol that was provided by manufacturer (Promega,Madison, WI).
Flow Cytometry
After virus infection, podocytes were cultured under nonpermissiveconditions for 10 d and then incubated with atRA (10 µM)or vehicle for 4 additional days. Cells were trypsinized, washedwith 1x PBS, and fixed with ice-cold 80% ethanol. Cells werepelleted, resuspended in 1x PBS, and passed through a 25-G needleto create a single suspension. RNase (1 mg/ml) and propidiumiodine (400 µg/ml) were added, and cells were incubatedfor 30 min at 37°C. Cell-cycle analysis was performed ona FACSCalibur flow cytometer, and data were analyzed with CellQuestsoftware (Becton Dickinson, Mountain View, CA).
Apoptosis Study
Apoptosis was determined by Annexin V FITC apoptosis detectionkit (BD Bioscience, San Jose, CA). This combined staining usingAnnexin V and propidium iodide allowed us to distinguish earlyapoptotic cells from necrotic or later apoptotic cells. Afterstaining, apoptotic cells were measured by flow cytometry.
Northern Blot Analysis
Total RNA was extracted using Trizol (Life Technologies BRL,Grand Island, NY). Probes were generated by reverse transcriptase–PCRof RNA that were isolated from glomeruli of a normal mouse.The cDNA probes were radiolabeled with [32P-] dCTP by randomoligonucleotide priming and hybridized either with Hybrisol(Intergen, Purchase, NY) or with ULTRAhyb (Ambion, Austin, TX).
Real-Time PCR
Real-time PCR was performed with a Roche LightCycler with aQiagen QuantiTect One Step RTPCR SYBR green kit (Qiagen, Valencia,CA) according to the manufacturer’s instructions. Validatedprimer sets for nephrin and tubulin were obtained from Qiagen.Melting-curve analysis was performed to check for a single ampliconthat was verified by size using gel electrophoresis. LightCycleranalysis software was used for determining crossing points usingthe second derivative method. Data were analyzed by the 2–CT method as described by Livak and Schmittgen (30) and arepresented as fold increase normalized to housekeeping genes.
Assay for cAMP
Intracellular cAMP was measured using a cAMP Biotrak enzymeimmunoassay system (Amersham Biosciences, Piscataway, NJ). Afterbeing serum-starved overnight, cells were stimulated with differentanalogs of retinoids (0.1 to 10 µM) with or without rolipram(10 µM) for 2, 5, 10, and 30 min as indicated. Cells werelysed, and 100 µl of cell lysates was used for the assay.A nonacetylation enzyme immunoassay procedure was used to measuredintracellular cAMP production with a standard curve in a rangefrom 12.5 to 3200 fmol/well.
Western Blot
Podocytes were lysed with a buffer that contained 1% NP40, aprotease inhibitor cocktail and tyrosine and serine-threoninephosphorylation inhibitors. Cell lysates were subjected to Westernblot analysis using the following specific antibodies: Anti–phospho-MAPK1,2,anti-MAPK1,2, anti–phospho-Stat3, and anti-Stat3 fromCell Signaling Laboratory (Beverly, MA). Anti-Nef antibody wasobtained from FIT Biotech Oyj Plc (Tampere, Finland). Anti-podocinantibody was a gift from Dr. Peter Mundel (Mount Sinai Schoolof Medicine, NY).
In Vivo Studies Experimental Design.
Tg26 and their littermates were given intraperitoneal injectionof atRA at 16 mg/kg or vehicle alone (corn oil) three timesper week from 10 d of age to 4 mo of age (five mice per group).
Urine Analysis.
Urine was collected at 20-d intervals. Urine was assayed forprotein excretion using sulfosalicylic acid and creatinine excretionusing a colorimetric microplate assay based on the Jaffe reaction(Oxford Biomedical Research, Oxford, MI).
Histology and Sclerosis Scoring.
Kidney tissue was collected at 120 d and fixed in formalin ormethyl Carnoy fixed tissue. Periodic acid-Schiff staining wasperformed. A glomerulosclerosis index and interstitial diseaseindex were measured on tissue from all animals. A total of 50glomeruli were observed per tissue sample, and the percentageof segmental sclerosis was recorded as 0, 0 to 25, 25 to 50,50 to 75, or 75 to 100%. Collapsed glomerular tufts also wererecorded. The degree of interstitial disease, mostly tubulardilation, was recorded as an overall impression of the biopsywith the same percentage range.
