Inhibition of apoptosis signal-regulating kinase 1 mitigates the pathogenesis of human immunodeficiency virus-associated nephropathy.

BACKGROUND: Chronic kidney disease (CKD) is a common cause of morbidity and mortality in human immunodeficiency virus (HIV)-positive individuals. Among the HIV-related kidney diseases, HIV-associated nephropathy (HIVAN) is a rapidly progressive renal disease characterized by collapsing focal glomerulosclerosis (GS), microcystic tubular dilation, interstitial inflammation and fibrosis. Although the incidence of end-stage renal disease due to HIVAN has dramatically decreased with the widespread use of antiretroviral therapy, the prevalence of CKD continues to increase in HIV-positive individuals. Recent studies have highlighted the role of apoptosis signal-regulating kinase 1 (ASK1) in driving kidney disease progression through the activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase and selective ASK-1 inhibitor GS-444217 was recently shown to reduce kidney injury and disease progression in various experimental models. Therefore we examined the efficacy of ASK1 antagonism by GS-444217 in the attenuation of HIVAN in Tg26 mice. METHODS: GS-444217-supplemented rodent chow was administered in Tg26 mice at 4 weeks of age when mild GS and proteinuria were already established. After 6 weeks of treatment, the kidney function assessment and histological analyses were performed and compared between age- and gender-matched control Tg26 and GS-444217-treated Tg26 mice. RESULTS: GS-444217 attenuated the development of GS, podocyte loss, tubular injury, interstitial inflammation and renal fibrosis in Tg26 mice. These improvements were accompanied by a marked reduction in albuminuria and improved renal function. Taken together, GS-4442217 attenuated the full spectrum of HIVAN pathology in Tg26 mice. CONCLUSIONS: ASK1 signaling cascade is central to the development of HIVAN in Tg26 mice. Our results suggest that the select inhibition of ASK1 could be a potential adjunctive therapy for the treatment of HIVAN.

Role of SIRT1 in HIV-associated kidney disease.

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.

Tubular HIPK2 is a key contributor to renal fibrosis.

We previously used global Hipk2-null mice in various models of kidney disease to demonstrate the central role of homeodomain-interacting protein kinase 2 (HIPK2) in renal fibrosis development. However, renal tubular epithelial cell-specific (RTEC-specific) HIPK2 function in renal fibrogenesis has yet to be determined. Here, we show that modulation of tubular HIPK2 expression and activity affects renal fibrosis development in vivo. The loss of HIPK2 expression in RTECs resulted in a marked diminution of renal fibrosis in unilateral ureteral obstruction (UUO) mouse models and HIV-associated nephropathy (HIVAN) mouse models, which was associated with the reduction of Smad3 activation and downstream expression of profibrotic markers. Conversely, WT HIPK2 overexpression in RTECs accentuated the extent of renal fibrosis in the setting of UUO, HIVAN, and folic acid-induced nephropathy in mice. Notably, kinase-dead HIPK2 mutant overexpression or administration of BT173, an allosteric inhibitor of HIPK2-Smad3 interaction, markedly attenuated the renal fibrosis in these mouse models of kidney disease, indicating that HIPK2 requires both the kinase activity and its interaction with Smad3 to promote TGF-ß-mediated renal fibrosis. Together, these results establish an important RTEC-specific role of HIPK2 in kidney fibrosis and further substantiate the inhibition of HIPK2 as a therapeutic approach against renal fibrosis.

Notch4 activation aggravates NF-κB-mediated inflammation in HIV-1-associated nephropathy.

