Sickle Cell Disease Nephropathy Research Articles

Time-restricted feeding does not improve nephropathy in humanized sickle cell disease mice

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a point mutation in the hemoglobin gene. This substitution results in a “sickling” of red blood cells and subsequent vascular and organ damage. Current treatments, while increasing the lifespan of sickle cell patients, has resulted in a higher prevalence of chronic complications like SCD nephropathy. Our group has previously shown that time-restricted feeding (TRF) in chronic high fat fed mice reduces kidney damage. Given the increased kidney damage in SCD, we hypothesize that TRF in humanized SCD mice will attenuate sickle cell nephropathy. To test this hypothesis, we studied 18-22-week-old male SCD mice. Mice underwent 3 weeks of TRF, only having access to food during the active, dark period (Zeitgeber Time (ZT) 12 to 24). The control group had access to food ad libitum (ad-lib). At the end of the 3 weeks, at 21-25 week old ages, 12 hour urine was collected in metabolic cages as well as kidneys and plasma collection at ZT3 (inactive period). Excretion of urinary kidney damage markers (albumin and NGAL-1) showed no difference between TRF and ad-lib in both the active and inactive collection periods. Urinary excretion of endothelin-1 is known to be upregulated in SCD patients; however, TRF did not change this marker in SCD mice. TRF did not change plasma levels of cytokines or soluble adhesion molecules. Histological analysis of kidneys was conducted as an additional marker of kidney damage. We analyzed iron levels in the kidney with Prussian blue staining as well as fibrosis with Masson’s trichrome. TRF did not change kidney iron accumulation or kidney fibrosis. Immunohistochemical staining for CD3+ T cells did not show a difference in glomerular T cell infiltration in the TRF group compared to the ad-lib group. In conclusion, TRF does not appear to have an effect on renal inflammation or kidney function in SCD mice, but future studies need to be completed. R01 DK134562. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nontargeted Plasma Proteomic Analysis of Renal Disease and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by red blood cell sickling, vaso-occlusion, hemolytic anemia, damage to multiple organ systems, and, as a result, shortened life expectancy. Sickle cell disease nephropathy (SCDN) and pulmonary hypertension (pHTN) are common and frequently co-occurring complications of SCD; both are associated with markedly accelerated mortality. To identify candidate circulating biomarkers of SCDN and pHTN, we used mass spectrometry to quantify the relative abundance of >1000 proteins in plasma samples from 189 adults with SCD from the Outcome Modifying Genes in SCD (OMG-SCD) cohort (ProteomeXchange identifier PXD048716). Forty-four proteins were differentially abundant in SCDN, most significantly cystatin-C and collagen α-1(XVIII) chain (COIA1), and 55 proteins were dysregulated in patients with SCDN and pHTN, most significantly insulin-like growth factor-binding protein 6 (IBP6). Network analysis identified a module of 133 coregulated proteins significantly associated with SCDN, that was enriched for extracellular matrix proteins, insulin-like growth factor binding proteins, cell adhesion proteins, EGF-like calcium binding proteins, and several cadherin family members. Collectively, these data provide a comprehensive understanding of plasma protein changes in SCDN and pHTN which validate numerous studies of chronic kidney disease and suggest shared profiles of protein disruption in kidney dysfunction and pHTN among SCD patients.

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Genome-wide meta-analysis identifies new candidate genes for sickle cell disease nephropathy.

Sickle cell disease nephropathy (SCDN), a common sickle cell disease (SCD) complication, is strongly associated with mortality. Polygenic risk scores calculated from recent trans-ethnic meta-analyses of urinary albumin-to-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) trended toward association with proteinuria and eGFR in SCD but the model fit was poor (R2<0.01), suggesting that there are likely unique genetic risk factors for SCDN. Therefore, we performed genome-wide association studies (GWAS) for two critical manifestations of SCDN, proteinuria and decreased eGFR, in two well-characterized adult SCD cohorts, representing the largest SCDN sample to date. Meta-analysis identified six genome-wide significant associations (false discovery rate [FDR] q≤0.05): three for proteinuria (CRYL1, VWF, ADAMTS7) and three for eGFR (LRP1B, linc02288, and FPGT-TNNI3K/TNNI3K). These associations are independent of APOL1 risk and represent novel SCDN loci, many with evidence for regulatory function. Moreover, GWAS SNPs in CRYL1, VWF, ADAMTS7, and linc02288 are also associated with gene expression in kidney and pathways important to both renal function and SCD biology, supporting the hypothesis that SCDN pathophysiology is distinct from other forms of kidney disease. Together, these findings provide new targets for functional follow-up that could be tested prospectively and potentially used to identify SCD patients at risk prior to kidney dysfunction onset.

Emerging Therapies and Advances in Sickle Cell Disease with a Focus on Renal Manifestations.

The underlying mechanisms of disease in sickle cell disease (SCD) contribute to a multifaceted nephropathy, commonly manifested as albuminuria. In severe SCD genotypes ( e.g. , Hemoglobin SS [HbSS]), albuminuria and CKD are major predictors of mortality in this population. Therefore, the monitoring and management of renal function is an intrinsic part of comprehensive care in SCD. Management of nephropathy in SCD can be accomplished with SCD-directed therapies and/or CKD-directed therapies. In the past 5 years, novel disease-modifying and palliative therapies have been approved in SCD to target aspects of the disease, such as anemia, inflammation, and vasculopathy. Along with conventional hydroxyurea and chronic transfusion, l -glutamine, crizanlizumab, and voxelotor have all been shown to mitigate some adverse effect of SCD, and their effect on nephropathy is being investigated. CKD-directed therapies such as renin-angiotensin-aldosterone system blockers have long been used in SCD nephropathy; however, more complete long-term studies on benefits are needed. Given the effect of renal disease on survival, further assessment of the mechanisms and efficacy of these SCD-directed or CKD-directed therapeutic agents is essential.