Immunohistochemical analysis for nuclear cell proliferation–associatedantigen (Ki67) to assess proliferation was performed with mouseanti-Ki67 1:100 (BD Pharmingen, San Diego, CA) and followedby biotinylated horse anti-mouse IgG antibodies (1:100; bothfrom Vector Laboratories, Burlingame, CA). The ABC elite kitwas used to enhance the signal, and staining was visualizedby reaction with 3,3'-diaminobenzidine. The number of Ki67-positivenuclei per glomerulus was scored in a total of 50 glomeruliper mouse.
Statistical Analyses
For the cell proliferation and cAMP assays, the data are means± SD of the mean. For Northern blot and Western blotanalysis, all experiments were repeated at least three times.Representative experiments are shown below. Statistically significantdifferences between the means were determined by unpaired ttest or the Mann-Whitney U test where appropriate. Significancewas defined as a P 0.05.
RA Inhibits Proliferation and Restores Differentiation Markers in HIV-1–Infected Podocytes
We found that atRA (10 µM) markedly decreased cell proliferationrate in both control and HIV-1–infected podocytes (Figure 1A).There was no nuclear fragmentation in the cells that were treatedwith up to 10 µM atRA, suggesting that cell toxicity orincreased apoptosis was not responsible for these findings.This was confirmed further by analysis of apoptosis cells usingAnnexin V and propidium iodide staining. The antiproliferativeeffects of atRA were reversible because HIV-infected podocytesrecovered a high growth rate when atRA was removed from culturemedium (data not shown). atRA (10 µM) also inhibited thecontact-independent growth of podocytes that was induced byHIV-1 infection (Figure 1B). Podocytes that were treated withatRA were enlarged and grew in a monolayer, suggesting thatthese cells had converted back to a more differentiated state.For further exploration of the effective dosage of atRA on podocyteproliferation, both control and HIV-infected podocytes wereincubated with atRA at different concentrations. We found thatatRA significantly inhibited HIV-induced proliferation at 0.5µM (Figure 1C).
Figure 1. (A) All-trans retinoic acid (atRA) inhibits HIV-1–induced podocyte proliferation. After differentiation at 37°C for 10 d, Mock or HIV-1–infected podocytes were plated on collagen-coated six-well plates at 20,000 cells/well with or without atRA (10 µM) for 7 d. Bars represent mean cell number ± SD of six samples. (B) atRA inhibits HIV-induced loss of contact inhibition. Mock- or HIV-1–infected podocytes were plated on collagen-coated six-well plates at 80% confluence. Cells were cultured further with or without atRA (10 µM). Photographs were taken on day 7. C. Dosage response of retinoids on podocyte proliferation. After differentiation, control podocytes () and HIV-infected podocytes () were incubated in collagen-coated dishes for 3 d with or without atRA at concentrations of 0.1, 0.5, 1, or 10 µM. Cell number was counted. Means ± SD of three independent experiments are shown. *P < 0.001 verus cells without atRA treatment. (D) Effects of atRA on mRNA expression levels of WT-1, synaptopodin, cyclin A, cyclin E, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Normal podocytes (Mock) or HIV-1–infected podocytes (HIV) were cultured for 3 d with or without atRA (10 µM). Total RNA (10 µg/lane) was analyzed by Northern blotting. This is a representative blot of three independent experiments. (E) Dosage response of retinoids on synaptopodin expression. HIV-infected podocytes were treated with various concentrations (0.1 to 10 µM) of atRA or 9-cis-RA for 3 d. Total RNA (10 µg/lane) was analyzed by Northern blotting for synaptopodin and GAPDH. This is a representative blot of three independent experiments. (F) Effects of atRA on podocin expression. HIV-infected podocytes were treated with atRA (1 µM) for 3 d. Podocin expression was determined by Western blot using anti-podocin antibody. This is a representative blot of three independent experiments. (G) Effects of atRA on nephrin expression. HIV-infected podocytes were treated with atRA (1 µM) for 3 d. Nephrin/tubulin ratios were determined by real-time PCR. The fold of increased as compared with control podocytes are expressed. *P < 0.05 versus control (CL); **P < 0.05 versus HIV-infected podocytes; n = 3. Magnification, x200.
