Substitutions for Hydrophobic Amino Acids in the N-terminal Domains of IGFBP-3 and -5 Markedly Reduce IGF-I Binding and Alter Their Biologic Actions

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I

Associations with age and glomerular filtration rate in a referred population with chronic kidney disease: Methods and baseline data from a UK multicentre cohort study (NURTuRE-CKD)

BACKGROUND: Chronic kidney disease (CKD) is common but heterogenous and is associated with multiple adverse outcomes. The National Unified Renal Translational Research Enterprise (NURTuRE)-CKD cohort was established to investigate risk factors for clinically important outcomes in persons with CKD referred to secondary care. METHODS: Eligible participants with CKD stages G3-4 or stages G1-2 plus albuminuria > 30 mg/mmol were enrolled from 16 nephrology centres in England, Scotland and Wales from 2017 to 2019. Baseline assessment included demographic data, routine laboratory data and research samples. Clinical outcomes are being collected over 15 years by the UK Renal Registry using established data linkage. Baseline data are presented with subgroup analysis by age, sex and estimated GFR (eGFR). RESULTS: 2996 participants were enrolled. Median (interquartile range) age was 66 (54 to 74) years, 58.5% were male, eGFR 33.8 (24.0 to 46.6) ml/min/1.73m2 and UACR 209 (33 to 926) mg/g. 1883 participants (69.1%) were in high-risk CKD categories. Primary renal diagnosis was CKD of unknown cause in 32.3%, glomerular disease in 23.4% and diabetic kidney disease in 11.5%. Older participants and those with lower eGFR had higher systolic blood pressure and were less likely to be treated with renin-angiotensin system inhibitors (RASi) but were more likely to receive a statin. Female participants were less likely to receive a RASi or statin. CONCLUSIONS: NURTuRE-CKD is a prospective cohort of persons who are at relatively high risk of adverse outcomes. Long-term follow-up and a large biorepository create opportunities for research to improve risk prediction and investigate underlying mechanisms to inform new treatment development

National Unified Renal Translational Research Enterprise: Idiopathic Nephrotic Syndrome (NURTuRE-INS) study

BackgroundIdiopathic Nephrotic Syndrome (INS) is a heterogenous disease and current classification is based on observational responses to therapies or kidney histology. The National Unified Renal Translational Research Enterprise (NURTuRE)-INS cohort aims to facilitate novel ways of stratifying INS patients to improve disease understanding, therapeutics, and design of clinical trials.MethodsNURTuRE-INS is a prospective cohort study of children and adults with INS with a linked biorepository. All recruits had at least one sampling visit collecting serum, plasma, urine and blood for RNA and DNA extraction, frozen within 2 hours of collection. Clinical histology slides and biopsy tissue blocks were also collected.ResultsIn total, 739 participants were recruited from 23 centres to NURTuRE-INS, half of whom were diagnosed in childhood (n = 365, 49%). The majority were white (n = 525, 71%) and the median age at recruitment was 32 (interquartile range 12-54). Steroid-sensitive nephrotic syndrome (SSNS) was the most common clinical diagnosis (n = 518, 70%). Of patients diagnosed in childhood who underwent a kidney biopsy - for SSNS (n=103), 76 demonstrated minimal change disease (MCD); whereas for steroid-resistant nephrotic syndrome (n=80), 21 had MCD. Almost all patients diagnosed in adulthood had a kidney biopsy (n = 352, 94%); 187 MCD and 162 focal segmental glomerulosclerosis.ConclusionsNURTuRE-INS is a prospective cohort study with high-quality biosamples and longitudinal data that will assist research into the mechanistic stratification of INS. Samples and data will be available through a Strategic Access and Oversight Committee

Multivariate canonical correlation analysis identifies additional genetic variants for chronic kidney disease

