Induction of Hepcidin Expression in the Renal Cortex of Sickle Cell Disease Mice.

In patients with sickle cell disease (SCD), chronic hemolysis and frequent blood transfusions cause iron overload and accumulation in the kidneys. The iron deposition is found in the renal cortex and correlates with the severity of hemolysis. In this study, we observed a significant accumulation of iron in the renal cortex of a mouse model of SCD, and assessed the expression of the proteins involved in maintaining renal iron homeostasis. Despite the intracellular iron accumulation, the levels of the transferrin receptor in the kidneys were increased, but the levels of the iron exporter ferroportin were not altered in SCD mice. Ferroportin is regulated by hepcidin, which binds to it and promotes its degradation. We found reduced serum hepcidin levels but increased renal hepcidin production in SCD mice. Furthermore, we observed significant macrophage infiltration and increased expression of intercellular adhesion molecule 1 in the endothelial cells of the kidneys in SCD mice. These observations correlated with elevated levels of proinflammatory cytokines IL-1ß and IL-6, which can potentially stimulate hepcidin expression. Taken together, our results demonstrate that in individuals with SCD, a renal inflammation state induces renal hepcidin production that blocks the upregulation of ferroportin levels, resulting in dysregulation of iron homeostasis in the kidney and iron deposition in the renal cortex.

Urinary Kringle Domain-Containing Protein HGFL: A Validated Biomarker of Early Sickle Cell Anemia-Associated Kidney Disease.

INTRODUCTION: Chronic kidney disease (CKD) is a prevalent complication of sickle cell anemia (SCA). Hyperfiltration that delayed detection of CKD is common in SCA patients. Identification of novel urinary biomarkers correlating with glomerular filtration rates may help to detect and predict progression of renal disease. METHODS: Reanalysis of mass spectra of urinary samples obtained from University of Illinois at Chicago identified kringle domain-containing protein HGFL. RESULTS: HGFL levels correlated with hyperfiltration, were significantly reduced at CKD stage 1 compared to stage 0, negatively correlated with progression of CKD and were suitable for differentiation of stage 1. Better prediction of CKD progression to stage 2 was observed for HGFL-based risk prediction compared to the estimated glomerular filtration rate (eGFR)-based prediction. Results from a Howard University patient cohort supported the utility of HGFL-based test for the differentiation of stage 1 of CKD. CONCLUSION: Urinary HGFL may contribute additional information beyond eGFR and improve diagnosis of early-stage CKD in SCA patients.

Protein Phosphatase 1-Targeting Small-Molecule C31 Inhibits Ebola Virus Replication.

Background: Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results: High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion: C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.

RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice.

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease.

Angiopoietin-1 prevents severe bleeding complications induced by heparin-like drugs and fibroblast growth factor-2 in mice.

Critically ill children can develop bleeding complications when treated with heparin-like drugs. These events are usually attributed to the anticoagulant activity of these drugs. However, previous studies showed that fibroblast growth factor-2 (FGF-2), a heparin-binding growth factor released in the circulation of these patients, could precipitate intestinal hemorrhages in mice treated with the heparin-like drug pentosan polysulfate (PPS). Yet very little is known about how FGF-2 induces bleeding complications in combination with heparin-like drugs. Here, we examined the mechanisms by which circulating FGF-2 induces intestinal hemorrhages in mice treated with PPS. We used a well-characterized mouse model of intestinal hemorrhages induced by FGF-2 plus PPS. Adult FVB/N mice were infected with adenovirus carrying Lac-Z or a secreted form of recombinant human FGF-2, and injected with PPS, at doses that do not induce bleeding complications per se. Mice treated with FGF-2 in combination with PPS developed an intestinal inflammatory reaction that increased the permeability and disrupted the integrity of submucosal intestinal vessels. These changes, together with the anticoagulant activity of PPS, induced lethal hemorrhages. Moreover, a genetically modified form of the endothelial ligand angiopoietin-1 (Ang-1*), which has powerful antipermeability and anti-inflammatory activity, prevented the lethal bleeding complications without correcting the anticoagulant status of these mice. These findings define new mechanisms through which FGF-2 and Ang-1* modulate the outcome of intestinal bleeding complications induced by PPS in mice and may have wider clinical implications for critically ill children treated with heparin-like drugs.

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.

Adenoviral E4 gene stimulates secretion of pigmental epithelium derived factor (PEDF) that maintains long-term survival of human glomerulus-derived endothelial cells.

Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region also supported survival of human glomerulus-derived endothelial cells in serum-free media. Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT-PCR, Western blot and ELISA analysis. PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells.

Antiviral response and HIV-1 inhibition in sickle cell disease.