FACS analysis was performed on HIV-1–and mock-infectedpodocytes that were treated with atRA for 7 d. As shown in Table 1,the percentage of HIV-1–infected podocytes in the G0/G1phase was one third that of mock-infected podocytes (22.1 versus61%). Treatment of HIV-1–infected podocytes with atRAresulted in an increase in the percentage of cells in G1 to53.6% with a decrease in the fraction of cells in the S phase.These results demonstrate that atRA inhibits the proliferationof HIV-1–infected podocytes by inducing G1 arrest.
RA has been shown to inhibit G1-to-S transition by regulatingthe expression of cell-cycle proteins (31,32). We found thatatRA inhibited the transcription of cyclin A and cyclin E inHIV-1–infected podocytes (Figure 1D). These findings suggestthat atRA blocks HIV-1–induced G1S progression throughdownregulation of cyclin A and cyclin E expression.
Because HIV-1 infection also is known to suppress expressionof podocyte differentiation markers, we examined whether atRAcould reverse these effects as well. HIV-1–infected podocyteswere treated with atRA (10 µM) or vehicle for 3 d. Asshown by Northern blot, atRA significantly increased the mRNAlevels of WT-1 and synaptopodin in HIV-infected podocytes (Figure 1D).The effect of atRA and 9-cis RA on synaptopodin expression wasdosage dependent, as shown in Figure 1E. By real-time PCR, wefound that nephrin mRNA levels also increased after atRA treatmentin both control and HIV-infected podocytes (Figure 1E). Similarly,Western blot analysis showed that atRA stimulated podocin expressionin both control and HIV-infected podocytes (Figure 1F). Thesedata suggest that treatment with atRA induces podocyte differentiation.
RA Induces a Rapid Increase in Intracellular cAMP in Podocytes through RAR
Recently, it was shown that atRA stimulates intracellular cAMPgeneration in leukemia cells, suggesting a critical cytosolicpathway for myeloid differentiation (26). Because activationof the cAMP pathway generally is considered protective of injuryin podocytes (33), we examined whether cAMP mediates the effectsof atRA on podocytes. We found that atRA induced a three- tofour-fold increase in intracellular cAMP production in controlpodocytes. This stimulation was amplified when cells were pretreatedwith rolipram, a phosphodiesterase 4 (PDE4) inhibitor (Figure 2A).A similar pattern of stimulation was observed in HIV-1–infectedpodocytes (Figure 2B). Once again, stimulation of cAMP productionby atRA and 9-cis RA were dosage dependent (Figure 2, C andD).
Figure 2. (A and B) atRA stimulates intracellular cAMP production. After differentiation at 37°C, control podocytes (A) and HIV-infected podocytes (B) were cultured on collagen-coated six-well dishes and were stimulated with atRA (10 µM) for 2, 5, 10, 20, and 30 min with or without preincubation with rolipram (100 µM). Intracellular cAMP levels were determined using ELISA. The representative experiment of three independent assays is shown. (C and D) Dosage response of atRA and 9-cis-RA on cAMP production. Control podocytes were cultured on collagen-coated six-well dishes and were stimulated with various concentration of atRA or 9-cis-RA for 2, 5, and 10 min after preincubation with rolipram (100 µM). The representative experiment of three independent assays is shown.
Next, we investigated whether podocytes have retinoid receptorsand whether these receptors mediate the actions of atRA on HIV-infectedpodocytes. By Northern blot, we found that podocytes expressRAR, RAR, RAR, RXR, and RXR (Figure 3A). We also found thatHIV infection significantly suppressed expression of two majorreceptors in podocytes (RAR and RXR; Figure 3A). Using receptor-specificagonists and antagonists, we evaluated the roles of these receptorsin mediating the effects of RA on podocytes. The RAR antagonist(Ro-41-5253) blocked atRA-induced cAMP production (Figure 3B).Conversely, the RXR agonist (Ro-25-7386) had only a minor effecton atRA-induced cAMP production (Figure 3C). These data suggestthat RAR but not RXR mediates atRA-induced cAMP production.The mechanism by which this occurs remains to be determined.We examined whether RAR also mediates the phenotypic effectsof atRA on podocytes. As shown in Figure 3D, RAR agonists (Am580and Ro-23-4217) but not RXR agonist (Ro-25-7386) significantlyinhibited HIV-induced proliferation, similar to atRA and 9-cis-RA.Furthermore, the RAR antagonist Ro-41-5253 blocked the inhibitoryeffects of atRA. The RAR agonists also restored expression ofsynaptopodin in HIV-infected podocytes similar to what was observedwith atRA, and the RAR antagonist blocked the effect of atRAon synaptopodin expression (Figure 3E). Therefore, althoughthe expression is suppressed partially in HIV-infected podocytes,RAR still plays a critical role in mediating the effects ofretinoids on podocytes.