Natural Course of Albuminuria in Pediatric and Murine Sickle Cell Disease

Sickle cell disease (SCD), an inherited blood disorder, is associated with prevalent chronic kidney disease (CKD), evidenced by persistent albuminuria. It is critical to understand the natural history of albuminuria and albumin to creatinine (ACR) ratio variance to optimally identify, diagnose and treat SCD pediatrics prior to CKD. Thus, we investigated natural course of albuminuria in pediatrics and murine SCD, and identify ACR level predictive of persistent albuminuria.Basic science study: As hyperfiltration occurs at 12 weeks of age in male and 20 weeks of age in female humanized sickle cell (HbSS) mice, we performed metabolic cage studies in 1-week intervals for 4 weeks before (4 weeks old mice, n=21) and after hyperfiltration phase (males: 16 weeks (n=10), females: 20 weeks old (n=8)), and measured albuminuria using GenWay Biotech ELISA kit. Clinical study: We performed a retrospective study of the UAB Pediatric Sickle Cell Kidney Cohort. We identified all participants with HbSS and HbSB0 thalassemia ≥ 5 years of age with ACR measurements performed at a steady-state outpatient clinic visit. We identified the date and age of participants with at least 1 ACR level ≥100mg/g to compare outcomes for albuminuria and to determine the natural course of ACR after an ACR measurement &gt;100mg/g.We observed a significant difference in the trajectory of albuminuria over time prior and post hyperfiltration (p=0.02) in HbSS mice. Young HbSS mice presented with downward trend over time, with average values of albuminuria 47.6±27.2 (week 1), 32.6±20.3 (week 2), 32.0±24.3 (week 3), 31.3±16.1mg/24h (week 4), respectively. After hyperfiltration, older HbSS mice had an upward trend in albuminuria over time (week 1: 49.7±23.6, week 2: 74.5±66.9, week 3: 55.9±82.2, week 4: 73.0±86.4 mg/24h, respectively). Finally, prior to hyperfiltration the average albuminuria over a time of 4 weeks was 35.9±7.8 mg/24h (±21.7%), while following hyperfiltration it was 63.3±12.4 mg/24h (±19.6%). These findings mirror human data in which pediatric patients present with similar degree of natural variability in ACR. We identified 62 patients (17%) with persistent albuminuria, 46 (13%) with intermittent albuminuria, and 247 (70%) with not yet having albuminuria. Out of 107 patients with at least 1 abnormal ACR, 45 had 1 measurement &gt;100mg/g, and 43 of these participants were categorized with persistent albuminuria (50x higher odds ratio than patients with ACR&lt;100mg/g). Assessment of natural history of albuminuria after an initial ACR &gt;100mg/g showed significant variability. Regarding 1-year clinical trial design, the change in ACR showed a decline from 175 to 131 mg/g, accounting for 23% decline in albuminuria without any intervention.The natural course of albuminuria is an important characteristic of renal phenotype in SCD and should be considered while designing and assessing effectiveness of novel therapeutic approaches in preclinical and clinical studies targeting SCD nephropathy. Funded by NIH R25 DK 112731 and NHLBI–R00HL144817 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RNA-Sequencing of Isolated Cell Populations Expressing Human APOL1 Risk Variants Reveals Molecular Correlates of Sickle Cell Nephropathy in Zebrafish Podocytes

