Potential Therapeutic Value of Urotensin II Receptor Antagonist in Chronic Kidney Disease and Associated Comorbidities.

Chronic kidney disease (CKD) remains a common disorder, leading to growing health and economic burden without curative treatment. In diabetic patients, CKD may result from a combination of metabolic and nonmetabolic-related factors, with mortality mainly driven by cardiovascular events. The marked overactivity of the urotensinergic system in diabetic patients implicates this vasoactive peptide as a possible contributor to the pathogenesis of renal as well as heart failure. Previous preclinical studies with urotensin II (UII) antagonists in chronic kidney disease were based on simple end points that did not reflect the complex etiology of the disease. Given this, our studies revisited the therapeutic value of UII antagonism in CKD and extensively characterized 1-({[6-{4-chloro-3-[3-(dimethylamino)propoxy]phenyl}-5-(2-methylphenyl)pyridin-2-yl]carbonyl}amino) cyclohexanecarboxylic acid hydrochloride (SAR101099), a potent, selective, and orally long-acting UII receptor competitive antagonist, inhibiting not only UII but also urotensin-related peptide activities. SR101099 treatment more than halved proteinurea and albumin/creatinine ratio in spontaneously hypertensive stroke-prone (SHR-SP) rats fed with salt/fat diet and Dahl-salt-sensitive rats, respectively, and it halved albuminuria in streptozotocin-induced diabetes rats. Importantly, these effects were accompanied by a decrease in mortality of 50% in SHR-SP and of 35% in the Dahl salt-sensitive rats. SAR101099 was also active on CKD-related cardiovascular pathologies and partly preserved contractile reserve in models of heart failure induced by myocardial infarction or ischemia/reperfusion in rats and pigs, respectively. SAR101099 exhibited a good safety/tolerability profile at all tested doses in clinical phase-I studies. Together, these data suggest that CKD patient selection considering comorbidities together with new stratification modalities should unveil the urotensin antagonists' therapeutic potential. SIGNIFICANCE STATEMENT: Chronic kidney disease (CKD) is a pathology with growing health and economic burden, without curative treatment. For years, the impact of urotensin II receptor (UT) antagonism to treat CKD may have been compromised by available tools or models to deeper characterize the urotensinergic system. New potent, selective, orally long-acting cross-species UT antagonist such as SAR101099 exerting reno- and cardioprotective effects could offer novel therapeutic opportunities. Its preclinical and clinical results suggest that UT antagonism remains an attractive target in CKD on top of current standard of care.

Interactive dependency curves for resilience management.

Physical dependencies are a fundamental consideration when assessing the resilience of an organisation and, ultimately, the resilience of a region. Every organisation needs specific resources for supporting its operations. A disruption in the supply of these resources can severely impact business continuity. It is important to characterise dependencies thoroughly when seeking to reduce the extent an organisation is directly affected by the missions, functions and operations of other organisations. The general protocol when addressing each critical resource is to determine the use for the resource, whether there are redundant services providing the resource, and what protections, backup equipment and arrangements are in place to maintain service. Finally, the criticality of the resource is determined by estimating the time it will take for the facility to experience a severe impact once primary service is lost and what percentage of facility operations can be maintained without backup service in place, as well as identifying whether any external regulations/policies are in place that require shutdown of the facility because of service disruption owing to lack of a critical resource. All of this information can be presented in the form of interactive dependency curves that help anticipate and manage the effect(s) of a disruption on critical resources supply.

5-Chlorothiophene-2-carboxylic acid [(S)-2-[2-methyl-3-(2-oxopyrrolidin-1-yl)benzenesulfonylamino]-3-(4-methylpiperazin-1-yl)-3-oxopropyl]amide (SAR107375), a selective and potent orally active dual thrombin and factor Xa inhibitor.