Notch pathway activation plays a central role in the pathogenesis of many glomerular diseases. We have previously shown that Notch4 expression was upregulated in various renal cells in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) patients and rodent models of HIVAN. In this study, we examined whether the Notch pathway can be distinctly activated by HIV-1 gene products and whether Notch4, in particular, can influence disease progression. Using luciferase reporter assays, we did not observe activation of the NOTCH4 promoter with the HIV protein Nef in podocytes. Further, we observed upregulated expression of a gamma secretase complex protein, presenilin 1, but not Notch4, in podocytes infected with an HIV-1 expression construct. To assess the effects of Notch4 on HIVAN disease progression, we engineered Tg26 mice with global deletion of the Notch4 intracellular domain (Notch4dl ), which is required for signaling function. These mice (Notch4d1/Tg26+ ) showed a significant improvement in renal function and a significant decrease in mortality compared to Tg26 mice. Histological examination of kidneys showed that Notch4d1/Tg26+ mice had overall glomerular, tubulointerstitial injury and a marked decrease in interstitial inflammation. A significant decrease in the proliferating cells was observed in the tubulointerstitial compartments of Notch4d1/Tg26+ mice. Consistent with the diminished inflammation, kidneys from Notch4d1/Tg26+ mice also showed a significant decrease in expression of the inflammatory cytokine transcripts Il-6 and Ccl2, as well as the master inflammatory transcription factor NF-κB (Nfkb1 transcripts and p65 protein). These data identify Notch4 as an important mediator of tubulointerstitial injury and inflammation in HIVAN and a potential therapeutic target.

The HIV protease inhibitor darunavir prevents kidney injury via HIV-independent mechanisms.

HIV-associated nephropathy (HIVAN) is a rapidly progressive kidney disease that is caused by HIV infection of renal epithelial cells with subsequent expression of viral genes, including vpr. Antiretroviral therapy ameliorates HIVAN without eradicating HIV from the kidneys and the mechanism by which it protects kidneys is poorly understood. Since HIV protease inhibitors have "off target" cellular effects, we studied whether darunavir, the most commonly prescribed protease inhibitor, protects kidneys from HIV-induced injury via mechanisms independent of HIV protease and viral replication. Renal epithelial cells were transduced with lentiviruses encoding HIV (lacking protease and reverse transcriptase), Vpr, or vector control. Darunavir attenuated HIV and Vpr-induced activation of Stat3, Src, Erk, and cytokines, which are critical for HIVAN pathogenesis. We then studied HIV-transgenic mice, which develop HIVAN in the absence of HIV protease or reverse transcriptase. Mice were treated with darunavir, zidovudine, darunavir + zidovudine, or control. Darunavir and darunavir + zidovudine reduced albuminuria and histologic kidney injury and normalized expression of dysregulated proteins. RNA-seq analyses demonstrated that darunavir suppressed HIV-induced upregulation of immune response genes in human kidney cells. These data demonstrate that darunavir protects against HIV-induced renal injury via mechanisms that are independent of inhibition of HIV protease.

Tyro3 is a podocyte protective factor in glomerular disease.

Our previous work demonstrated a protective role of protein S in early diabetic kidney disease (DKD). Protein S exerts antiinflammatory and antiapoptotic effects through the activation of TYRO3, AXL, and MER (TAM) receptors. Among the 3 TAM receptors, we showed that the biological effects of protein S were mediated largely by TYRO3 in diabetic kidneys. Our data now show that TYRO3 mRNA expression is highly enriched in human glomeruli and that TYRO3 protein is expressed in podocytes. Interestingly, glomerular TYRO3 mRNA expression increased in mild DKD but was suppressed in progressive DKD, as well as in focal segmental glomerulosclerosis (FSGS). Functionally, morpholino-mediated knockdown of tyro3 altered glomerular filtration barrier development in zebrafish larvae, and genetic ablation of Tyro3 in murine models of DKD and Adriamycin-induced nephropathy (ADRN) worsened albuminuria and glomerular injury. Conversely, the induction of TYRO3 overexpression specifically in podocytes significantly attenuated albuminuria and kidney injury in mice with DKD, ADRN, and HIV-associated nephropathy (HIVAN). Mechanistically, TYRO3 expression was suppressed by activation of TNF-α/NF-κB pathway, which may contribute to decreased TYRO3 expression in progressive DKD and FSGS, and TYRO3 signaling conferred antiapoptotic effects through the activation of AKT in podocytes. In conclusion, TYRO3 plays a critical role in maintaining normal podocyte function and may be a potential new drug target to treat glomerular diseases.