Background: Kidney failure occurs in 5-13% of individuals with sickle cell disease (SCD), and is associated with early mortality. We have shown previously that genetic variation in apolipoprotein L1 (APOL1) is associated with increased risk for developing SCD nephropathy (Ashley-Koch et al, 2011). Two APOL1 alleles (G1 and G2) have been identified as risk factors for SCD nephropathy, as well as chronic kidney disease (CKD) in African Americans without SCD (Limou et al, 2014). Both risk alleles are highly prevalent in individuals of African descent. Despite the strong association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased CKD risk remain poorly understood. However, the observation of podocyte effacement in CKD patients suggests that podocytes and/or endothelial cells are likely implicated in pathogenesis. We established zebrafish models of these human risk alleles previously for the purpose of better understanding the underlying pathology contributing to SCD nephropathy (Anderson et al, 2015). Here, we have utilized these models to explore further the cell-type specific transcriptomic perturbations contributing to nephropathy.Methods: To investigate the effects of the APOL1 G2 risk variant on cell-type specific transcriptomes, we injected human APOL1 mRNA into embryos from 3 different zebrafish lines expressing cell-type specific reporters to permit the isolation of podocytes (pod:NTR-mCherry), endothelial cells (fli1:egfp), or peripheral neurons (hb9:gfp; control). Three populations from each line were utilized: (1) uninjected controls; (2) embryos injected with canonical human APOL1 (G0); and (3) embryos injected with human APOL1 (G2) mRNA. Larvae were dissociated at 4 days post-fertilization (4 dpf), and fluorescent cell types were isolated via fluorescence activated cell-sorting (FACS). Messenger RNA was extracted from sorted cell populations (n=3 FACS sorted cell populations per condition) and subsequently amplified to obtain a sufficient amount of RNA for sequencing. The amplified RNA was prepared into cDNA libraries, and sequenced on an Illumina HiSeq 2500. We performed pairwise comparisons between uninjected and G0 injected larvae in each cell type to ascertain the effects of canonical human APOL1 on the zebrafish kidney transcriptome. Additionally, to measure the effects of APOL1 G2 expression on the transcriptome, we compared APOL1 G0 and APOL1 G2 samples. Transcripts were considered to be differentially expressed if they achieved an FDR-adjusted q-value of less than 0.05. To understand better the biological processes altered by injection condition, we conducted gene ontology enrichment analyses (GOEA) on sets of differentially expressed genes.Results: Our analysis of uninjected and APOL1 G0 libraries identified 38, 154, and 204 differentially expressed (DE) genes in endothelial cells, podocytes, and motor neurons, respectively. No significant enrichment of gene ontology terms was detected, suggesting that canonical G0 APOL1 did not have a substantial impact on the zebrafish transcriptome. In contrast, the comparison of APOL1 G0 and APOL1 G2 samples found 95, 7400 (Figure below), and 652 DE genes in endothelial cells, podocytes, and motor neurons, respectively. GOEA of the podocyte DE gene list showed enrichment for several autophagy-associated terms, including “Lysosome” and “Phagosome”. Close inspection of these autophagy-associated genes revealed that the majority are up-regulated in G2 relative to G0 (68/75 genes for “Lysosome” and 52/78 genes for “Phagosome”). Thus, the G2 allele appears to induce transcription of genes involved in autophagy in podocytes.Summary: Taken together, these data suggest that podocytes are the predominant cell type affected by the expression of APOL1 G2. The comparison of podocytes injected with APOL1 G0 or APOL1 G2 yielded an order of magnitude more DE transcripts than any other comparison. The GOEA results of the podocyte comparison are consistent with a recent study of transgenic mice expressing the G1 or G2 APOL1 high risk variants, which showed alterations in endocytic vesicle content and autophagic flux (Beckerman et al 2017). Ongoing work includes molecular analysis to characterize the effects of the APOL1 G1 allele. This work will continue to shed light on the molecular pathology of SCD nephropathy and provide insight into new therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Management of Renal Dysfunctions in Escort-HU Study

▪BACKGROUNDRenal impairment is a progressive chronic complication of sickle cell disease (SCD) that begins in childhood and may progress until renal failure. It is responsible for mortality in 12% of SS SCD adults (Tehseen S, 2017).Causes of renal damages in SCD patients are multiple: hemolysis, vaso-occlusive episodes which lead to renal injuries, papillary necrosis, medullary fibrosis and focal segmental glomerulosclerosis (Rees D, 2010).Hydroxycarbamide (hydroxyurea or HU) is a myelosuppressive drug marketed since 1968 for the treatment of hematological malignancies, and authorized since 2007 in Europe as an orphan medicinal product for the prevention of recurrent vaso-occlusive crises and acute chest syndrome in adults and children older than 2 years with SCD. HU also seems to protect kidney function in SCD by decreasing proteinuria (Bartolucci P, 2015).As renal excretion is a pathway of HU elimination (60% of dose excreted in urine), consideration should be given to decrease the dose of HU in patients with renal impairment.We propose to study SCD patients with renal diseases treated with hydroxycarbamide (Siklos) and enrolled in ESCORT-HU (European Sickle Cell Disease COhoRT - HydroxyUrea), European multicenter prospective non interventional study.METHODSFrom January 2009 to June 2017, 1841 patients were enrolled in the ESCORT-HU study. 1016 patients were adults and 824 were children (1 date of birth missing). Among them, renal lesions have been reported in the study database in 220 patients (194 adults, 25 children, 1 date of birth missing).At inclusion the renal status of patients are reported in the electronic Case Report Form (e-CRF) using different clinical approaches. It allows to distinguish patients with a medical history of renal impairment (i.e. patient with renal insufficiency of etiology not reported, with an irreversible and progressive reduction in renal function) from patients with a history of sickle cell (SC) nephropathy (patients with any renal alteration related to SCD and not necessarily irreversible and progressive).RESULTSCompared to the global cohort without any kidney involvement, patients with renal lesions were older (35.8 years vs 21.0 years) as expected, with less children (11.4% vs 50.2%) and mainly with SS genotype (94.5% vs 82.2%) (Table 1).64.1% of patients with renal lesions were already treated with HU other than Siklos before their enrollment in the study, in line with the fact that kidney lesions are correlated with the ageing of the SCD population.HU was initially prescribed at lower doses in SCD patients with renal disease and naïve of HU treatment as recommended. For patients already treated with HU other than Siklos (the dose at enrolment ~ maintenance dose), even if we observed an increase of the dose until maintenance dose, this subpopulation received HU at lower daily doses (Table 2).SCD Patients with kidney diseases reported more frequent and more severe adverse events not related to SCD (AENOTSCD) than patients without renal lesions (2.30 AE per patient vs 1.49 AE per patients and 15.4% of severe AE vs 11.2%) (Figure 1). However these events are less serious (15% of serious AE vs 25% in patients without renal lesions) (Figure 2). Similarly, when focusing on AENOTSCD causally related to HU (as judged by the investigators), patients with renal lesions reported more AE especially in patients with history of renal impairment.As recommended by some guidelines (Habibi 2015, Sharpe 2014), renin-angiotensin system (RAS) inhibitors may be used is SCD patients with renal damage in order to limit the kidney disease progression. In ESCORT-HU study, RAS inhibitors were used in 47.3% of patients with renal lesions whereas it was used in 3.0% of patients without renal lesions. Consistently RAS inhibitors are mainly used in patients with a history of SCD nephropathy (54.54% vs 21.15% in the population with renal impairment) probably because damages in this subpopulation were irreversible (Table 3).CONCLUSION12% of SCD patients, mainly adults, enrolled in the ESCORT-HU study had renal dysfunctions at enrolment. As expected this subpopulation is older than the global population of the cohort. Although HU is prescribed at lower doses, these patients go on to have more adverse events and this justifies a close monitoring of them. Controlled trials are required to evaluate the role of HU on the renal failure. [Display omitted] DisclosuresBartolucci:Novartis US: Membership on an entity's Board of Directors or advisory committees; Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding. Ribeil:Addmedica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lobitz:Addmedica: Membership on an entity's Board of Directors or advisory committees. Galacteros:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Engulfment and cell motility 1 (ELMO1) and apolipoprotein A1 (APOA1) as candidate genes for sickle cell nephropathy.

