*2.2.2. Endothelin-A receptor antagonismo retards the progression of sickle cell nephopathy*

Endothelin-1 (ET-1) is a signaling peptide produced by diverse cell types that exerts its physiologic and pathophysiologic actions by binding to two receptor subtypes, ETA and ETB. ETA receptor activation induces vasoconstriction, inflammation and nociception which is abolished by ETB activation in some tissues. ETA receptor signaling produces oxidant stress and the release of cytokines such as NF-kB activate and promote the production of ET-1, the agonist for the ETA receptor. A number of studies have reported increased production of ET-1 in SCD thus promoting sickling and tissue injury. Kasztan et al. reported that by blocking ETA receptor, the progression of SCN in a murine model of SCD was abolished [21]. The introduction of sickle RBCs into murine endothelial cells induces ET-1, leading to ETA-dependent vasoconstriction [22, 23]. It has also been shown that plasma ET-1 levels are elevated in patients with SCD during steady state periods as well as during acute vaso-occlusive crisis. Conversely, plasma ET-1 levels are decreased in SCD patients treated with hydroxyurea [24]. Elevated ET-1 level in SCD is associated with endothelial dysfunction and albuminuria in patients with SCD [25]. Bosentan, a dual ETA/ETB receptor antagonist used in murine models, decreases hypoxia-related injury to renal vessels and lung inflammation [26]. As it has been shown by Kasztan' s studies, administration of ambrisentan (an ETA receptor antagonist), at the time of weaning and continued for 10 weeks, prevented glomerular dysfunction, tubulointerstitial inflammation and fibrosis [24]. The observations of Kasztan et al. support this speculation as the markedly elevated plasma ET-1 levels that occur in this model are normalized by chronic administration of ambrisentan [21]. Long-term administration of ambrisentan significantly reduced the degree of iron deposit in renal tubules. The reduction in tubular iron deposits suggests that ambrisentan reduces hemolysis in murine models. Free heme is a well-known promoter of oxidative stress and the generation of proinflammatory species, for example, ROS. Free heme also stimulates the production of placenta growth factor (PlGF), an angiogenic growth factor that is implicated in the pathogenesis of tissue injury in SCD as well as the production of ET-1 [27–29]. NO is a suppressor of ET-1 synthesis and vascular ET-1 production may also increase when the vascular system is depleted due to NO binding to HbS plasma. ET-1 causes RBC dehydration by activating the Gardos channel present in the plasma membrane of RBCs [30, 31].

### *2.2.3. Mouse models*

in the patients with FSGS-type lesions showed irregular staining for IgM and C3 in areas of sclerosis [15, 16, 18]. Complement deposition occur in the glomeruli coinciding with various

Hemolysis in SCD leads to release of arginase 1, asymmetric dimethylarginine and adenine nucleotides, these promote vasomotor dysfunction and proliferative vasculopathy. Circulating hemoglobin and heme both referred to as erythrocytic danger-associated molecular pattern (eDAMP) molecules activate endothelial inflammatory and angiogenesis. Hemolysis in SCD therefore leads to anemia, increased superoxide anion and reactive oxygen species (ROS) production and low ROS scavenging enzymes activity promote oxidative stress-induced vascular complications. Itokua et al. reported in their study [20] that albuminuria was associated with increased white blood cell (WBC) count and LDH enzyme levels. Oxidative damage may alter both the structure and the function of the glomerulus due to its effects on mesangial and endothelial cells. Activated circulating white blood cells and platelets express adhesion glycoproteins leading to endothelial cell adhesion molecules and endothelial dysfunction.

*2.2.2. Endothelin-A receptor antagonismo retards the progression of sickle cell nephopathy*