Chronic kidney diseases (CKD) have genetic associations with kidney function. Univariate genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with estimated glomerular filtration rate (eGFR) and blood urea nitrogen (BUN), two complementary kidney function markers. However, it is unknown whether additional SNPs for kidney function can be identified by multivariate statistical analysis. To address this, we applied canonical correlation analysis (CCA), a multivariate method, to two individual-level CKD genotype datasets, and metaCCA to two published GWAS summary statistics datasets. We identified SNPs previously associated with kidney function by published univariate GWASs with high replication rates, validating the metaCCA method. We then extended discovery and identified previously unreported lead SNPs for both kidney function markers, jointly. These showed expression quantitative trait loci (eQTL) colocalisation with genes having significant differential expression between CKD and healthy individuals. Several of these identified lead missense SNPs were predicted to have a functional impact, including in SLC14A2. We also identified previously unreported lead SNPs that showed significant correlation with both kidney function markers, jointly, in the European ancestry CKDGen, National Unified Renal Translational Research Enterprise (NURTuRE)-CKD and Salford Kidney Study (SKS) datasets. Of these, rs3094060 colocalised with FLOT1 gene expression and was significantly more common in CKD cases in both NURTURE-CKD and SKS, than in the general population. Overall, by using multivariate analysis by CCA, we identified additional SNPs and genes for both kidney function and CKD, that can be prioritised for further CKD analyses.</p

National Unified Renal Translational Research Enterprise: Idiopathic Nephrotic Syndrome (NURTuRE-INS) study

Background Idiopathic Nephrotic Syndrome (INS) is a heterogenous disease and current classification is based on observational responses to therapies or kidney histology. The National Unified Renal Translational Research Enterprise (NURTuRE)-INS cohort aims to facilitate novel ways of stratifying INS patients to improve disease understanding, therapeutics, and design of clinical trials. Methods NURTuRE-INS is a prospective cohort study of children and adults with INS with a linked biorepository. All recruits had at least one sampling visit collecting serum, plasma, urine and blood for RNA and DNA extraction, frozen within 2 hours of collection. Clinical histology slides and biopsy tissue blocks were also collected. Results In total, 739 participants were recruited from 23 centres to NURTuRE-INS, half of whom were diagnosed in childhood (n = 365, 49%). The majority were white (n = 525, 71%) and the median age at recruitment was 32 (interquartile range 12-54). Steroid-sensitive nephrotic syndrome (SSNS) was the most common clinical diagnosis (n = 518, 70%). Of patients diagnosed in childhood who underwent a kidney biopsy - for SSNS (n=103), 76 demonstrated minimal change disease (MCD); whereas for steroid-resistant nephrotic syndrome (n=80), 21 had MCD. Almost all patients diagnosed in adulthood had a kidney biopsy (n = 352, 94%); 187 MCD and 162 focal segmental glomerulosclerosis. Conclusions NURTuRE-INS is a prospective cohort study with high-quality biosamples and longitudinal data that will assist research into the mechanistic stratification of INS. Samples and data will be available through a Strategic Access and Oversight Committee

Associations with age and glomerular filtration rate in a referred population with chronic kidney disease: methods and baseline data from a UK multicentre cohort study (NURTuRE-CKD)

Background: Chronic kidney disease (CKD) is common but heterogenous and is associated with multiple adverse outcomes. The National Unified Renal Translational Research Enterprise (NURTuRE)-CKD cohort was established to investigate risk factors for clinically important outcomes in persons with CKD referred to secondary care.Methods: eligible participants with CKD stages G3-4 or stages G1-2 plus albuminuria &gt; 30 mg/mmol were enrolled from 16 nephrology centres in England, Scotland and Wales from 2017 to 2019. Baseline assessment included demographic data, routine laboratory data and research samples. Clinical outcomes are being collected over 15 years by the UK Renal Registry using established data linkage. Baseline data are presented with subgroup analysis by age, sex and estimated GFR (eGFR).Results: 2996 participants were enrolled. Median (interquartile range) age was 66 (54 to 74) years, 58.5% were male, eGFR 33.8 (24.0 to 46.6) ml/min/1.73m2 and UACR 209 (33 to 926) mg/g. 1883 participants (69.1%) were in high-risk CKD categories. Primary renal diagnosis was CKD of unknown cause in 32.3%, glomerular disease in 23.4% and diabetic kidney disease in 11.5%. Older participants and those with lower eGFR had higher systolic blood pressure and were less likely to be treated with renin-angiotensin system inhibitors (RASi) but were more likely to receive a statin. Female participants were less likely to receive a RASi or statin.Conclusions: NURTuRE-CKD is a prospective cohort of persons who are at relatively high risk of adverse outcomes. Long-term follow-up and a large biorepository create opportunities for research to improve risk prediction and investigate underlying mechanisms to inform new treatment development