Sickle cell disease (SCD) and apolipoprotein L1 (APOL1) G1/G2 variants increase chronic kidney disease (CKD) risk in African Americans by poorly understood mechanisms. We applied bioinformatics to identify new candidate genes associated with SCD-related CKD. An interaction network demonstrated APOA1 connecting haemoglobin subunit β (HBB) and APOL1 with 36 other candidate genes. Gene expression revealed upregulation of engulfment and cell motility 1 (ELMO1) and downregulation of APOA1 in the kidney cortex of SCD versus non-SCD mice. Analysis of candidate genes identified ELMO1 rs10951509 to be associated with albuminuria and APOA1 rs11216132 with haemoglobinuria in patients with SCD. A bioinformatic approach highlights ELMO1 and APOA1 as potentially associated with SCD nephropathy.

Nocturnal Hypertension Associated with Stroke and Silent Cerebral Infarcts in Children with Sickle Cell Disease

Background: Strokes and silent cerebral infarcts (SCIs) lead to significant morbidity and mortality in children with sickle cell disease (SCD). Discovering modifiable risk factors is paramount to decreasing the incidence and progression of these devastating cerebrovascular outcomes. Higher systolic blood pressures have previously been shown to increase risk for stroke and SCIs however patients with SCD often have lower clinic blood pressures than the general population. 24 hour ambulatory blood pressure monitoring (ABPM) allows for more robust examination of blood pressure values. Previous studies have investigated associations with abnormal ABPM parameters and end-organ dysfunction, mainly SCD nephropathy. Associations with cerebrovascular events have not yet been investigated. This study aimed to determine if there is an association between blood pressure abnormalities on ABPM with stroke and silent cerebral infarcts. Methods: A cross-sectional study was performed. Children with SCD were enrolled and completed a 24-hour ABPM. Of this cohort, children with HbSS or HbSβ0 with a documented MRI brain within a year of the ABPM were included in the analysis. SCIs were defined as discrete, infarct-like T2 hyperintense lesions measuring at least 3mm in diameter on two orthogonal imaging sequences. Bivariate analyses were performed to identify the association between ambulatory blood pressure parameters with cerebrovascular outcomes specifically SCI or history of stroke. Results: A final cohort of 42 children with a median age of 13 years (10, 17) was included in the analysis. Seven (17%) had a history of stroke, 7 (17%) had documented SCIs, and the remaining 28 (67%) had no history of stroke or SCIs. Masked hypertension was seen in 5% of subjects and nocturnal hypertension was seen in 25%. Abnormal overnight dipping noted in 85% of subjects. The presence of nocturnal hypertension was significantly higher in the SCI/stroke group (55% vs 12%, p = 0.01). Sensitivity analyses were performed to determine if blood pressure abnormalities continued to show an association with cerebrovascular findings when stroke patients were removed. Nocturnal hypertension remained significantly associated with the presence of SCIs (p = 0.006). The median systolic load (percentage of blood pressures above the 95th percentile on ABPM), while below the cutoff for hypertension in both cohorts, was higher in the stroke/SCI group (18.6) compared to the group without SCIs or stroke (6.1), but did not meet statistical significance (p = 0.07). Conclusions: This study reveals an association between nocturnal hypertension and a higher prevalence of SCI and stroke in children with SCD. Our findings also confirm the high prevalence of nocturnal hypertension and impairment of nocturnal dipping seen in this population. Adequately powered, prospective cohorts of ABPM in SCD patients are needed to further evaluate causation between nocturnal hypertension and cerebrovascular outcomes. Disclosures No relevant conflicts of interest to declare.

The Effect of the Soluble Guanylate Cyclase Stimulator Olinciguat on Renal Function and Biomarkers of Inflammation and Renal Injury in Sickle Cell Mice