Figure 3. (A) HIV reduces expression of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Northern blotting of RAR, RAR, RAR, RXR, and RXR in control podocytes (Mock) and HIV-1–infected podocytes (HIV) that were treated with or without atRA (10 µM). HIV reduces the expression of both RAR and RXR. Twenty micrograms per lane of total RNA was loaded. The representative blots of three independent experiments are shown. (B) Effects of RAR antagonist on atRA-induced cAMP production. After differentiation at 37°C, podocytes were cultured on collagen-coated six-well dishes and stimulated with atRA (1 µM) for 2, 5, and 10 min with or without preincubation with 10 µM of RAR antagonist (Ro-41-5253). DMSO served as a control. All cells were pretreated with rolipram. Intracellular cAMP level was determined using ELISA. A representative experiment from three independent assays is shown. (C) Effect of RXR agonist on intracellular cAMP production. Podocytes were cultured on collagen-coated six-well dishes and stimulated with RXR agonist (Ro-25-7186; 0.1 and 1 µM) for 2, 5, and 10 min after preincubation with rolipram. Intracellular cAMP level was determined using ELISA. A representative experiment from three independent assays is shown. (D) Effects of RAR and RXR agonists and RAR antagonist on HIV-induced podocyte proliferation. After differentiation, control podocytes and HIV-infected podocytes were incubated in collagen-coated dishes for 3 d with DMSO (control), atRA (1 µM), RAR agonists (Am580; 1 µM), RXR agonist (Ro-25-7386; 1 µM), 9-cis-RA (1 µM), or both atRA and RAR antagonist (Ro-41-5253). Cell number was counted. Mean ± SD of three independent experiments is shown. *P < 0.001. (E) Effects of RAR and RXR agonists and RAR antagonist on synaptopodin expression in HIV-infected podocytes. HIV-infected podocytes were treated with atRA (1 µM), 4-hydroxyphenylretinamide (poor activator of RAR; 1 µM), Am580 (RAR agonist; 1 µM), Ro-23-4272 (RAR agonist; 1 µM), or Ro-25-7386 (RXR agonist; 1 µM) for 3 d. Additional cells were preincubated with Ro-41-5253 (RAR antagonist; 10 µM) and then treated with atRA (1 µM). Total RNA (10 µg/lane) was analyzed by Northern blotting for synaptopodin and GAPDH. This is a representative blot of three independent experiments.
Activation of the cAMP/PKA Pathway by atRA Reverses HIV-1–Induced Proliferation and Dedifferentiation
Because atRA induces cAMP production in podocytes, we investigatedwhether activation of the cAMP/PKA pathway was sufficient toreverse the effects of HIV-1 on podocytes. HIV-1 podocytes weretreated with forskolin (to stimulate adenylyl cyclase) or exogenous8-bromo-cAMP (a cAMP analogue). Both compounds inhibited HIV-inducedpodocyte proliferation and restored the expression of synaptopodin(Figure 4). These data suggest that activation of the cAMP/PKApathway could be the mechanism by which atRA reverses the effectsof HIV-1 on podocytes Therefore, we examined whether the actionsof atRA were mediated by activation of the cAMP/PKA pathway.Rp-cAMP (a competitor of endogenous cAMP) significantly diminishedthe inhibitory effect of atRA on HIV-1–induced podocyteproliferation as assessed by the MTS cell proliferation assay(Figure 5A) and cell counting (Figure 5B). Treatment of cellswith both rolipram and atRA to increase cAMP production wereadditive in inhibiting podocyte proliferation (Figure 5C). Wealso found that Rp-cAMP blocked the ability of atRA to stimulatethe expression of synaptopodin in HIV-infected podocytes, whereasthe effects of atRA were enhanced by rolipram (Figure 5D). Thesedata indicate that the activation of the cAMP pathway mediatesthe effects of atRA on podocytes.