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress.

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap-/- model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2-) that promotes endothelin-1 synthesis. Plg via O2- also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a "second hit" in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

Characterization of neuropathology in the HIV-1 transgenic rat at different ages.

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

BACKGROUND: HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). METHODS: Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). RESULTS: Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. CONCLUSIONS: We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

HIV Promotes NLRP3 Inflammasome Complex Activation in Murine HIV-Associated Nephropathy.

Dysregulated growth and loss of podocytes are important features of HIV-associated nephropathy. Recently, HIV was reported to induce a new type of programed cell death, pyroptosis, in T lymphocytes through induction of Nod-like receptor protein 3 (NLRP3) inflammasome complexes. We evaluated the role of HIV in podocyte NLRP3 inflammasome formation both in vivo and in vitro. Renal cortical sections of HIV-transgenic mice (Tg26) displayed increased expression of NLRP3, ASC (a CARD protein), caspase-1, and IL-1ß proteins, confirming NLRP3 inflammasome complex formation in podocytes of Tg26 mice. Renal tissues of Tg26 mice also displayed enhanced mRNA levels and protein expressions of inflammasome markers (NLRP3, ASC, and caspase-1, and IL-1ß). Serum of Tg26 mice also showed elevated concentrations of IL-1ß cytokine compared with FVBN mice. HIV induced pyroptosis in a dose- and time-dependent manner within podocytes, a phenotype of inflammasome activation. Caspase-1 inhibitor not only attenuated podocyte expression of caspase-1 and IL-1ß but also provided protection against pyroptosis, suggesting that HIV-induced podocyte injury was mediated by caspase-1 activation. Interestingly, HIV-induced podocyte pyroptosis could be partially inhibited by Tempol (a superoxide dismutase-mimetic agent) and by glyburide (an inhibitor of potassium efflux). These findings suggest that generation of reactive oxygen species and potassium efflux contribute to HIV-induced pyroptosis and NLRP3 inflammasome activation in podocytes.

The National NeuroAIDS Tissue Consortium (NNTC) Database: an integrated database for HIV-related studies.

We herein present the National NeuroAIDS Tissue Consortium-Data Coordinating Center (NNTC-DCC) database, which is the only available database for neuroAIDS studies that contains data in an integrated, standardized form. This database has been created in conjunction with the NNTC, which provides human tissue and biofluid samples to individual researchers to conduct studies focused on neuroAIDS. The database contains experimental datasets from 1206 subjects for the following categories (which are further broken down into subcategories): gene expression, genotype, proteins, endo-exo-chemicals, morphometrics and other (miscellaneous) data. The database also contains a wide variety of downloadable data and metadata for 95 HIV-related studies covering 170 assays from 61 principal investigators. The data represent 76 tissue types, 25 measurement types, and 38 technology types, and reaches a total of 33,017,407 data points. We used the ISA platform to create the database and develop a searchable web interface for querying the data. A gene search tool is also available, which searches for NCBI GEO datasets associated with selected genes. The database is manually curated with many user-friendly features, and is cross-linked to the NCBI, HUGO and PubMed databases. A free registration is required for qualified users to access the database.

Hyperglycemia enhances kidney cell injury in HIVAN through down-regulation of vitamin D receptors.