Introduction: Nephropathy, one of the most common complications of sickle cell disease (SCD), shortens life expectancy and accounts for 16-18% of SCD-associated deaths. SCD nephropathy is caused by recurrent vaso-occlusions (Am. J. Hematol. 2014;89:907) and by hemolytic anemia, which result in chronic tissue hypoxia, inflammation, and ultimately organ damage (Haematologica. 2012;97(2):201; Pediatr. Nephrol. 2014;29(10):1997). Reduced bioavailability of the gasotransmitter nitric oxide (NO) is one of the consequences of red blood cell hemolysis in SCD. NO insufficiency dysregulates the NO-soluble guanylyl cyclase (sGC)- cyclic guanosine monophosphate (cGMP) signaling pathway, promoting vasoconstriction, inflammation, and assembly of hetero cellular aggregates on the vascular wall, which occlude venous microcirculation. Olinciguat, a stimulator of soluble guanylate cyclase, increases NO-induced synthesis of the second messenger cGMP. In preclinical models, olinciguat has been shown to induce vasorelaxation and elicit anti-inflammatory and reno-protective effects. In an SCD mouse model of vaso-occlusive crisis (VOC), olinciguat reduced leukocyte/endothelial cell interactions, improved blood flow, and increased survival time (EHA 2019, PS1521). Here we evaluated the effect of chronic olinciguat treatment on biomarkers of inflammation and renal injury in an SCD mouse model. Methods: 18-week-old Townes non-disease control (HbAA) and SCD mice (HbSS) were studied. SCD mice were twice daily orally administered with either vehicle or olinciguat (20 mg/kg/day) for 8 weeks. Animals were placed in metabolic cages at 8 weeks for 24-hour urine collection. At the end of the study, plasma and kidneys were collected for biomarker analysis. Results: Plasma concentrations of the inflammatory biomarkers serum amyloid P component (SAP), serum amyloid A (SAA), interleukin 6 (IL-6), and plasminogen activator inhibitor 1 (PAI-1) were higher in SCD mice compared to non-disease control mice. SCD mice treated with olinciguat had significantly (p&amp;lt;0.05) lower levels of SAP, SAA, IL-6, and PAI-1 as compared to SCD control mice. Plasma levels of soluble adhesion receptors were also higher in SCD mice than non-disease control mice which indicate endothelial activation. SCD mice that were treated with olinciguat had significantly lower levels of plasma sE-selectin and sP-selectin compared to SCD control mice. There was no effect of treatment on sICAM-1. Cystatin C, a plasma biomarker of renal function, was significantly higher in SCD mice as compared to non-disease control mice; while mice treated with olinciguat had significantly lower levels compared to SCD control mice. Compared to non-disease control mice, SCD mice had proteinuria and albuminuria along with increased urine output and water consumption. Urine protein and urine albumin were lower in mice treated with olinciguat yet these data did not reach statistical significance (p=0.053 and p=0.065, respectively). Urine excretion was significantly lower in SCD mice treated with olinciguat as compared to SCD control mice. Olinciguat-treated mice also consumed significantly less water than mice from the SCD control mice. As compared to healthy control mice, SCD mice had significantly lower urine osmolality indicating tubular dysfunction while olinciguat treatment had no effect on urine osmolality. Analysis of biomarkers of kidney injury demonstrated that, relative to healthy control mice, urinary excretion of NAG, NGAL and KIM-1 was significantly higher in SCD mice. SCD mice treated with olinciguat had significantly less urinary excretion of NAG and NGAL as compared to SCD control mice. KIM-1 was not affected by olinciguat treatment. Kidney weight was significantly increased in sickle cell mice as compared to healthy control mice. Olinciguat-treated SCD mice had significantly lower kidney weight than SCD mice from vehicle control group. Conclusions: In summary, treatment of SCD mice with the sGC stimulator olinciguat elicited lower levels of plasma biomarkers of inflammation and endothelial cell activation and biomarkers of kidney injury. Disclosures Tchernychev: Cyclerion Therapeutics: Employment, Equity Ownership. Liu:Cyclerion Therapeutics: Employment, Equity Ownership. Hall:Cyclerion Therapeutics: Employment, Equity Ownership. Graul:Cyclerion Therapeutics: Employment, Equity Ownership. Masferrer:Cyclerion Therapeutics: Equity Ownership.

Sickle cell disease nephropathy: an update on risk factors and potential biomarkers in pediatric patients.

One of the major chronic complications of sickle cell disease (SCD) is sickle cell nephropathy. The aim of this review is to discuss the pathophysiology, natural history, clinical manifestations, risk factors, biomarkers and therapeutic approaches for sickle cell nephropathy, focusing on studies with pediatric patients. The earliest manifestation of renal disease is an increase in the glomerular filtration rate. A finding that may also be observed in early childhood is microalbuminuria. Nephrin, KIM-1, VGFs, chemokines and renin-angiotensin system molecules have emerged as potential early markers of renal dysfunction in SCD. In regards to a therapeutic approach, renin-angiotensin system inhibitors and angiotensin receptor blockers seem to be effective for the control of albuminuria in adults with SCD, although new studies in children are needed. The precise moment to begin renoprotection in SCD patients who should be treated remains to be determined.

RNA sequencing of isolated cell populations expressing human APOL1 G2 risk variant reveals molecular correlates of sickle cell nephropathy in zebrafish podocytes

Kidney failure occurs in 5–13% of individuals with sickle cell disease and is associated with early mortality. Two APOL1 alleles (G1 and G2) have been identified as risk factors for sickle cell disease nephropathy. Both risk alleles are prevalent in individuals with recent African ancestry and have been associated with nephropathic complications in other diseases. Despite the association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased risk remain poorly understood. Previous work in zebrafish models suggest that the G2 risk allele functions as a dominant negative, whereas the G1 allele is a functional null. To understand better the cellular pathology attributed to APOL1 G2, we investigated the in vivo effects of the G2 risk variant on distinct cell types using RNA sequencing. We surveyed APOL1 G2 associated transcriptomic alterations in podocytes and vascular endothelial cells isolated from zebrafish larvae expressing cell-type specific reporters. Our analysis identified many transcripts (n = 7,523) showing differential expression between APOL1 G0 (human wild-type) and APOL1 G2 exposed podocytes. Conversely, relatively few transcripts (n = 107) were differentially expressed when comparing APOL1 G0 and APOL1 G2 exposed endothelial cells. Pathway analysis of differentially expressed transcripts in podocytes showed enrichment for autophagy associated terms such as “Lysosome” and “Phagosome”, implicating these pathways in APOL1 G2 associated kidney dysfunction. This work provides insight into the molecular pathology of APOL1 G2 nephropathy which may offer new therapeutic strategies for multiple disease contexts such as sickle cell nephropathy.

