Driving Discoveries 2023: abstracts

Professor Maria Grazia De Angelis

Authors and affiliations:  
Thomas Fabiani¹, Maria Grazia De Angelis¹, Simone Dimartino¹, Cristiana Boi², Eleonora Ricci^3
1University of Edinburgh
²University of Bologna
³ National Center for Scientific Research Demokritos, Athens

Background:
A wearable artificial kidney (WAK) is the holy grail of hemodialysis. WAKs would improve the lifestyle and health of patients, especially those with comorbidities, as dialysis would be performed more gently and even at night, reducing restrictions on patients' lives. Furthermore, it would enable huge water savings in a water-intensive process. The biggest technological obstacle to making a WAK is the purification and recirculation of the dialysate, which is the wastewater containing the molecules removed from the blood. Previous authors have used a biochemical reaction to deplete the more abundant uremic toxins, e.g. urea, but those clinical trials failed because the byproducts formed from the reaction are toxic to patients.

Methods:  
Our goal is to eliminate the bottleneck of WAK by synthesizing a material that can safely and completely deplete uremic toxins from dialysate without releasing byproducts. To this end, we performed molecular simulations and experimental tests to screen a large database of materials capable of removing uremic toxins, particularly urea, from water.

Results:  
Commercially available materials do not provide the purification capacity needed for a miniaturized system. After examining hundreds of existing and new materials, we realized that some porous materials with ordered nanopores (Covalent Organic Framework) could get good removal of urea from water, if they were heavily loaded with fluorine atoms. Two materials were studied in more detail.
  
Conclusion:  
Our calculations allowed us to sift through a large database of porous materials and pre-select those suitable for WAK. We were also able to understand the physical and chemical mechanism behind their performance.

Lay summary  
Lay title: Redefining hemodialysis with materials science and data-driven innovation: towards the wearable artificial kidney

Background:  
Our goal is to create a wearable artificial kidney, i.e., a self-supporting dialysis system that can function in the absence of a water supply. Ideally these would allow for more continuous and less invasive treatment for patients, nocturnal dialysis and would be less life-shattering than home dialysis systems, even the most advanced ones.

Your methodology - what have done in your study?
From the study of the previous works carried out and from our engineering expertise, we have identified the main bottleneck in the realization of the WAK in the materials for the purification of water. Therefore, we are focusing on this section of the machine, looking for materials that can safely and permanently capture uremic toxins from the water circulating in the device. We have carried out experimental tests on some commercial materials and calculations on a family of about 500 candidate materials suitable for our process.

Results:  What have you discovered? 
Commercial materials do not have the ability to remove all uremic toxins. Some new materials have the potential to capture urea from water in large quantities. Their chemical nature appears to play a large part in this, especially their fluorine atom content.

What are the potential benefits that this research could bring to patients? 
Patients could perform hemodialysis overnight, travel easily, and perform some light physical activity during treatment. They may be far from a water source. The treatment itself would be much less invasive, require less water and be more accessible to populations with limited access to water or medical facilities.

Thandi Ferris

Authors and affiliations:
Thandi Ferris ¹, Katarzyna Szymanska¹, Colin A Johnson^1
1University of Leeds

Background:  
Autosomal recessive polycystic kidney disease (ARPKD) is a rare, severe disorder characterized by polycystic kidneys during the perinatal period. Mutations in the PKHD1 gene, encoding the ciliary-associated protein fibrocystin, are responsible for ARPKD. Several studies have presented compelling evidence that mutations in the PKHD1 gene, resulting in reduced or absent fibrocystin, correlated to dysregulated cilia and the cystic phenotype. However, a definitive pathomechanism establishing a relationship between these factors remains unknown.

Methods: 
Here, using an eSpCas9GFP/gRNA system, three (SCTi003-A) human iPSC lines were generated, two carrying compound heterozygous mutations; c.[106_107insC];[106_112delACGTGGA], c.[99_100insA];[98_106delCAGGGGGAA], and one with the heterozygous mutation, c.106_113insTCACTGTG all in exon 2 of PKHD1. These cell lines were characterized and validated for pluripotency cell markers Oct-4 and SSEA-3/4 using immunocytochemistry/immunofluorescence and confocal microscopy.

Mutation analysis was done with Sanger sequencing. The mutational consequences on the PKHD1 gene product, fibrocystin, were analyzed with Western blot. RT-PCR analysis is to be done to further assess the mutation consequences on cDNA. To characterize the impact of these mutations on fibrocystin localization to primary cilia, iPSCs will be stained with antibodies specific for cilia (ARL13B) and fibrocystin (binding to residues 143-235 on the 2nd TIG domain) and immunofluorescence assessed via confocal microscopy. Karyotyping of the cell lines will be completed in due course.

Results:  
iPSCs expressed normal immunofluorescence for SSEA-3/4 and Oct-4. Mutation analysis revealed no off-target events, and the targeted mutations were present in each cell line.

Western blot analysis indicated the presence of potential fibrocystin isoforms within the 75- 150kDa range.

Conclusion:  
These iPSCs mutant PKHD1 cell lines expressed pluripotency cell markers and therefore markers and, therefore, have the potential to be differentiated into suitable ARPKD kidney organoid models.
 