Sickle cell disease (SCD) is characterized by hemolysis, vaso-occlusion, and ischemia. HIV-1 infection was previously shown to be suppressed in SCD PBMCs. Here, we report that HIV-1 suppression is attributed to the increased expression of iron, hypoxia, and interferon-induced innate antiviral factors. Inhibition of upregulated antiviral genes, HMOX-1, CDKN1A, and CH25H, increased HIV-1 replication in SCD PBMCs, suggesting their critical role in HIV-1 suppression. Levels of IFN-ß were elevated in SCD patients. Sickle cell hemoglobin (HbS) treatment of THP-1-derived and primary monocyte-derived macrophages induced production of IFN-ß, upregulated antiviral gene expression, and suppressed HIV-1 infection. Infection with mouse-adapted EcoHIV was suppressed in the SCD mice that also exhibited elevated levels of antiviral restriction factors. Our findings suggest that hemolysis and release of HbS leads to the induction of IFN-ß production, induction of cellular antiviral state by the expression of iron and IFN-driven factors, and suppression of HIV-1 infection.

Ebola Virus NP Binding to Host Protein Phosphatase-1 Regulates Capsid Formation.

The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.

Expression of a protein phosphatase 1 inhibitor, cdNIPP1, increases CDK9 threonine 186 phosphorylation and inhibits HIV-1 transcription.

CDK9/cyclin T1, a key enzyme in HIV-1 transcription, is negatively regulated by 7SK RNA and the HEXIM1 protein. Dephosphorylation of CDK9 on Thr(186) by protein phosphatase 1 (PP1) in stress-induced cells or by protein phosphatase M1A in normally growing cells activates CDK9. Our previous studies showed that HIV-1 Tat protein binds to PP1 through the Tat Q(35)VCF(38) sequence, which is similar to the PP1-binding RVXF motif and that this interaction facilitates HIV-1 transcription. In the present study, we analyzed the effect of expression of the central domain of nuclear inhibitor of PP1 (cdNIPP1) in an engineered cell line and also when cdNIPP1 was expressed as part of HIV-1 pNL4-3 in place of nef. Stable expression of cdNIPP1 increased CDK9 phosphorylation on Thr(186) and the association of CDK9 with 7SK RNA. The stable expression of cdNIPP1 disrupted the interaction of Tat and PP1 and inhibited HIV-1 transcription. Expression of cdNIPP1 as a part of the HIV-1 genome inhibited HIV-1 replication. Our study provides a proof-of-concept for the future development of PP1-targeting compounds as inhibitors of HIV-1 replication.

Role of circulating fibroblast growth factor-2 in lipopolysaccharide-induced acute kidney injury in mice.

Fibroblast growth factor-2 (FGF-2) is an angiogenic growth factor involved in renal growth and regeneration. Previous studies in rodents revealed that single intrarenal injections of FGF-2 improved the outcome of acute kidney injury (AKI). Septic children usually show elevated plasma levels of FGF-2, and are at risk of developing AKI. However, the role of circulating FGF-2 in the pathogenesis of AKI is not well understood. We have developed a mouse model to determine how FGF-2 released into the circulation modulates the outcome of AKI induced by lipopolysaccharide (LPS). Young FVB/N mice were infected with adenoviruses carrying a secreted form of human FGF-2 or control LacZ vectors. Subsequently, when the circulating levels of FGF-2 were similar to those seen in septic children, mice were injected with a non-lethal dose of LPS or control buffer. All mice injected with LPS developed hypotension and AKI, from which they recovered after 5 days. FGF-2 did not improve the outcome of AKI, and induced more significant renal proliferative and apoptotic changes during the recovery phase. These findings suggest that circulating FGF-2 may not necessarily prevent the development or improve the outcome of AKI. Moreover, the renal accumulation of FGF-2 might cause further renal damage.

Glomerular filtration rate abnormalities in sickle cell disease.

Sickle cell disease (SCD) is a group of inherited blood disorders affecting the ß-globin gene, resulting in the polymerization of hemoglobin and subsequent sickling of the red blood cell. Renal disease, the most common complication in SCD, begins in childhood with glomerular hyperfiltration and then progresses into albuminuria, a fast decline of glomerular filtration, and renal failure in adults. This mini-review focuses on glomerular filtration abnormalities and the mechanisms of hyperfiltration, explores genetic modifiers and methods of estimating glomerular filtration rates, and examines novel biomarkers of glomerular filtration in SCD.

Iron chelators of the di-2-pyridylketone thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series inhibit HIV-1 transcription: identification of novel cellular targets--iron, cyclin-dependent kinase (CDK) 2, and CDK9.