Endothelin-1 (ET-1) is a signaling peptide produced by diverse cell types that exerts its physiologic and pathophysiologic actions by binding to two receptor subtypes, ETA and ETB. ETA receptor activation induces vasoconstriction, inflammation and nociception which is abolished by ETB activation in some tissues. ETA receptor signaling produces oxidant stress and the release of cytokines such as NF-kB activate and promote the production of ET-1, the agonist for the ETA receptor. A number of studies have reported increased production of ET-1 in SCD thus promoting sickling and tissue injury. Kasztan et al. reported that by blocking ETA receptor, the progression of SCN in a murine model of SCD was abolished [21]. The introduction of sickle RBCs into murine endothelial cells induces ET-1, leading to ETA-dependent vasoconstriction [22, 23]. It has also been shown that plasma ET-1 levels are elevated in patients with SCD during steady state periods as well as during acute vaso-occlusive crisis. Conversely, plasma ET-1 levels are decreased in SCD patients treated with hydroxyurea [24]. Elevated ET-1 level in SCD is associated with endothelial dysfunction and albuminuria in patients with SCD [25]. Bosentan, a dual ETA/ETB receptor antagonist used in murine models, decreases hypoxia-related injury to renal vessels and lung inflammation [26]. As it has been shown by Kasztan' s studies, administration of ambrisentan (an ETA receptor antagonist), at the time of weaning and continued for 10 weeks, prevented glomerular dysfunction, tubulointerstitial inflammation and fibrosis [24]. The observations of Kasztan et al. support this speculation as the markedly elevated plasma ET-1 levels that occur in this model are normalized by chronic administration of ambrisentan [21]. Long-term administration of ambrisentan significantly reduced the degree of iron deposit in renal tubules. The reduction in tubular iron deposits suggests that ambrisentan reduces hemolysis in murine models. Free heme is a well-known promoter of oxidative stress and the generation of proinflammatory species, for example, ROS. Free heme also stimulates the production of placenta growth factor (PlGF), an angiogenic growth factor that is implicated in the

degrees of proteinuria including nephrotic syndrome [19].

**2.2. Risk factors for renal impairment in sickle cell disease**

*2.2.1. Hemolysis and vasculopathy*

158 Hematology - Latest Research and Clinical Advances

Mouse models provide opportunities to explore the mechanisms of globin gene regulation and the feasibility of gene therapy for this condition and the molecular basis of end-organ damage, including SCN [32]. The use of established mouse models is of invaluable help to investigate the pathogenesis of SCD-associated multiple organ complications and to identify targets for prevention and therapy.

Several murine models have been developed to mimic human SCD.Of these, the Berkeley model (BERK mice) has targeted deletions of murine α and β globins (α−/−, β−/−) with a transgene containing human α, β<sup>s</sup> , Aγ, Gγ and β globins (Hba0/0 Hbb0/0 TG (Hu-miniLCRα1GγAγδβS) (α−/−, β−/−, transgene +); thus, these mice almost exclusively express human sickle hemoglobin [33]. The BERK mouse model exhibits a wide spectrum of hematologic and histopathologic findings that are similar to those found in humans with SCD. Erythrocyte sickling is significant in BERK mice, and erythrocyte survival is very short resulting in massive amounts of heme being released into the plasma. As seen in humans with SCD, BERK mice showed a wide spectrum of kidney pathologies such as increased cortical hypertrophy, gross and microscopic infarcts, iron deposition, enlarged glomeruli associated with mesangial cell and mononuclear cell hypercellularity are observed in kidneys from BERK mice [34].

Another mouse model of SCD, the transgenic SAD mouse bears the human α-globin gene and the HbS mutation, β<sup>S</sup> , as well as βAntilles and βD−Punjab which greatly enhance the tendency of its hemoglobin to polymerize [35]. The SAD mice display renal hemosiderosis, microvascular occlusions, vascular thrombosis, cortical infarcts and papillary necrosis. Most mice show glomerular hypertrophy and mesangial sclerosis. The glomerular damage is associated with abnormal function, characterized by increased blood urea nitrogen levels and proteinuria [35]. The glomerular lesions of SAD mice faithfully mimic sickle cell glomerulosclerosis, the most severe renal complication observed in individuals with SCD. Therefore, the SAD mouse constitutes a valuable model to investigate the pathophysiology of the thrombotic and glomerulosclerotic complications of human SCD. Ischemic injury contributes to end-organ damage and other complications of SCD. Increased sensitivity of tissues in SCD to ischemic insults has been demonstrated in SCD mice. As it has been showed by Nath et al., after induction of bilateral renal ischemia, transgenic SCD mice exhibited massive vascular congestion, sickling of red blood cells and more prominent capillary congestion in the lungs and heart compared to control mice [36]. These results demonstrated increased susceptibility to vascular congestion and to ischemia in tissues from SCD mice, suggesting that ischemic episodes may contribute to the renal complications observed in SCD. Abnormal leukocyte-endothelium attachment associated with endothelial activation was observed in SCD mice, showing interesting parallels between the vascular injury after reperfusion and kidney damage. In addition, this study suggested that allopurinol, that prevents ischemia-reperfusion generation of reactive oxygen species, might be a potential therapy for SCD [37]. The anti-sickling property of fetal hemoglobin was also demonstrated in SCD mice [38]. Patients with SCD suffer from painful crises associated with vaso-occlusion. Increased circulating erythrocyte membrane microparticles (MPs) have been associated with occlusion of capillaries. Interestingly, MPs triggered immediate renal vaso-occlusion in mice. In vitro studies showed that MPs stimulate the production of reactive oxygen species by endothelial cells, stimulate RBC adhesion and induce endothelial apoptosis. This work introduced a novel concept that associates the shedding of MPs from sickled RBC with vascular disease [39]. An interaction of free heme with TLR4 receptor was shown to mediate the nephrotoxicity of heme, in particular, the effects of heme on renal blood flow and inflammatory responses [40].