Lay summary  
Lay title: Modelling ARPKD Compound Heterozygous Mutations in Human Induced Pluripotent Stem Cell Line

Autosomal recessive polycystic kidney disease (ARPKD) is a rare form of polycystic kidney disease that is typically diagnosed during the perinatal period and early childhood. This inherited disease causes cysts to grow in the kidneys. This often leads to kidney failure, where patients require dialysis or kidney transplants since there are no specific drugs for ARPKD.

ARPKD is caused by mutations in the PKHD1 gene encoding for the ciliary-associated protein fibrocystin. Cilia are hair-like structures that protrude from the apical surface of cells and are actively involved in several cellular processes, including kidney organogenesis.

Studies have demonstrated a link between PKHD1 mutations and dysfunctional cilia in animal models that implicates their role in cystic kidney disease. However, the exact mechanism causing cyst formation is poorly understood. Although animal models have provided substantial insights into ARPKD disease mechanisms, they fail to effectively recapitulate human phenotypes.

This study characterizes and validates human induced pluripotent stem cells (iPSCs) with induced compound mutations comparable to ARPKD mutations. These cell lines can potentially be differentiated into 3D kidney organoid ARPKD models to further investigate the molecular mechanisms of cyst formation and develop therapeutic strategies to abrogate the cystic kidney phenotype.

Magdalena Karwatka

Authors and affiliations:  
Magdalena Karwatka¹, Joe Cockburn¹, Steve Evans¹, John Ladbury¹, Colin A Johnson¹, Michelle Peckham¹; Rowan Taylor^2
1University of Leeds
²Human Centric Drug Discovery, Oxford

Background: 
Primary cilia are microtubule-based organelles protruding from the surface of most mammalian cells. They are important signaling hubs with essential roles in vertebrate development. Defects in cilia cause ciliopathies that range from non-syndromic inherited retinal diseases to severe multi-organ disorders, invariably presenting with renal cystic dysplasia.
CEP290 (Centrosomal protein of 290kDa) is a key regulator of ciliary structure and ciliary vesicular trafficking. CEP290 mutations are a major cause of ciliopathies. Nearly half of CEP290 coding exons are in-frame “skippable” exons that can be spliced from the transcript without altering the reading frame. This can remove the frame-disrupting segment of mRNA, restoring the reading frame and producing a truncated protein that retains some or all function.

Methods:  
Using CRISPR-Cas9, we knocked-in frame-disrupting pathogenic mutations in exon 7, 25 and 36 of CEP290 in induced pluripotent stem cells (iPSC). Kidney organoids derived from iPSCs will be imaged and assessed for key cellular phenotypes. We will test splice-switching antisense oligonucleotides (ssAONs) targeting exons 7, 25 and 36 for exon-skipping efficacy in cells by qRT-PCR and western blotting. The ssAONs will then be encapsulated in liposomes attached to microbubbles to assess deliverability and accessibility in organoids following bubble rupture using ultrasound.

Results: 
We have generated CRISPR-edited iPSC lines with mutations in exon 36 of CEP290 and have used these to generate organoid models of CEP290-related disease. We show that exon 36 is skipped in kidney but not retinal organoids, exon-skipping does not affect primary ciliogenesis, but a frameshift mutant markedly reduces cilia size in iPSCs.

Conclusion:   
We have developed models and a workflow for assessing exon-skipping as a potential therapy for CEP290-related forms of renal cystic dysplasia. We will use ssAONs in kidney cells to trigger skipping of exons carrying frame-disrupting mutations and assess the suitability of microbubbles as a carrier for targeted ssAON delivery.

Lay summary  
Lay title: Early testing of oligonucleotide therapies as potential treatments for cystic kidney diseases

Background: 
Cilia are antennae-like structures on cells that receive and transduce signals from their surroundings during human development. Defects in cilia cause a range of human developmental disorders called “ciliopathies”. CEP290 (Centrosomal Protein 290kDa) is an essential molecule in the cilium, and mutations are the most frequent cause of ciliopathies, invariably associated with cystic kidney disease. This project aims to identify and develop therapeutics for patients with these “untreatable” conditions. CEP290 contains many “in-frame” exons. If a mutation occurs within an in-frame exon, cellular processes (“splicing”) can remove this segment (“exon-skipping”), avoiding the detrimental effects of the mutation.

Your methodology - what have done in your study?  
We have edited different types of CEP290 mutations into induced pluripotent stem cells (iPSCs) from which we can grow 3D human cell models (“organoids”) of kidney disease. We will assess if new therapeutic agents called antisense oligonucleotides will force cellular processes to perform exon-skipping on an exon containing a mutation, which has the potential to relieve disease symptoms.

Results:  What have you discovered?  
We have shown that exon-skipping occurs for only some types of CEP290 mutations in kidney but not retina cells, providing the basis for development of new disease models.

What are the potential benefits that this research could bring to patients?
Using antisense splice-switching oligonucleotides to induce exon-skipping is a potential pre-clinical therapeutic approach that targets CEP290 mutations causing renal ciliopathies.