Hyperfiltration predicts long-term renal outcomes in humanized sickle cell mice

AbstractWe previously reported that humanized sickle cell (HbSS) mice develop spontaneous nephropathy, a major cause of morbidity and mortality in sickle cell disease (SCD). Because sex-dependent protective mechanisms in SCD have been reported, we examined the course of nephropathy in male and female HbSS mice to determine contributors and/or predictors of disease severity. In male HbSS mice, glomerular filtration rate was characterized by a rapid onset of hyperfiltration and subsequent progressive decline of renal function over 20 weeks. Early tubular injury presented with increased excretion of kidney injury marker 1 (KIM-1), progressive loss of tubular brush border, and interstitial fibrosis that preceded the onset of glomerular damage, suggesting a tubuloglomerular mechanism of kidney injury in these mice. Additionally, we observed a strong association between the magnitude of hyperfiltration and the degree of long-term kidney injury in male HbSS mice. Unlike males, female HbSS mice did not demonstrate a significant loss of renal function or severe kidney damage during the time course of the study. These results suggest that magnitude of hyperfiltration predicts the onset of chronic kidney damage in male HbSS mice, whereas protective mechanisms in female HbSS mice delay the onset of SCD nephropathy.

Glomerular hyperfiltration predicts the onset of chronic kidney disease in humanized sickle cell mice

Kidney disease occurs in 68% of adults with sickle cell disease (SCD), and accounts for 30% mortality. Although a high prevalence of hyperfiltration occurs in SCD, its role as a direct determinant of chronic kidney disease remains unknown. Further, while potential sex‐dependent protective mechanisms of chronic SCD complications have been reported, whether sex differences exist in SCD‐associated kidney disease is unknown. The aim of the study was to perform a longitudinal study to determine the course of renal phenotype and examine potential predictors of long‐term renal outcomes in humanized sickle cell mice (HbSS). Time course analysis of structural and functional markers of kidney injury were performed in 4‐week intervals for 24 weeks starting at 8 weeks of age in male and female HbSS and genetic controls (HbAA) mice. An early and rapid hyperfiltration occurring at 12 weeks of age in male HbSS vs. HbAA mice (GFR: 262±9 vs. 225±3 μl/min; p&lt;0.05) was followed by a progressive decline of renal function (GFR: 139±7 vs. 229±4 μl/min; p&lt;0.0001), and proteinuria (3.62±0.28 vs. 1.46±0.24 mg/24h; p&lt;0.0001). Unlike male, female HbSS mice had a diffuse hyperfiltration pattern with a peak at 20 weeks of age (GFR: 229±18 μl/min), followed by mild decline in kidney function (177±7 μl/min) and proteinuria (1.70±0.33 mg/24h) at 32 weeks of age. Following hyperfiltration male HbSS mice developed progressive glomerular damage presented with albuminuria (319±22 μg/24h) and glomerulopathy, including glomerular sclerosis, congestion, hypertrophy, basement membrane of Bowman's capsule thickening, and glomerular loss. Female HbSS mice manifested with mild albuminuria (51±9 μg/24h) and a minor degree of glomerular injury. Male HbSS mice had more severe podocyte damage subsequent to hyperfiltration when compared to age‐matched females. Interestingly, elevated levels of urinary kidney injury marker 1 (at 8 weeks of age), a gradual brush border loss, progressive extension of interstitial fibrosis and tubular iron deposition were observed in both sexes of HbSS mice throughout the study course, suggesting that early tubular dysfunction preceded the onset of glomerular damage in these mice. Moreover, female HbSS mice had significantly lower mRNA expression of hypoxia inducible factor 1α when compared to males. In male HbSS mice, hyperfiltration strongly correlated with proteinuria (R2=0.57, p&lt;0.0003), albuminuria (R2=0.4, p&lt;0.003), decreased GFR (R2=0.44, p&lt;0.004), and elevated plasma creatinine (R2=0.27, p&lt;0.03) at 32 weeks of age. Additionally, the magnitude of hyperfiltration greater than 60 μl/min was associated with the greater degree of kidney damage in male HbSS mice. Unlike male, female HbSS mice did not demonstrate with a significant loss of renal function nor severe kidney damage during the time course of the study. These data suggest that hyperfiltration predicts the development of nephropathy in male HbSS mice, while protective mechanisms existing in female HbSS mice, may delay the onset of SCD nephropathy.Support or Funding InformationSupport provided by an NIH‐funded U01 HL117684 to D.M.P, R00 HL127178 to J.S.S and ASN Ben J. Lipps Research Fellowship for M.K.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clinical and metabolomic risk factors associated with rapid renal function decline in sickle cell disease

Sickle cell disease (SCD) nephropathy and lower estimated glomerular filtration rate (eGFR) are risk factors for early mortality. Furthermore, rate of eGFR decline predicts progression to end-stage renal disease in many clinical settings. However, factors predicting renal function decline in SCD are poorly documented. Using clinical, laboratory, genetic, and metabolomic data, we evaluated predictors of renal function decline in a longitudinal cohort of 288 adults (mean age 33.0 years). In 193 subjects with 5-year follow-up data, mean rate of eGFR decline was 2.35 mL/min/1.73 m2 /year, nearly twice that of African American adults overall. Hyperfiltration was prevalent at baseline (61.1%), and 36.8% of subjects experienced rapid eGFR decline (≥3 mL/min/1.73 m2 /year). Severe Hb genotype; proteinuria; higher platelet and reticulocyte counts, and systolic BP; and lower Hb level and BMI were associated with rapid decline. A risk scoring system was created using these 7 variables and was highly predictive of rapid eGFR decline, with odds of rapid decline increasing 1.635-fold for every point increment (P < 0.0001). Rapid eGFR decline was also associated with higher organ system severity score and peak creatinine. Additionally, two metabolites (asymmetric dimethylarginine and quinolinic acid) were associated with rapid decline. Further investigation into longitudinal SCD nephropathy (SCDN) trajectory, early markers of SCDN, and tools for risk stratification should inform interventional studies targeted to slowing GFR decline and improving SCD outcomes.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Acute kidney injury during a pediatric sickle cell vaso-occlusive pain crisis.