Figure 4. (A) Forskolin (FK) and cAMP inhibit HIV-induced podocyte proliferation. After differentiation, control podocytes and HIV-infected podocytes were incubated in collagen-coated dishes with or without FK (30 µM) and cAMP (100 µM) for 3 d. Cell number was counted. Mean ± SD of five independent experiments is shown. *P < 0.001 versus HIV-infected cells. (B) FK and cAMP restore synaptopodin expression. The HIV-infected cells that were treated with FK and cAMP were subjected to Northern blot analysis for synaptopodin and GAPDH. A representative blot of three independent experiments is shown.
Figure 5. (A and B) Rp-cAMP (Rp) blocks antiproliferative effect of atRA on podocytes. Differentiated podocytes were pretreated with Rp-cAMP (100 µM) for 30 min and then were stimulated with or without atRA (10 µM) for another 3 d. Cells were lysed for MTS assay using the Promega kit (A) or for counting cell number (B). Mean ± SD of four independent experiments is shown. *P < 0.001. (C) A synergic effect is observed between atRA and rolipram. Podocytes were pretreated with rolipram (10 µM) for 30 min and then stimulated with atRA (10 µM) for another 3 d. The cell number was counted. Mean ± SD of three independent experiments is shown. *P < 0.01. (D) atRA induces synaptopodin expression in a protein kinase A (PKA)-dependent pathway. Under the same culture conditions as above, cells were used for Northern blot analysis for synaptopodin and GAPDH. A representative blot of three independent experiments is shown.
atRA Inhibits HIV-Induced MAPK1,2 and Stat3 Phosphorylation
We reported previously that HIV stimulates the Ras/Raf/MAPK1,2pathway, leading to podocyte proliferation through Nef (10).We examined whether atRA used a cross-talk mechanism with cAMPto affect MAPK1,2 phosphorylation in podocytes. It is interestingthat both atRA and 9-cis RA decreased HIV-induced MAPK1,2 phosphorylationin a dosage-dependent manner (Figure 6A). Forskolin and 8-bromo-cAMPalso inhibited HIV-induced MAPK1,2 phosphorylation (Figure 6B),whereas Rp-cAMP abolished the inhibitory effect of atRA on MAPK1,2activation (Figure 6C). Together, these data indicate that atRAinhibits MAPK1,2 phosphorylation through activation of the cAMPpathway. We also found that both atRA and 9-cis-RA suppressedphosphorylation of Stat3 (Tyr705; Figure 6D). As a control,we did not see changes in Nef expression in HIV-infected podocytesafter atRA treatment (Figure 6D).
Figure 6. atRA inhibits HIV-induced mitogen-activated protein kinase (MAPK) 1 and 2 and Stat3 phosphorylation. (A) HIV-infected podocytes were incubated with atRA or 9-cis-RA at 0.1 to 10 µM overnight in serum-free medium. Control podocytes served as a control (CL). Cells were lysed for Western blot of phospho and total MAPK1,2. (B) Control and HIV-infected podocytes were incubated with FK (30 µM), cAMP (100 µM), or vehicle overnight in serum-free medium. Cells then were lysed for Western blot of phospho and total MAPK1,2. (C) Cells were preincubated with Rp-cAMP (100 µM) for 30 min and then stimulated with or without atRA (10 µM) overnight in serum-free medium, and Western blots were performed for phospho and total MAPK1,2. A representative blot from a minimum of three independent experiments is shown for all experiments. (D) HIV-infected podocytes were treated with atRA (1 µM) overnight in serum-free medium. Control podocytes served as a control (CL). Cells were lysed for Western blot of phospho and total Stat3 and Nef. A representative blot from a minimum of three independent experiments is shown for all experiments.
In Vivo Study
To confirm our in vitro findings, we examined whether treatingHIV-1–transgenic mice (Tg26) with atRA ameliorates HIV-associatedkidney disease. As shown in Figure 7, Tg26 mice developed moreproteinuria and glomerulosclerosis than their littermates. WhenTg26 mice were treated with atRA, they exhibited significantlyreduced proteinuria, cell proliferation, and glomerulosclerosis,compared with nontreated Tg26 mice. These data indicate thatatRA exerts protective effects in vivo.