Cheuk Yan Man

Authors and affiliations:  
Cheuk Yan Man¹, Hortensja Ł Brzóska², Owen Williams², Michael G Robson³, Jonathan Elliott¹, David A Long², Elisavet Vasilopoulou¹

¹Comparative Biomedical Sciences, The Royal Veterinary College, London, UK
²Developmental Biology and Cancer Programme, UCL Great Ormond Street Institute of Child Health, London, UK
³ MRC Centre for Transplantation, King’s College London, London

Background:  
Glomerular disease involves both injury of glomerular cells and inflammatory responses that drive disease progression. Thymosin β4 (Tβ4) is a peptide that is expressed in podocytes and macrophages in the mouse kidney and has a protective role in glomerular disease. Tβ4-knockout mice develop exacerbated glomerular disease compared to wild-type controls, with impaired renal function, reduced podocyte number and increased macrophage accumulation. However, the precise contribution of leukocyte and resident kidney cell-produced Tβ4 to glomerular disease progression is unknown. We aimed to answer this question in our study.

Methods:
Transplantation of bone marrow cells from wild-type (WT) or Tβ4-knockout (KO) mice into irradiated recipient mice was carried out to generate four groups of chimeric mice; WT mice with WT bone marrow (WT/WT, n=9), WT mice with KO bone marrow (WT/KO, n=6), KO mice with WT bone marrow (KO/WT, n=6) and KO mice with KO bone marrow (KO/KO, n=7). Glomerulonephritis was induced ten weeks later by administration of nephrotoxic serum (NTS). Bone marrow reconstitution (flow cytometry), macrophage accumulation, podocyte density (immunohistochemistry) and albuminuria (ELISA) were assessed 21 days post-NTS.

Results:  
Bone marrow transplantation was efficient with >75% of circulating cells being donor-derived.  Macrophage accumulation in the periglomerular area was significantly lower in WT/WT mice (10.18±0.34 macrophages/glomerulus) compared with WT/KO (13.73±0.49, p<0.0001), KO/WT (15.24±0.46, p<0.0001), and KO/KO mice (18.26±0.47, p<0.0001). Podocyte density was highest in WT/WT mice (6.64±0.18 podocytes/100μm2) compared with WT/KO (5.54±0.19, p<0.0001), KO/WT (5.98±0.22, p=0.0083), and KO/KO mice (4.94±0.14, p<0.0001). Albuminuria was significantly lower in WT/WT (9,947±3,403 μg/24hours) compared with KO/KO mice (29,177±4,838, p=0.0070), but there were no statistical differences with WT/KO (13,468±3,056) and KO/WT (14,635±3,729) mice.

Conclusion:
Our findings show that Tβ4 expression by both bone marrow-derived and kidney-resident cells has a protective role in glomerular disease.

Lay summary  
Lay title: Investigating the protective effect of thymosin β4 in chronic kidney disease

Background:
One of the leading causes of end-stage kidney disease is damage to the glomeruli, the components of the kidney where the blood is filtered to make urine. This damage is caused by disruption of the structure of the glomeruli and by the build-up of white blood cells, which causes inflammation. Thymosin β4 is a naturally occurring protein that can reduce inflammation and the severity of glomerular disease in mice. The aim of this study is to understand how thymosin β4 exerts its protective effect in the kidney.

Your methodology - what have done in your study? 
Mice with selective deletion of thymosin β4 either in circulating white blood cells, or in the kidney, were generated followed by the initiation of glomerular disease.

Results:  What have you discovered? 
The results show that lack of thymosin β4 in either white blood cells or kidney cells, exacerbates glomerular damage and inflammation. Loss of thymosin β4 in both white blood cells and kidney cells had a cumulative effect.

What are the potential benefits that this research could bring to patients? 
These results show that thymosin β4 regulates both white blood cells and kidney cells to limit the effects of kidney injury. This work provides the rationale to harness this protective pathway to develop new therapies to slow the progression of glomerular disease.

Katarzyna Szymanska

Authors and affiliations:  
Katarzyna Szymanska¹, Claire Smith¹, Alice Lake¹, Andrew Streets², Albert Ong², Colin A Johnson^1
1Leeds Institute of Medical Research, University of Leeds
² University of Sheffield Medical School

Background:  
Polycystic kidney disease (PKD) and other syndromic forms of cystic kidney disease (CyKD) are a major cause of childhood morbidity and mortality in the UK, with approximately 5,000 patients in the UK needing either dialysis or transplantation every year due to kidney failure. Tolvaptan is a drug that slows the decline in renal function for autosomal dominant polycystic kidney disease (ADPKD). However, common (11 to 13%) side-effects of Tolvaptan mean that many patients do not tolerate treatment. In this work we aim to model a series of CyKD patient mutations (from severe syndromic ciliopathies such as Meckel-Gruber syndrome to non-syndromic PKD) in kidney organoids (KiO) and treat cystic phenotypes with ROCK inhibitors (fasudil and hydroxyfasudil), recently identified to rescue normal ciliogenesis in cell-based models and diminish cystogenesis in mouse model.

Methods:  
Patient mutations were introduced in MKS1, PKD2, BICC1 and PKHD1 in induced pluripotent stem cells (iPSCs) line SCTi003-A using CRISPR-Cas9 genetic engineering. Mini KiO, grown in non-adherent 96-well plates, were optimised for drug treatment to induce cellular cystic phenotypes using forskolin, blebbistatin and 8Br-cAMP.