*2.2.5. Proteinuria and chronic kidney disease*

*2.2.6. Chronic kidney disease and end-stage renal disease*

*2.2.7. Urinary concentration abnormalities*

HbF such as hydroxyurea and decitabine.

The prevalence of albuminuria in SCD is age dependent. It may be classified as moderately increased albuminuria (previous called microalbuminuria)—urine albumin concentration of 30–300 mg/g creatinine and severely increased albuminuria (macroalbuminuria)—urine albumin concentration of 300 mg/g creatinine. The prevalence of albuminuria in the first three decades of life is up to 27% increasing to 68% in older SCD patients [13]. The understanding of the evolution of CKD in SCD is evolving the extent to which moderate albuminuria progresses to severely increased albuminuria and the relationship with SCN. The development of SCN is likely due to complex interactions between SCD-related risk factors and non-SCD phenotype characteristics. Albuminuria is more likely to occur in patients who express specific single-nucleotide polymorphisms in the MYH9 and APOL1 genes, which are associated with an increased risk of CKD in African Americans [53]. On the other hand, microdeletions in the gene that encodes α-globin (reflecting a form of α-thalassemia trait) leads to a lower prevalence albuminuria [54]. Genetic polymorphisms of bone morphogenetic protein receptor 1B also influence GFR in SCD [55, 56]. SCD patients with albuminuria have increased levels of urinary excretion of markers of tubular injury (KIM-1 and NAG) [57]. The individual contribution of these phenomena to SCN is not clear.

Sickle Cell Nephropathy: Current Understanding of the Presentation, Diagnostic and…

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The reported prevalence of ESRD in SCD varies from 5 to 18% depending on the age of the cohort but reamins a significant cause of mortality [58, 59]. Similarly, CKD (defined based on eGFR) which is usually diagnosed between 30 and 40 years is also a risk factor death [47, 60]. In a recent study in Rio de Janeiro, Brazil, 4.3% of patients admitted with SCD had CKD [61]. A lower incidence was observed in a study from Senegal, where CKD was identified in 2.6% of 229 adults with SCD [62]. The manifestations of CKD in SCD include hypertension, proteinuria and anemia. Vaso-occlusive history, legs ulcers, osteonecrosis, retinopathy, proteinuria, hematuria, hypertension and severe anemia were all identified as predictive factors for CKD in SCD [58, 61, 63]. In a recent study from Nigeria, 50% of SCD patients with proteinuria had CKD [64]. Risk factors associated with progression of CKD to ESRD (**Table 1**) include increased blood pressure, low hemoglobin levels, haemolysis, leukocytosis, hematuria, prior vaso-occlusive crisis, the βS Central African Republic (CAR) haplotype, pulmonary hypertension, stroke, acute chest syndrome and infection with parvovirus B19 [65–78]. The mean survival of patients

with ESRD and SCD is estimated to be 4 years, even with dialytic treatment [79].

The onset of urinary concentration defects begins in early infancy (6–12 months) and may account for nocturia, polyuria and enuresis in later childhood. The defect in urinary concentration does not respond to vasopressin but it is reported to improve with chronic blood transfusions in young children [47, 63, 64, 80–83]. Further deterioration of the defect in urinary concentration is observed from the second decade of life due to the onset of medullary fibrosis and the loss of the collecting ducts system. High HBF levels are associated with better urinary concentration [84–86]. There may be a role for drugs therapy that enhance the production of