Compound 15 (SAR107375), a novel potent dual thrombin and factor Xa inhibitor resulted from a rational optimization process. Starting from compound 14, with low factor Xa and modest anti-thrombin inhibitory activities (IC50's of 3.5 and 0.39 µM, respectively), both activities were considerably improved, notably through the incorporation of a neutral chlorothiophene P1 fragment and tuning of P2 and P3-P4 fragments. Final optimization of metabolic stability with microsomes led to the identification of 15, which displays strong activity in vitro vs factor Xa and thrombin (with Ki's of 1 and 8 nM, respectively). In addition 15 presents good selectivity versus related serine proteases (roughly 300-fold), including trypsin (1000-fold), and is very active (0.39 µM) in the thrombin generation time (TGT) coagulation assay in human platelet rich plasma (PRP). Potent in vivo activity in a rat model of venous thrombosis following iv and, more importantly, po administration was also observed (ED50 of 0.07 and 2.8 mg/kg, respectively). Bleeding liability was reduced in the rat wire coil model, more relevant to arterial thrombosis, with 15 (blood loss increase of 2-fold relative to the ED80 value) compared to rivaroxaban 2 and dabigatran etexilate 1a.

Commitment to apoptosis in CD4(+) T lymphocytes productively infected with human immunodeficiency virus type 1 is initiated by lysosomal membrane permeabilization, itself induced by the isolated expression of the viral protein Nef.

Primary CD4(+) T lymphocytes, supporting in vitro human immunodeficiency virus type 1 (HIV-1) replication, are destined to die by apoptosis. We explored the initial molecular events that act upstream from mitochondrial dysfunction in CD4(+) T lymphocytes exposed to the HIV-1(LAI) strain. We tracked by immunofluorescence the cells expressing the p24 viral antigen and used Percoll density gradients to isolate a nonapoptotic CD4(+) T-cell subset with a high inner mitochondrial transmembrane potential (DeltaPsim) but no outer mitochondrial membrane (OMM) rupture. In most p24(+) (but not bystander p24(-)) cells of this subset, the lysosomes were undergoing limited membrane permeabilization, allowing the lysosomal efflux of cathepsins (Cat) to the cytosol. This was also induced by HIV-1 isolates from infected patients. Using pepstatin A to inhibit Cat-D enzymatic activity and Cat-D small interfering RNA to silence the Cat-D gene, we demonstrate that once released into the cytosol, Cat-D induces the conformational change of Bax and its insertion into the OMM. Inhibition of Cat-D activity/expression also conferred a transient survival advantage upon productively HIV-1-infected cells, indicating that Cat-D is an early death factor. The transfection of activated CD4(+) T lymphocytes with a Nef expression vector rapidly induced the permeabilization of lysosomes and the release of Cat-D, with these two events preceding OMM rupture. These results reveal a previously undocumented mechanism in which Nef acts as an internal cytopathic factor and strongly suggest that this viral protein may behave similarly in the context of productive HIV-1 infection in CD4(+) T lymphocytes.

Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase.

Poliovirus (PV) is the causal agent of paralytic poliomyelitis, a disease that involves the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through an apoptotic process. However, mechanisms by which PV induces cell death in neuronal cells remain unclear. Here, we demonstrate that PV infection of neuronal IMR5 cells induces cytochrome c release from mitochondria and loss of mitochondrial transmembrane potential, both of which are evidence of mitochondrial outer membrane permeabilization. PV infection also activates Bax, a proapoptotic member of the Bcl-2 family; this activation involves its conformational change and its redistribution from the cytosol to mitochondria. Neutralization of Bax by vMIA protein expression prevents cytochrome c release, consistent with a contribution of PV-induced Bax activation to mitochondrial outer membrane permeabilization. Interestingly, we also found that c-Jun NH(2)-terminal kinase (JNK) is activated soon after PV infection and that the PV-cell receptor interaction alone is sufficient to induce JNK activation. Moreover, the pharmacological inhibition of JNK by SP600125 inhibits Bax activation and cytochrome c release. This is, to our knowledge, the first demonstration of JNK-mediated Bax-dependent apoptosis in PV-infected cells. Our findings contribute to our understanding of poliomyelitis pathogenesis at the cellular level.

Death of CD4+ T cells from lymph nodes during primary SIVmac251 infection predicts the rate of AIDS progression.