Results:  
SCTi003-A iPSCs were successfully differentiated into KiO with nephron structures (expressing markers NPHS1 for podocytes, LTL for proximal tubules, and CDH1 for distal tubules) following an adaptation of existing protocols. We are currently optimising a protocol that differentiates KiO with both nephron and collecting duct structures. We have successfully developed iPSC lines with biallelic MKS1, PKD2, BICC1 and PKHD1 mutations. These will be characterised, differentiated into KiO and assessed for rescue of cellular cystic phenotypes by treatment with ROCK inhibitors.

Conclusion:  
We have developed pre-clinical iPSC-derived models of CyKD with a series of patient mutations for use in potential pre-clinical testing of ROCK inhibitors for this disease indication.

Lay summary  
Lay title: New treatments for cystic kidney disease.

Tolvaptan is a drug that slows the decline in renal function for autosomal dominant polycystic kidney disease (ADPKD). However, common (11 to 13%) side-effects of Tolvaptan mean that many patients do not tolerate treatment. Furthermore, there are no preventative treatments or new therapeutic interventions that may modify disease progression of patients with ADPKD or other types of cystic kidney disease. This has created an acute clinical need for the potential repurposing of existing drugs or the development of novel lead compounds that could treat cystic kidney disease. We identified and validated two approved drugs (fasudil and hydroxyfasudil) and now want to test these in cell-based disease models of kidney cysts. We will use kidney organoids (mini kidneys grown in a dish) that we can artificially produce from special “induced pluripotent stem cells” (iPSCs). We will model renal tubule formation and cyst formation in response to drug treatment in a physiologically relevant tissue, an essential step in drug efficacy testing. The overall aim is to re-purpose and translate a known drug into Phase II clinical trials in less than 5 years, significantly reducing the time and investment required to make the move from laboratory to the clinic.

Mannie Sher

Authors and affiliations:    
Mannie Sher¹, Francis Clay^1
1The Tavistock Institute of Human Relations

Background:  
There is a relatively low take-up of home dialysis in the UK.  This project will investigate the psycho-social, socio-technical and socio-ecological dynamics (the emotional, behavioural, technical and environmental properties) of home dialysis of patients with chronic kidney disease (CKD), their families, carers and hospital renal staff.

Methods:  
The method of investigation will focus on how patients relate to and feel about their illness, their patient role, and the impact of these on their identities. The method will consist of an investigation into personal, conscious, and unconscious factors and environmental contextual factors in determining individual and systemic behaviours in relation to CKD and treatments; it will seek to understand various influences in decision-making and patient resistance to facing and dealing with their illness and treatment.

In-depth interviews of up to 20 CKD patients will allow for the collection of rich, qualitative data. The patient, treatment, and context (environment, culture, educational, social and economic circumstances) will lie at the heart of this project.
The core research activity will comprise individual and group interviews. Attitudes and beliefs towards CKD and its treatment will be examined in-depth in patients and families, renal ward staff and policy makers. The questions will aim to answer practical questions like diet, the onset of the disease, symptoms, attitudes concerning health-promoting and health-diminishing behaviours. Patient life-cycle histories will be investigated, with reference to early childhood relationships and milestone events, cultural food habits, education and work histories, sport, accidents, inter-personal relationships, personality and identity-formation, sexual development, behaviour and relationships. Patients and their families will be asked about their in-the-moment experiences of dialysis and their expectations of home dialysis.

Results:   
The resulting material will be subject to rigorous content analysis to produce trends and detailed pictures of patient attitudes and experiences of hospital and home dialysis.

Conclusion:  
The research project is in the application-for-funding stage; therefore, no conclusions are available at present.

Lay summary  
Lay title:  An investigation into the emotions (feelings), attitudes and behaviours towards home dialysis of CKD patients and their families, hospital renal staff, policy makers. A primary aim of the research is to increase the take-up of home dialysis.

Background:  
The take up of home dialysis is about 1% of patients with kidney disease. This is a startling figure, given that home dialysis offers patients improved health, increased chances of survival, greater longevity, greater freedom and flexibility of lifestyle and treatment frequencies.  For the NHS too, there are advantages of home dialysis since hospital dialysis costs over 2.5 times more than home dialysis and produces a healthier population. Some obstacles to having dialysis at home are practical, e.g. lack of space, ageing and frailty, poor hand-eye coordination, clinically unsuitable; others are psychological and emotional, e.g. fear of needling oneself, fear of things going wrong. Health inequalities in society also factors regarding home dialysis, e.g. poverty, poor education levels, the rural/urban divide, disadvantaged families, immigrant families, transient populations.

Your methodology - what have you done in your study?  
The methodology of this study will involve new approaches to investigating decisions about home dialysis.

Results:  What have you discovered?  
The research project is at the application for funding stage; therefore, no results are available at present.

What are the potential benefits that this research could bring to patients? 
Home dialysis offers patients better health outcomes, increased chances of survival, greater longevity, greater freedom and flexibility of lifestyle and treatment frequencies.