Figure 7. atRA improves kidney disease in HIV-1–transgenic mice. (A) Mean urine protein/urine creatinine (Upro/Ucr) ratio measured at 20-d intervals between 40 and 120 d of age. HIV-Tg26 mice had a significant increase in Upro/Ucr ratio at day 60 when compared with wild type (WT) mice. atRA treatment decreased Upro/Ucr ratio in HIV-Tg26 to WT levels. (B) The number of glomeruli with segmental sclerosis or collapsed tufts was increased in HIV-Tg26 mice at day 120 when compared with WT mice. atRA treatment significantly decreased the number of glomeruli with segmental sclerosis and completely abrogated tuft collapse. (C) Mean average of Ki67-positive nuclei was increased in HIV-Tg26 mice at day 120 when compared with WT mice. atRA treatment decreased the average number of Ki67-positive nuclei to WT levels.
atRA Changes HIV-Infected Podocytes from a Pathologic Phenotype to a More Differentiated Phenotype
This study demonstrates that atRA can reverse the pathologicchanges that are observed in HIV-infected podocytes (cell dedifferentiationand proliferation) and in HIV-1–transgenic mice (proteinuriaand glomerulosclerosis). Recently, retinoids were shown to improvekidney diseases in several experimental animal models, includinganti–Thy-1 nephritis (13,14), puromycin aminonucleoside–inducednephrosis (15,16), lupus nephritis (34), and anti–glomerularbasement membrane nephritis (35,36). atRA prevents the decreasein synaptopodin, nephrin, and podocin staining in an anti–glomerularbasement membrane glomerular injury model (36).
HIV-associated nephropathy is characterized by podocyte proliferation,loss of contact inhibition, and dedifferentiation, similar tomalignant cells (7–10). In this study, we found that RAblocked HIV-1–induced proliferation and contact-independentgrowth primarily through G1 arrest and decreased expressionof cyclin A and cyclin E in HIV-1–infected podocytes.atRA also induced podocyte differentiation manifested by increasedexpression of WT-1, nephrin, podocin, and synaptopodin. Thesefindings provide a rationale for using RA to treat HIVAN andother diseases of podocyte proliferation, such as idiopathiccollapsing FSGS.
atRA Reverses Abnormal Phenotype of HIV-Infected Podocytes through Stimulation of Intracellular cAMP Production
Several studies demonstrated that atRA induces rapid cAMP productionand increases PKA activity in acute myeloblastic leukemia cells(26). Our results demonstrate significant stimulation of cAMPproduction within minutes when podocytes are treated with eitheratRA or 9-cis-RA. This stimulation was amplified when cellswere pretreated with rolipram, an inhibitor of PDE4. Podocytesexpress several G protein–coupled receptors (GPCR), includingthe 2-adrenergic receptor, the D1-dopamine receptor, and theprostaglandin IP and EP4 receptors (33,37). Activation of thesereceptors results in generation of intracellular cAMP. Inductionof the cAMP–PKA pathway in podocytes regulates cell morphology,actin assembly, and matrix production (33). In addition, cAMPseems to attenuate the effect of hormones that activate theCa2+/PKC pathway (38). Overall, the cAMP pathway seems to exerta protective effect on podocytes (33). In this study, we foundthat the cAMP/PKA pathway mediates the beneficial effects ofatRA on podocytes because Rp-cAMP blocked atRA-induced differentiationof podocytes. We also found that rolipram enhanced the atRAeffect. These data indicate that atRA exerts its effect on HIV-1–infectedpodocytes through activation of the cAMP pathway. Treatmentwith atRA alone or in combination with PDE inhibitors couldbe an effective treatment strategy (in combination with antivirals)for HIVAN.