Immunological and virological events that occur during the earliest stages of SIV infection are now considered to have a major impact on subsequent disease progression. In the present study, we demonstrate a clear correlation between progression to AIDS and the rate of in vitro CD4+ (but not CD8+) T cell death in lymph nodes. The dying CD4+ T cells were effector memory T cells, which are critical for the immune response to pathogens. However, there was no correlation between the rate of the viral replication within lymph nodes and the extent of Fas ligand-mediated death, despite the increased sensitivity of CD4+ T cells to death in response to recombinant human Fas ligand. CD4+ T cell death was caspase and apoptosis-inducing factor independent but was clearly associated with mitochondrion damage. Interestingly, higher expression levels of the active form of Bak, a proapoptotic molecule involved in mitochondrial membrane permeabilization, were observed in SIV-infected macaques progressing more rapidly to AIDS. Finally, we demonstrated that the strain of SIV we used requires CCR5 and BOB/GRP15 molecules as coreceptors and caused death of unstimulated noncycling primary CD4+ T cells. Altogether, these results demonstrate that CD4+ T cell death occurring early after SIV infection is a crucial determinant of progression to AIDS and that it is mediated by the intrinsic death pathway.

CD8+ T cell dynamics during primary simian immunodeficiency virus infection in macaques: relationship of effector cell differentiation with the extent of viral replication.

Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8(bright) T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8(bright) T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67(+)) CD8(bright) T cells, and with the pattern of CD8(bright) T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251Deltanef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8(bright) T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8(+) T cells, these changes are associated with a poor prognosis for disease progression to AIDS.

Mitochondria are sensors for HIV drugs.

Highly active anti-retroviral therapy (HAART) has drastically altered the course of HIV-1 infection, resulting in a major decrease in morbidity and mortality. However, adverse drug reactions and long-term toxicities associated with HAART are now a concern. A major toxicity that has been highlighted by the increased use of HAART is related to mitochondrial side-effects. At the same time, analysis of the biochemical pathways involved in programmed cell death has revealed that mitochondria are main sensors in this process. In this article, the regulation of mitochondrial damage following the use of nucleoside analogue reverse transcriptase inhibitors (NRTIs) and protease inhibitors is discussed, with a particular focus on the putative molecular mechanisms involved.

Interleukin 7 increases human immunodeficiency virus type 1 LAI-mediated Fas-induced T-cell death.

Fas-mediated T-cell death is known to occur during human immunodeficiency virus (HIV) infection. In this study, we found that HIV type 1 LAI (HIV-1(LAI)) primes CD8(+) T cells from healthy donors for apoptosis, which occurs after Fas ligation. This effect is counteracted by a broad caspase inhibitor (zVAD-fmk). Fas-mediated cell death does not depend on CD8(+) T-cell infection, because it occurred in the presence of reverse transcriptase inhibitors. However, purified CD8(+) T cells are sensitive to Fas only in the presence of soluble CD4. Finally, we found that interleukin 7 (IL-7) increases Fas-mediated CD4(+) and CD8(+) T-cell death induced by HIV-1(LAI). Since high levels of IL-7 are a marker of poor prognosis during HIV infection, our data suggest that enhancement of Fas-mediated T-cell death by HIV-1(LAI) and IL-7 is one of the mechanisms involved in progression to AIDS.

The density of coreceptors at the surface of CD4+ T cells contributes to the extent of human immunodeficiency virus type 1 viral replication-mediated T cell death.

Chemokine receptors serve as coreceptors for HIV-1 entry into CD4(+) T cells. Several reports have mentioned that density of CCR5 expression modulates in vitro viral replication and in vivo the course of the disease. Our goal was to investigate the impact of coreceptor density at the surface of a CD4(+) cell line on HIV-1 entry, replication, spreading, and programmed cell death. We engineered a CEM cell line that expresses constitutively CD4 and CXCR4 and CCR5 after transfection. This model allows us to compare the effect of the X4 and R5 strains to induce T cell death in the same T cell host. We show here that the extent of T cell death correlates with the rate of virus replication. X4 induces faster T cell death than R5 that depends at least in part on the higher density of CXCR4 compared to CCR5. Furthermore, sorting CEM populations expressing low, intermediate, and high densities of CCR5 molecules but constant amount of CD4, we found that the capacity to induce T cell death depends at least in part on the level of CCR5 when low amount of virus was used to infect the CEM cells. Moreover, viral transcription, assessed by cell-associated HIV-1 RNA/DNA ratio, was increased in CCR5high as compared to CCR5low cells, while inhibition of replication by zidovudine was more effective in CCR5low cells. Our data indicate that the density of chemokine receptors expressed on CD4(+) T cells may be a critical parameters for the cytopathic effect of HIV strains and may have major impact on CD4 T cell depletion during HAART.