Authors and affiliations:  
Praveen D Sudhindar¹, Elisa Molinari¹, Seamus M McLafferty¹, Colin A Johnson³, Colin Miles¹, John A Sayer^1
1Newcastle University
³University of Leeds

Renal ciliopathies are a heterogeneous class of disorders caused by dysfunctions of the primary cilia. They are often multisystem disorders characterized by extensive genetic heterogeneity and clinical variability with high levels of lethality and there is marked phenotypic overlap among distinct ciliopathy syndromes. Nephronophthisis (NPHP) is a typical renal ciliopathy phenotype that causes kidney failure often within early childhood, for which there are no curative treatments beyond dialysis and transplantation.

To identify novel therapeutics for NPHP, we designed a high-throughput ciliary phenotype-driven screening strategy to interrogate the Tocris® library of 1120 biologically active compounds, making use of the Operetta high-content imaging system with Harmony/Columbus software. Initially, 33 compounds were identified that restored ciliary phenotype in renal epithelial cells derived from Cep290 mutant mice. These compounds were subjected to a secondary screen using NPHP patient fibroblasts (P-BB), carrying compound heterozygous CEP290 mutations, including an allele that we had previously shown to be amenable to Anti-sense Oligonucleotide (ASO) mediated exon skipping. In this screen, 12 compounds either restored ciliogenesis or corrected cilia length defects.

A tertiary phenotypic screen of the 12 TOCRIS compounds was then carried out in human urine-derived renal epithelial cells (hURECs) from a NPHP patient (P-HB) carrying CEP290 mutation (p. (Thr832Asnfs*12) and p.(Gly1890*)) along with control hURECs from an unaffected sibling. The initial hUREC phenotypic screen of P-HB cells displayed a ciliogenesis defect with elongated cilia typically found in CEP290 patients, compared to the unaffected sibling who had normal cilia. Two of the Tocris® chemical compounds rescued the ciliary phenotype in patient (P-HB). We will employ RNAseq to identify the underlying molecular pathways using the Tocris® compounds which may reveal novel insights into the mechanisms underlying NPHP secondary to CEP290 mutations and explore this in additional genetic causes.

Lay summary  
Lay title: Small molecule drug treatments for inherited kidney disease: Nephronophthisis

Background:  
Cilia are slender hair like projections that extend from almost all types of eukaryotic cells. They are involved in cellular signalling and sensory roles which are important for maintaining the local cellular environment. Dysfunction of cilia leads to number of human diseases which are termed as ‘ciliopathies’. Nephronophthisis (NPHP) is a kidney ciliopathy, which is a rare genetic disorder with an estimated occurrence of 1 in 922,000 in the US, that causes kidney failure often within early childhood, for which there are no curative treatments beyond dialysis and transplantation.

Your methodology - what have done in your study? 
To identify novel therapeutics for NPHP we employed a screening strategy to interrogate a selection of biologically active chemical compounds using high resolution microscopic imaging. Primary, secondary, and tertiary screening were conducted in kidney epithelial cells derived from mouse models, patient fibroblast cells and patient kidney epithelial cells respectively.

Results: What have you discovered? 
The patient kidney epithelial cells had longer cilia and dramatically reduced ciliation rates compared to a healthy sibling. Treatment with selected compounds either restored the ciliogenesis or corrected the cilia length defects.

What are the potential benefits that this research could bring to patients? 
We will employ deep sequencing methodologies in the future to identify molecular mechanisms underlying NPHP. This study will provide valuable insights for future treatment of NPHP in affected patients.

Amarpreet Thind

Authors and affiliations:   
Dr Amarpreet K Thind*^1,2, Dr Michelle Willicombe^1,2 and Professor Edwina A Brown^1,2 on behalf of the KTOP Study Investigator Group^3-6.
¹Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London,
²Imperial College Renal and Transplant Centre, Imperial College Healthcare NHS Trust,
³Central London Community Healthcare NHS Trust,
⁴Department of Surgery and Cancer, Imperial College London,
⁵Institute of Health and Social Care, London South Bank University,
⁶The Centre for Health Services and Clinical Research, The University of Hertfordshire.

Background:  
Older people with kidney failure are vulnerable to developing frailty, which affects all aspects of kidney transplantation (KT). To better inform, guide, and support older people considering KT a holistic understanding of experiences is required.

Methods:   
The Kidney Transplantation in Older People (KTOP) study recruited KT candidates aged >60, assessed frailty (Edmonton Frail Scale), quality of life (QoL) (Short Form-12 V2), and clinical outcomes on the waitlist (WL) and post-KT from October 2019-June 2023. It was powered for QoL differences. Comparative and mixed-effect analysis determined variations by frailty.

Results:  
210 patients were recruited, with 118 transplanted. At recruitment 63.4% (118) were not frail, 19.4% (36) were vulnerable, and 17.2% (32) were frail. A trend towards poorer WL and KT outcomes in the vulnerable/frail was observed. Vulnerable/frail WL candidates were more likely to experience infection events (60% vulnerable/frail c.f. 24.4% not frail, p=0.001) and spend longer suspended (mean 434 days vulnerable/frail c.f. 307 not frail, p=0.025). Vulnerable/frail KT recipients were more likely to be hospitalised (73% vulnerable/frail c.f. 52% not frail, p=0.028), have longer admissions (mean 36.6 days vulnerable/frail c.f. 24.8 not frail, p=0.024), with a trend towards higher graft loss (29.4% vulnerable/frail c.f. 9.9% not frail, p=0.059) and mortality (18.2% vulnerable/frail c.f. 7% not frail, p=0.078).