RAR Mediates the Effects of atRA on HIV-Infected Podocytes
Although receptor-independent effects of retinoids have beendescribed, most studies have shown that RAR and/or RXR playa role in retinoid-induced cellular effects through either genomicor nongenomic pathways (18,24,26). It was surprising that RAR,which is suppressed in HIV-infected cells, still played a criticalrole in atRA’s effects on podocyte phenotype as supportedby our data using the RAR agonist and antagonist. How RAR mediatesatRA-induced cAMP production, however, remains unclear. Estrogencan induce cAMP production through activation of adenylyl cyclase(39), and a recent study suggested that estrogen binds to intracellularGPCR (40). Further studies are required to determine whetherthere are GPCR that bind atRA.
atRA Inhibits HIV-Induced MAPK1,2 and Stat3 Phosphorylation
In previous studies, we found that HIV Nef induced MAPK1,2 andStat3 activation in podocytes (10,41). Inhibition of MAPK1,2or Stat3 activation suppressed Nef-induced cell proliferationand dedifferentiation (10). Here, we found that atRA inhibitedboth MAPK1,2 and Stat3 phosphorylation in podocytes. atRA probablyreduces MAPK phosphorylation through activation of MAPK phosphatase1, as this was reported previously (42). It has been reportedthat atRA can inhibit Stat3 activation in leukemia cells (43).
We found that atRA inhibits proliferation and induces differentiationin HIV-infected podocytes through RAR-mediated cAMP/PKA activation.These data provide new insights into the underlying molecularmechanisms of the podocyte disease of HIVAN as well as a noveltherapeutic strategy for its treatment.
This work was supported by National Institutes of Health grantP01DK056492 (to P.K.) and grant GM-54508 (to R.I.). J.H. issupported by a K08 award (DK-65495). S.N. is supported by anindividual National Research Service Award grant (GM-65065).
We thank Rafael Mira Lopez for providing RAR and RXR agonistsand antagonist and for thoughtful discussion and suggestions.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
J.C.H. and T.-C.L. contributed equally to this work.
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Top
Abstract
Introduction
. RAR
antagonists blocked atRA-induced cAMP production and its antiproliferative and prodifferentiation effects on podocytes, suggesting that RAR
, and 
B (22). Activation of cytosolic signaling molecules by retinoids also has been reported as an important pathway to induce leukemia cell differentiation (23). Retinoids directly alter the activity of various protein kinases, including MAPK and protein kinase C isoforms (24,25). A recent study reported that atRA induced rapid cAMP production and increases protein kinase A (PKA) activity in acute myeloblastic leukemia cells, leading to cell differentiation (26). A synergistic effect between atRA and inhibitors of phosphodiesterase also was observed for myeloid differentiation (27). 
B, which contains the HIV-1 long-term repeat and EGFP. As the pilot study, we performed experiments in both freshly infected podocytes and a subset of established podocytes. We obtained similar results; therefore, we used here the established HIV-infected podocytes. In all experiments, cells were grown at 37°C on type 1 collagen–coated dishes for 10 d to inactivate the temperature-sensitive T antigen and to allow for differentiation. By Western blot, we confirmed that T antigen was absent in these cells. 
0.05. 
S progression through downregulation of cyclin A and cyclin E expression. 
) and HIV-infected podocytes (
) were incubated in collagen-coated dishes for 3 d with or without atRA at concentrations of 0.1, 0.5, 1, or 10 µM. Cell number was counted. Means ± SD of three independent experiments are shown. *P < 0.001 verus cells without atRA treatment. (D) Effects of atRA on mRNA expression levels of WT-1, synaptopodin, cyclin A, cyclin E, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Normal podocytes (Mock) or HIV-1–infected podocytes (HIV) were cultured for 3 d with or without atRA (10 µM). Total RNA (10 µg/lane) was analyzed by Northern blotting. This is a representative blot of three independent experiments. (E) Dosage response of retinoids on synaptopodin expression. HIV-infected podocytes were treated with various concentrations (0.1 to 10 µM) of atRA or 9-cis-RA for 3 d. Total RNA (10 µg/lane) was analyzed by Northern blotting for synaptopodin and GAPDH. This is a representative blot of three independent experiments. (F) Effects of atRA on podocin expression. HIV-infected podocytes were treated with atRA (1 µM) for 3 d. Podocin expression was determined by Western blot using anti-podocin antibody. This is a representative blot of three independent experiments. (G) Effects of atRA on nephrin expression. HIV-infected podocytes were treated with atRA (1 µM) for 3 d. Nephrin/tubulin ratios were determined by real-time PCR. The fold of increased as compared with control podocytes are expressed. *P < 0.05 versus control (CL); **P < 0.05 versus HIV-infected podocytes; n = 3. Magnification, x200.