HIV-1 triggers mitochondrion death.

Apoptosis, a phenotype of programmed cell death involved in development and tissue homeostasis of multicellular organisms, brings into two major pathways and implies a central sensor: the mitochondria. Abnormalities in the cell death control can lead to a variety of diseases and many pathogenic agents target the mitochondria, especially affecting its permeability in order to induce cell death. HIV infection is linked to progressive CD4 T cell depletion. Among the different hypothesis that may explain T cell depletion, apoptosis is one of the main described mechanisms. This review provides current knowledge in HIV-mediated mitochondrial damage due to (i) HIV-specific proteins, (ii) death-by-neglect and (iii) side effects of the HIV drugs.

Intrinsic and extrinsic pathways signaling during HIV-1 mediated cell death.

Infection with human immunodeficiency virus (HIV) is characterized by the gradual depletion of CD4+ T lymphocytes. The incorporation of the concept of apoptosis as a rationale to explain progressive T cell depletion has led to growing research in this field during the last 10 years. In parallel, the biochemical pathways implicated in programmed cell death have been extensively studied. Thus, the influence of mitochondrial control in the two major apoptotic pathways-the extrinsic and intrinsic pathways-is now well admitted. In this review, we summarized our current knowledge of the different pathways involved in the death of T cells in the course of HIV infection.

Mitochondria in HIV-1-induced apoptosis.

It is now well admitted that HIV infection leading to AIDS is associated with an abnormal susceptibility of T cells to undergo apoptosis. Recent progress in research into programmed cell death has resulted in the identification of the principal pathways involved in this process. Thus the "extrinsic" as well as the "intrinsic" pathways converge to the mitochondria considered as the main sensor of programmed cell death. This review summarizes our knowledge of the influence of mitochondrial control on T cell death during HIV and SIV infections.

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and gamma-irradiation.

The murine IL-3-dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin-3 (IL-3), a process that is suppressed or delayed by the constitutive expression of Bcl-2 or the Bcl-2-related Bcl-xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL-3 deprival-induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl-2 protein. We observed that E1B 55K, as Bcl-2, delays the cell death caused by either IL-3-deprivation or DNA damage induced by gamma-irradiation. Cell-cycle analysis after IL-3 deprivation indicated that surviving Bcl-2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD-fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL-3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl-2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl-serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL-3 deprivation.

Effects of antiretroviral drugs on human immunodeficiency virus type 1-induced CD4(+) T-cell death.

Apoptosis of peripheral blood T cells plays an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. In this study, we found that HIV type 1 (HIV-1) primes CD4(+) T cells from healthy donors for apoptosis, which occurs after CD95 ligation or CD3-T-cell receptor (TCR) stimulation. CD95-mediated death did not depend on CD4 T-cell infection, since it occurred in the presence of the reverse transcriptase inhibitor didanosine (ddI). In contrast, apoptosis induced by productive infection (CD3-TCR stimulation) is prevented by both CD95 decoy receptor and ddI. Our data suggest that HIV-1 triggers at least two distinct death pathways: a CD95-dependent pathway that does not require viral replication and a viral replication-mediated cell death independent of the CD95 pathway. Further experiments indicated that saquinavir, a protease inhibitor, at a 0.2 microM concentration, decreased HIV-mediated CD95 expression and thus cell death, which is independent of its role in inhibiting viral replication. However, treatment of peripheral blood mononuclear cells from healthy donors with a higher concentration (10 microM) of an HIV protease inhibitor, saquinavir or indinavir, induced both a loss in mitochondrial membrane potential (DeltaPsim) and cell death. Thus, protease inhibitors have the potential for both beneficial and detrimental effects on CD4(+) T cells independent of their antiretroviral effects.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.