WL physical component scores (PCS) remained stable in not frail candidates but declined in vulnerable/frail. Post-KT, not frail recipients PCS declined initially and then recovered, whilst PCS stabilised in vulnerable/frail. WL mental component scores (MCS) improved in both groups. Post-KT MCS initially worsened and then improved in not frail recipients but declined in the vulnerable/frail.

Conclusion:  
WL and KT clinical outcomes were poorer in vulnerable/frail older people, however QoL experiences were mixed. KT did not drastically change QoL in either group. Assessing frailty is crucial to older peoples’ care, enabling tailored understanding, risk assessment, counselling, and interventions to be implemented.

Lay summary  
Lay title: Waitlist and Kidney Transplant Experiences in Older People Living with Frailty

The number of older people waiting for and receiving transplants is increasing. Older people with kidney failure are at risk of frailty, which can affect their ability to cope and manage after transplantation. The Kidney Transplantation in Older People study has used questionnaires to measure frailty and quality of life in older people on the waitlist and after a transplant. Medical events that occurred were also counted to see how these varied with different levels of frailty.

210 older people have been included in the study, 118 of whom have been transplanted. Vulnerable/frail older people had worse medical events both on the waitlist (major infection events and longer suspended from the waitlist) and after a transplant (more and longer hospital admissions). In the vulnerable/frail older people physical quality of life stabilised after a transplant but mental quality of life worsened. Across all older people however, transplantation did not lead to drastic changes in quality of life. This research provides more detailed information on how experiences vary in older people and why these variations exist (frailty). Consequently, more accurate, realistic information can be given to older people and now we can find specific ways to support older people during this time.

Priscilla Smith

Authors and affiliations: 
P Smith¹, K Dalrymple¹, K Clarke¹, Y Wang¹, T Harris², A Webb¹, L Chappell¹, K Bramham¹

¹King’s College London
²PKD Charity

Background: 46% of women with moderate-severe chronic kidney disease (CKD) will require dialysis or lose at least 25% of kidney function within six months of delivery with no development of preventative treatments.

Methods: Biological samples, longitudinal and outcome data will be collected in a prospective cohort and eligible women identified to participate in ORCHARD-BEET randomised controlled feasibility trial. Inclusion criteria: singleton pregnancies;<25 wks gestation; CKD (pre-pregnancy eGFR<90mls/min/m2 or pregnancy Cr>70µmol/l). Randomisation:1 to 1. Standard care or daily beetroot juice supplement (nitrate 400mg). Primary outcome: recruitment rate; secondary outcomes: tolerability, acceptability, eGFR change 6 months postpartum.

Results: 118 pregnant persons consented to participate to the cohort and 104 were randomised for ORCHARD-BEET trial.  Cohort maternal baseline characteristics are presented in Table 1.

Conclusion: To our knowledge this is the largest prospective cohort study with embedded pragmatic feasibility trial with concurrent biobanking of pregnant participants with CKD (close April 2024), which is representative of women with moderate and severe CKD. Findings will be used to inform future intervention trials to prevent pregnancy-associated progression of kidney disease.

Lay summary 

Lay title: ORCHARD study of kidney disease in pregnancy

Background  
Kidney disease affects 3% of women of childbearing age. Many women will lose kidney function or need to start dialysis during or after pregnancy. We have no treatments to prevent this and there have been no improvements in the last 20 years.

We hope to find out whether drinking a small amount of beetroot juice each day can help to protect kidney function in pregnancy. Beetroot juice contains ‘dietary nitrate’, which has been shown in non-pregnant and pregnant individuals to lower blood pressure and improve blood flow by relaxing the blood vessels.

Your methodology - what have done in your study? 
Women with kidney disease pre-pregnancy were asked to participate if under 25 weeks pregnant and not on dialysis. If randomly allocated to intervention arm, they were asked to drink a 70ml Beetroot Juice concentrate “shot” daily along with usual clinical care. They were seen up to 3 times during pregnancy and twice post-partum; biological samples were collected, blood pressure checked and those taking the beet shot were asked about any missed doses or problems with the drink.

Results:  What have you discovered? 
We have been able to recruit women with moderate to severe CKD to an interventional trial during pregnancy and meet our recruitment target overall (See Table 1). We will report full results once all follow-ups have been completed.

What are the potential benefits that this research could bring to patients? 
Potential therapy to prevent worsening of kidney function during or following pregnancy.

Maryline Fresquet

Authors and affiliations: 

Maryline Fresquet¹-Patrick Hamilton¹, Bernard Davenport¹, Emily Williams¹, Alex Mironov¹, Wayne Lim¹, Ronan o’cualain¹, Craig Lawless¹, Alex Nyström², Paul Brenchley³, Rachel Lennon¹

¹Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health Sciences, School of Biological Sciences, University of Manchester, Manchester, UK.
²Department of Dermatology, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany.
³Central Manchester University Hospitals NHS Foundation Trust and Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK.