In the present study, we evaluated the effect of short term hyperglycemia on renal lesions in a mouse model (Tg26) of HIV-associated nephropathy (HIVAN). Control and Tg26 mice in groups (n=6) were administered either normal saline (FVBN or Tg) or streptozotocin (FVBN+STZ or Tg26+STZ). After two weeks, biomarkers were collected and kidneys were harvested. FVBN+ STZ and Tg26+STZ displayed elevated serum glucose levels when compared to FVBN and Tg26 respectively. Tg26+STZ displayed elevated (P<0.05) blood urea nitrogen (BUN) levels (P<0.05) and enhanced (P<0.01) proteinuria when compared to Tg26. Tg26+STZ displayed enhanced (P<0.001) number of sclerotic glomeruli and microcysts vs. Tg26. Renal tissues of Tg26 displayed down regulation of vitamin D receptor (VDR) expression and enhanced Ang II production when compared to FVBN mice. Hyperglycemia exacerbated down regulation of VDR and production of Ang II in FVBN and Tg mice. Hyperglycemia increased kidney cell reactive oxygen species (ROS) production and oxidative DNA damage in both FVBN and Tg26 mice. In in vitro studies, HIV down regulated podocyte VDR expression and also enhanced renin angiotensin system activation. In addition, both glucose and HIV stimulated kidney cell ROS generation and DNA damage and compromised DNA repair; however, tempol (superoxide dismutase mimetic), losartan (Ang II blocker) and EB1089 (VDR agonist) provided protection against DNA damaging effects of glucose and HIV. These findings indicated that glucose activated the RAS and inflicted oxidative stress-mediated DNA damage via down regulation of kidney cell VDR expression in HIV milieu both in vivo and in vitro.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²âº overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the µ-opioid receptor antagonist CTAP and were not observed in neurons cultured from µ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via µ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of µ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

The basic domain of HIV-tat transactivating protein is essential for its targeting to lipid rafts and regulating fibroblast growth factor-2 signaling in podocytes isolated from children with HIV-1-associated nephropathy.

Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN.

Inhibition of Notch pathway attenuates the progression of human immunodeficiency virus-associated nephropathy.

The Notch pathway is an evolutionarily conserved signaling cascade that is critical in kidney development and has also been shown to play a pathogenetic role in a variety of kidney diseases. We have previously shown that the Notch signaling pathway is activated in human immunodeficiency virus-associated nephropathy (HIVAN) as well as in a rat model of the disease. In this study, we examined Notch signaling in the well established Tg26 mouse model of HIVAN. Notch signaling components were distinctly upregulated in the kidneys of these mice as well as in immortalized podocytes derived from these mice. Notch1 and Notch4 were upregulated in the Tg26 glomeruli, and Notch4 was also expressed in tubules. Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like 4 were all upregulated in the tubules of Tg26 mice, but glomeruli showed minimal expression of Notch ligands. To examine a potential pathogenetic role for Notch in HIVAN, Tg26 mice were treated with GSIXX, a gamma secretase inhibitor that blocks Notch signaling. Strikingly, GSIXX treatment resulted in significant improvement in both histological kidney injury scores and renal function. GSIXX-treated Tg26 mice also showed diminished podocyte proliferation and dedifferentiation, cellular hallmarks of the disease. Moreover, GSIXX blocked podocyte proliferation in vitro induced by HIV proteins Nef and Tat. These studies suggest that Notch signaling can promote HIVAN progression and that Notch inhibition may be a viable treatment strategy for HIVAN.

Nef interaction with actin compromises human podocyte actin cytoskeletal integrity.

The HIV-1 accessory protein Nef is considered to play an important role in the development of a podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering the podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45 kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef-actin and Nef-zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had a decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projections (filopodia). Nef/CIHP displayed an enhanced cortical F-actin score index (P<0.001) and thus indicated a reorganization of F-actin in the cortical regions. Microarray analysis showed that Nef enhanced the expression of Rac1, syndecan-4, Rif, and CDC42 and attenuated the expression of syndecan-3 and syntenin. In addition, Nef/CIHPs displayed a diminished sphingomyelinase (ASMase) activity. Functionally, Nef/CIHPs displayed diminished attachment and enhanced detachment to their substrate. These findings indicate that Nef interaction with actin compromises the podocyte cytoskeleton integrity.

Rapamycin-induced modulation of HIV gene transcription attenuates progression of HIVAN.

HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg, every other day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.