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 glomerular filtration rate (eGFR). RESULTS: A total of 2996 participants was enrolled. Median (interquartile range) age was 66 (54-74) years, eGFR 33.8 (24.0-46.6) mL/min/1.73 m2 and urine albumin to creatinine ratio 209 (33-926) mg/g; 58.5% were male. Of these participants, 1883 (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 to investigate underlying mechanisms to inform new treatment development.

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.

A Whole Genome-Wide Arrayed CRISPR Screen in Primary Organ Fibroblasts to Identify Regulators of Kidney Fibrosis.

Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat.

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

Targeting the podocyte to treat glomerular kidney disease.

The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics.

Isolation and characterization of circulating fragments of the insulin-like growth factor binding protein-3.

Proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP-3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP-3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non-glycosylated N-terminal fragments spanning residues Gly1-Ala98 of IGFBP-3. Both the circulating forms and those secreted from IGFBP-3(1-98) overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP-3(139-157) and IGFBP-3(139-159)) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP-3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Dsl1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motif to interact with different subunits of the COPI vesicle coat.

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with delta-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and delta-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to delta-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with delta-COP but also with alpha-COP. Strong interaction with alpha-COP requires the presence of comparable amounts of epsilon-COP or beta' -COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.