Scientific summary  

Background: 
Membranous nephropathy (MN) is an autoimmune disease affecting the kidney. 80% of MN patients have autoantibodies to the phospholipase A2 receptor found on podocytes and IgG-PLA2R immune complexes deposit in the glomerular basement membrane (GBM). We currently have limited understanding about the consequences of the antibody deposition in the filter. In this study, we defined the ultrastructural and compositional changes of the disorganised GBM in patients with MN.

Methods: 
A MN human biopsy was stained and analysed by volume electron microscopy. The acquired images were used to model segment containing the immune deposits in the GBM using the IMOD image processing software.
Glomerular sections from 5 MN patient biopsies and 5 control samples were collected. ~75 glomeruli/patient were isolated by laser-microdissection and prepared for proteomic analysis.

Results:  
The 3D model of the glomerulus highlighted novel ultrastructural features of the disorganised filtration barrier in MN patients. We observed a thickened appearance of the overall GBM due to the presence of large immune complex deposits, formation of spikes between the deposits and visualised vesicles in the immune complex. We found adhesions sites formed between the podocyte foot process and the basement membrane, possibly to resist mechanical force caused by the deposits.
Proteomic analysis identified enriched pathways; regulation of immune response and complement activation, hallmarks of MN pathology. Additional analysis showed an increased level of proteins in the MN glomeruli, namely PLA2R, COL7A1, COL12A1, FBLN5 and EFEMP1, all basement membrane proteins. These proteins are involved in collagen fibril organisation, extracellular matrix assembly and dysregulation of the GBM and remodelling. We validated these proteins using immunofluorescence and found PLA2R, COL7A1 and COL12A1 specifically enriched within the MN patient glomeruli.

Conclusion:  
This study improves our understanding about the pathobiology associated with GBM defects. These findings have now the potential to identify new therapies that target GBM repair.

Lay summary  

Lay title: Investigating the basement membrane in the autoimmune kidney disease, membranous nephropathy.

Background:  
Membranous nephropathy (MN) is an auto-immune disease that affects the filters (glomeruli) of the kidney and can cause protein in the urine.  Patients have circulating antibodies in their blood which accumulate in the filter, then become inflamed. We currently have limited understanding about the consequences of the antibody deposition in the filter.

Your methodology - what have done in your study?  
In this study we sought to define the changes in structure and composition of healthy and diseased glomeruli. We used MN patient kidney biopsies and analysed them using electron microscopy and mass spectrometry.

Results: What have you discovered? 
We described novel ultrastructural features of the glomerular filtration barrier, which appeared very abnormal in MN, thickened, with immune complex deposits. We found key proteins present in patient samples potentially involved in the dysregulation of the filter.

What are the potential benefits that this research could bring to patients? 
Improving the understanding of the disease mechanism will help in finding treatments targeting the removal of autoantibodies from the kidney.

Richard Naylor

Authors and affiliations:  
Richard W. Naylor¹, Rachel Lennon^1
1University of Manchester

Scientific summary  

Background:  
>90% of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD) are caused by mutations in the PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and PC2, respectively. The polycystins form an ion channel that is important for proper ciliary function. Much effort has been made to investigate the molecular mechanisms driving pathology in ADPKD. However, a detailed analysis of the proteome, using up-to-date cutting edge mass spectrometers, in ADPKD has not been performed.

Methods: 
Using the latest mass spectrometry approaches, proteomics analysis was performed on kidneys taken from P28 wild-type and Pkd1nl/nl (a Pkd1 hypomorphic mouse model of ADPKD). 4 biological replicates were assessed, and data processing was performed using MaxQuant and msqrob2 differential analysis.

Results:  
Differential protein intensity analysis showed a number of previously identified markers of PKD are up-regulated in the Pkd1nl/nl model. This includes Lcn2, Acta2, multiple claudins, markers of leukocytes (such as Itgam), downstream effectors of the Yap/Taz Hippo pathway, and multiple markers of fibrosis (TgfB, Col1a1, Postn). Novel proteins were also detected that are likely to be important in pathogenesis, including multiple RhoGEFs, Thy1, Grb2 and CD44. We have a particular interest in cell-matrix interactions and how these underpin disease. Of note, we find multiple integrins are more highly expressed in ADPKD, with integrin alpha1, alpha3, beta1 and beta5 particularly up-regulated. Given loss of integrin beta1 is known to preclude cystogenesis, we are currently investigating further the roles of integrin alpha subunits in ADPKD, with the specific aim to generate therapeutics against these more desirable targets.

Conclusion:  
Improved proteomics analyses have enabled greater understanding of ADPKD kidneys and highlights the power of the modern and cutting-edge mass spectrometers available to researchers. We have found multiple molecules that are likely to be important in disease progression in ADPKD and are developing ways to disrupt the function of these proteins with the aim of slowing down disease progression.