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670-7675, 2006; J Med Chem 50:3716-3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-κB-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs.

Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1.

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.

Circulating fibroblast growth factor-2 precipitates HIV nephropathy in mice.

People of African ancestry living with the human immunodeficiency virus-1 (HIV-1) are at risk of developing HIV-associated nephropathy (HIVAN). Children with HIVAN frequently show high plasma fibroblast growth factor-2 (FGF-2) levels; however, the role of circulating FGF-2 in the pathogenesis of childhood HIVAN is unclear. Here, we explored how circulating FGF-2 affected the outcome of HIVAN in young HIV-Tg26 mice. Briefly, we demonstrated that FGF-2 was preferentially recruited in the kidneys of mice without pre-existing kidney disease, precipitating HIVAN by activating phosphorylated extracellular signal-regulated kinase (pERK) in renal epithelial cells, without inducing the expression of HIV-1 genes. Wild-type mice injected with recombinant adenoviral FGF-2 (rAd-FGF-2) vectors carrying a secreted form of human FGF-2 developed transient and reversible HIVAN-like lesions, including proteinuria and glomerular enlargement. HIV-Tg26 mice injected with rAd-FGF-2 vectors developed more-significant proliferative and pro-fibrotic inflammatory lesions, similar to those seen in childhood HIVAN. These lesions were partially reversed by treating mice with the FGF/VEGF receptor tyrosine kinase inhibitor PD173074. These findings suggest that high plasma FGF-2 levels may be an independent risk factor for precipitating HIVAN in young children.

Macrophage HIV-1 Gene Expression and Delay Resolution of Inflammation in HIV-Tg Mice.

While antiretroviral therapy increases the longevity of people living with HIV (PLWH), about 30% of this population suffers from three or more concurrent comorbidities, whose mechanisms are not well understood. Chronic activation and dysfunction of the immune system could be one potential cause of these comorbidities. We recently demonstrated reduced macrophage infiltration and delayed resolution of inflammation in the lungs of HIV-transgenic mice. Additionally, trans-endothelial migration of HIV-positive macrophages was reduced in vitro. Here, we analyze macrophages' response to LPS challenge in the kidney and peritoneum of HIV-Tg mice. In contrast to the lung infiltration, renal and peritoneal macrophage infiltrations were similar in WT and HIV-Tg mice. Higher levels of HIV-1 gene expression were detected in lung macrophages compared to peritoneal macrophages. In peritoneal macrophages, HIV-1 gene expression was increased when they were cultured at 21% O2 compared to 5% O2, inversely correlating with reduced trans-endothelial migration at higher oxygen levels in vitro. The resolution of macrophage infiltration was reduced in both the lung and the peritoneal cavity of HIV-Tg mice. Taken together, our study described the organ-specific alteration of macrophage dynamics in HIV-Tg mice. The delayed resolution of macrophage infiltration might constitute a risk factor for the development of multiple comorbidities in PLWH.

HIV-1 Transcription Inhibitor 1E7-03 Restores LPS-Induced Alteration of Lung Leukocytes' Infiltration Dynamics and Resolves Inflammation in HIV Transgenic Mice.

Human immunodeficiency virus (HIV)-infected individuals treated with anti-retroviral therapy often develop chronic non-infectious lung disease. To determine the mechanism of HIV-1-associated lung disease we evaluated the dynamics of lung leukocytes in HIV-1 transgenic (Tg) mice with integrated HIV-1 provirus. In HIV-Tg mice, lipopolysacharide (LPS) induced significantly higher levels of neutrophil infiltration in the lungs compared to wild-type (WT) mice. In WT mice, the initial neutrophil infiltration was followed by macrophage infiltration and fast resolution of leukocytes infiltration. In HIV-Tg mice, resolution of lung infiltration by both neutrophils and macrophages was significantly delayed, with macrophages accumulating in the lumen of lung capillaries resulting in a 45% higher rate of mortality. Trans-endothelial migration of HIV-Tg macrophages was significantly reduced in vitro and this reduction correlated with lower HIV-1 gene expression. HIV-1 transcription inhibitor, 1E7-03, enhanced trans-endothelial migration of HIV-Tg macrophages in vitro, decreased lung neutrophil infiltration in vivo, and increased lung macrophage levels in HIV-Tg mice. Moreover, 1E7-03 reduced levels of inflammatory IL-6 cytokine, improved bleeding score and decreased lung injury. Together this indicates that inhibitors of HIV-1 transcription can correct abnormal dynamics of leukocyte infiltration in HIV-Tg, pointing to the utility of transcription inhibition in the treatment of HIV-1 associated chronic lung disease.