Patients who develop sickle cell disease (SCD) nephropathy are at a high risk for mortality. The pathophysiology of vaso-occlusive pain crisis may contribute to acute kidney injury (AKI). Non-steroidal anti-inflammatory drugs, known inducers of AKI, are used to treat pain crises. Multiple gaps exist in the knowledge about the impact of AKI in SCD. We conducted a 2-year retrospective review of AKI events in patients admitted for vaso-occlusive crisis. AKI was defined by an increase of ≥0.3mg/dL or 50% in serum creatinine from baseline. Laboratory values and ketorolac administration by days and dose (mg/kg) were identified from hospital records. A generalized mixed effects model for binary outcomes evaluated AKI based on laboratory variables and ketorolac administration. A generalized mixed Poisson effects model analyzed the association of AKI with hospital length of stay. Thirty-three out of 197 admissions for vaso-occlusive pain crisis (17%) were associated with AKI. Fifty-two percent of the cases presented to the Emergency Room (ER) with AKI. Every one unit decrease in hemoglobin from baseline to admission increased the risk of AKI by 49%. Among patients who received ketorolac for pain, both total days and doses of ketorolac were associated with AKI. Finally, patients with pain and AKI required longer periods of hospitalization than patients without AKI. Acute kidney injury during sickle cell pain crisis is common and may be an important modifiable risk factor for developing chronic kidney disease (CKD). Further studies are needed to determine the impact of nephrotoxic medications on progressive SCD nephropathy.

GWAS Meta-Analysis of Glomerular Filtration Rate in Three Cohorts of Sickle Cell Disease Patients and In Vivo Functional Analysis Reveals Potential Nephropathy Candidate Genes

▪BACKGROUND: Sickle cell disease nephropathy (SCDN) is a common complication of sickle cell disease (SCD) associated with risk for early mortality (Platt et al., 1994; Elmariah et al, 2014). To identify potential genetic risk factors for SCDN, we performed genome-wide association studies (GWAS) for glomerular filtration rate (GFR) in three well-characterized SCD cohorts and performed in vivo functional analysis of one of the candidate genes in zebrafish.METHODS: Three previously described SCD cohorts were utilized in this analysis: Outcome Modifying Genes in Sickle Cell Disease (OMG-SCD) (Elmariah et al, 2014), Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) (Machado et al, 2011) and Pulmonary Hypertension and the Hypoxic Response in Sickle Cell Disease (PUSH) (Minniti et al, 2009). Patients less than 16 years old were excluded from PUSH. GFR was estimated using the ’Modification of Diet in Renal Disease’ (MDRD) study definition (Levey et al, 1999) and, in the OMG-SCD and Walk-PHaSST cohorts, dichotomized at the clinically relevant threshold of 90 ml/min/1.73m2. 1064 patients with complete data were included in the analysis (Table 1). Genotyping was performed using the Illumina Human610-Quad BeadChip (Illumina, San Diego, CA). Linear regression was utilized to test for association between each SNP and GFR, controlling for genome-wide principle components using PLINK (Purcell et al, 2008). Logistic regression was utilized for the analysis of GFR<90. A total of 479,389 SNPs in common across all three cohorts were tested in a meta-analysis using METAL (Willer et al, 2010). False discovery rate (FDR) p-values were generated using PROC MULTTEST in SAS (SAS Systems, Cary, NC). To target and functionally evaluate xylt1 in vivo, a morpholino (MO) was injected into zebrafish embryos at the 1-4 cell stage. A wild-type (WT) XYLT1 ORF clone (GenBank: BC156039) was transferred to the pCS2+ vector, and used as template to transcribe capped mRNA. XYLT1 mRNA was co-injected with xylt1-MO to rescue the phenotype and demonstrate assay specificity. Proteinuria was assessed in zebrafish models by quantification of GFP leakage as described (Zhou and Hildebrandt, 2012). Larva were scored at 5 days-post-fertilization (dpf) for cardiac or yolk-sac edema.RESULTS: For the meta-analysis of continuous GFR, the SNP with the most evidence for association was rs7553158 in the TNNI3K-FPGT locus (p= 7.1E-7). For the meta-analysis of GFR<90, the most significant association within a gene was rs7201659 in XYLT1 (p=1.7E-5). None of these findings met FDR significance. Nonetheless, knockdown of xylt1 in zebrafish embryos resulted in pericardial edema, a phenotype associated previously with glomerular filtration defects, which was confirmed by the detection of GFP leakage of an albumin equivalent protein in the surrounding media. Importantly, complementation of xylt1 morphants with WT human XYLT1 mRNA rescued significantly these filtration defects in developing embryos.DISCUSSION:The absence of mechanistic knowledge about SCDN has led to limited therapeutic options for patients. The present study seeks to identify genetic risk factors for SCDN and represents the largest GWAS of GFR in SCD patients to date. The GWAS was likely still underpowered, however, as none of the findings met FDR significance. Nonetheless, the most significant genes identified are excellent biologic candidates for kidney function. Fucose-1-Phosphate Guanylyltransferase (FPGT) converts GTP and β-l-fucose-1-phosphate to GDP-l-fucose, a process essential in the kidney to reutilize L-fucose from the turnover of glycolipids. It is also highly expressed in the porcine and human kidney (Pastuszak et al, 1998; GTEx, Broad Institute). Xylosyltransferase 1 (XYLT1, also known as XT-1) encodes the enzyme responsible for biosynthesis of heparin sulfate proteoglycans, which affect permeability of the glomerular basement membrane. Furthermore, the c.343G>T polymorphism in XYLT1 has been implicated in diabetic nephropathy (Schön et al, 2006). Here, we have identified putative novel nephropathy loci associated with kidney decline in the largest combined cohort of SCD patients to date. These results further our understanding of the underlying pathology of SCDN and have uncovered genetic markers that could be used for identification of at-risk SCD patients prior to the onset of kidney dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factors Related to the Progression of Sickle Cell Disease Nephropathy

BACKGROUNDAs life expectancy in sickle cell disease (SCD) has continued to increase, chronic diseases are playing a bigger role in morbidity and mortality in this population. SCD nephropathy is a known risk factor for early mortality, and risk of death increases with worsening glomerular filtration rate (GFR) (Elmariah et al. 2014; Platt et al. 1994). Glomerular ischemia, hyperfiltration, and hyposthenuria characterize the renal dysfunction found in SCD, but the natural history of and risk factors contributing to progression of disease remain unclear. Potential markers of SCD nephropathy, such as anemia severity, hemolytic rate, blood pressure (BP), and albuminuria, have been studied with varied conclusions (Aoki and Saad 1995; Asnani et al. 2016; Elmariah et al. 2014; Falk et al. 1992; Guasch et al. 2006; Gurkan et al. 2010). Our recent work also identified two variant alleles (“G1” and “G2”) of the APOL1 gene as risk factors for SCD nephropathy (Ashley-Koch et al. 2011). We sought to evaluate clinical, laboratory, and genetic predictors of SCD nephropathy progression in a longitudinal SCD cohort. METHODSWe performed a chart review of 280 adults (age ≥18 at enrollment) with SCD followed regularly at the Duke University Medical Center and enrolled in a previously well-described study (Elmariah et al. 2014). All outpatient laboratory data were collected using the data extraction tool DEDUCE (Duke Enterprise Data Unified Content Explorer), and descriptive data were collected at time of enrollment and at most recent outpatient follow-up. APOL1 variants were genotyped as previously described (Ashley-Koch et al. 2011). Estimated GFR was calculated using both the extended MDRD and the CKD-EPI equations (Levey et al. 1999; Levey et al. 2009). CKD and hyperfiltration were defined as GFR <60 and >130 mL/min/1.73 m2, respectively. Statistical analysis was performed using SAS (SAS Systems, Cary, NC). RESULTSMean age at enrollment was 33.3 years, and mean baseline GFR was 148.6 mL/min/1.73 m2 by MDRD and 127 mL/min/1.73 m2 by CKD-EPI (see Table 1 for baseline characteristics). Results from the two equations correlated well for GFRs in the low to normal range but diverged for GFRs over ~150 mL/min/1.73 m2, with MDRD producing a wider range (Fig. 1). Given the high prevalence of hyperfiltration in our cohort, we chose to perform further analyses using CKD-EPI. Baseline GFR was associated with baseline proteinuria (p<0.0001), hemoglobinuria (p<0.0001), Hb level (p=0.03), platelet count (p<0.0001), reticulocyte count (p=0.03), bilirubin (p<0.0001), systolic BP (p=0.0003), diastolic BP (p<0.0001), and the APOL1 G1 allele (p=0.02). To characterize the pattern of progression of SCD nephropathy, we then identified 183 subjects with at least 5 years of follow-up and ≥3 outpatient GFR measurements in order to calculate a rate of GFR decline. There were no statistically significant differences in baseline characteristics for the two cohorts (Table 1). The mean rate of GFR decline was 2.5 ± 5.6 mL/min/1.73 m2 per year, higher than the 1.27 mL/min/1.73 m2 estimated annual GFR decline in African Americans in the general population (Young et al. 2016).We then defined a composite outcome of SCD nephropathy as either rapid rate of GFR decline (>3 mL/min/1.73 m2 per year, (Peralta et al. 2013; Young et al. 2016)), or incident CKD during the follow-up period. Seventy-three subjects (40%) developed nephropathy as thus defined. Using multivariable logistic regression modeling, with baseline GFR as a covariate, we found that lower baseline Hb (p=0.008), baseline proteinuria (p=0.01), higher reticulocyte count (p=0.03), and higher LDH (p=0.02) were all positive predictors of nephropathy. Baseline hemoglobinuria, white blood cell count, platelet count, bilirubin, BP, BMI, and APOL1 variants did not predict nephropathy. Subjects with hyperfiltration physiology at baseline also did not have a higher incidence of nephropathy over 5 years of follow up. DISCUSSIONOur results highlight the rapid rate of GFR decline and the high prevalence of hyperfiltration in adults with SCD, while not showing a correlation between the two. Proteinuria and markers of a higher hemolytic rate, among other measures, were most predictive of a rapid GFR decline. Additional investigation into the longitudinal trajectory of SCD nephropathy and potential interventions targeted to slowing the rate of GFR decline may improve outcomes in SCD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.