Lay summary  

Lay title: Understanding the changes in protein content of PKD kidneys highlights new insights into the disease

Background: 
Many researchers have looked at how kidneys change in terms of their mRNA transcripts. However, mRNA is not the functional readout of the tissue. In all cells, mRNA is converted into protein, which performs a specific function in the cell. How quickly the protein is degraded or altered (such as by interactions with other proteins or by being modified) is more important with respect to how it is able to change cell behavior. What this ultimately means is that the level of mRNA does not always correspond to the level/activity of the protein. Given this, we have directly analyzed all the proteins in the kidneys of a mouse model of ADPKD and compared them to normal kidneys to get a more refined understanding of how the kidney changes in ADPKD.

Your methodology - what have done in your study? 
We used a machine that detects proteins, called a mass spectrometer, and compared non-ADPKD to ADPKD kidneys to see which proteins are changed in terms of their amounts.

Results:  What have you discovered? 
We have discovered a number of proteins that are likely to be important in the progression of ADPKD and are currently developing methods to stop these changes in order to develop new therapies.

What are the potential benefits that this research could bring to patients? 
The main aim of this research is to discover new targets that I will use to apply for further funding to develop as potential novel therapies. This includes commercialization of new technologies targeting candidates in order to more speedily impact patients in a clinical setting.

Authors and affiliations:  
Ivan Hartling¹, Sarah Fawaz², Rebecca Vaughan^2,3, Ioannis Michelakis^2,4, Rutger Ploeg^2,3, Philip Charles¹, Cecilia Lindgren¹, and Maria Kaisar^2,3
1 Big Data Institute, Nuffield Department of Medicine, University of Oxford, United Kingdom
² Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom
³ Research and Development, NHS Blood and Transplant, Bristol, United Kingdom
⁴ Onassis Cardiac Surgery Center, Athens, Greece

Background: 
Kidney transplantation is a life-saving treatment for end stage kidney disease, but every year hundreds of patients with kidney failure die while waiting for a transplant. Donor organ acceptance criteria have been expanded in an effort to decrease organ shortage; however, current acceptance criteria depend heavily on immune matching and donor age and lack granularity. This study aimed to identify donor circulating protein signatures to improve granularity of assessing donor kidneys, predicting kidney transplant outcomes.

Methods:  
We analysed deceased donor plasma samples that were linked to complete donor and recipient metadata obtained from the QUOD biobank. A selection of 49 analytes were measured at three timepoints in the plasma of 132 brain death donors (DBD) and at two timepoints in the plasma of 119 circulatory death (DCD) donors. These measurements, along with donor age, height, and sex, were used to construct separate DBD and DCD 10-fold cross-validated lasso regression models using 12-m recipient averaged eGFR as outcome end point. Models were compared using root mean squared error (RMSE) which is a measure of prediction accuracy.

Results:  
Lasso regression identified unique protein signatures in both DBD and DCD donors consisting of 20 and 22 proteins respectively with 8 proteins common to both models. The DBD model achieved a RMSE of 18.7 mL/min/1.73 m2 while the DCD model RMSE was 19.0 mL/min/1.73 m2. Both models performed considerably better than models containing clinical variables alone which had RMSEs of 21.7 mL/min/1.73 m2 and 22.7 mL/min/1.73 m2 for DBD and DCD respectively.

Conclusion: 
This study identified protein signatures in DBD and DCD kidney donor plasma that could improve prediction of post-transplant outcome compared to using clinical variables alone. Identified protein signatures will be validated in a follow-up study on 1000 donors to develop a predictive score which could be used to improve transplant outcome prediction.

Lay summary  

Lay title: Improving assessment of donated kidneys

Background:  
Kidney transplantation is a life-saving treatment of end stage kidney disease, but every year hundreds of patients die waiting for a transplantation. To save more lives, transplant clinicians accept kidneys from higher risk donors; however, these organs may have poor function following transplantation. To improve assessment of donor organs, this study used blood markers and machine learning techniques to improve methods for selecting donor organs that will function well in the recipients.

Your methodology - what have done in your study? 
We analysed blood samples collected during donor management to identify markers that can predict how well grafts will function after transplantation. We combined blood markers with clinical variables and used machine learning models to select the most useful markers and variables. We then determined how much improvement was provided by the blood markers.

Results:  What have you discovered?
We identified unique markers that can improve the selection of donor kidneys. We found that using these markers along with donor age, height, and sex had better accuracy than using age, height, and sex alone.

What are the potential benefits that this research could bring to patients, whether that be in the near or distant future ? 
These markers will now be used in a large cohort study to develop a predictive score to help clinicians to better select organs for transplantation. This will help to both increase the number of donor organs and improve the outcomes of kidney transplantation.

Authors and affiliations:  
Griffiths, JD¹; Streets, AJ¹; Johnson, CA²; Smythe, E³; Ong, ACM¹

¹ Kidney Genetics Group, Academic Unit of Nephrology, Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK
² The School of Medicine, University of Leeds, Leeds LS2 9JT, UK
³ Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, UK.

Background:
Autosomal dominant polycystic kidney disease (ADPKD) is the most  common monogenic kidney disease in man and a leading cause of end-stage renal failure.  In the majority of cases, germline mutations in PKD1 or PKD2, encoding Polycystin-1 (PC1) and Polycystin-2 (PC2) are causative. Whilst the mechanisms underlying cyst development remain unclear, recent work demonstrates abnormal actin organisation modulated by Rho GTPases. We have identified the RhoGAP protein, SH3BP1, as a novel interactor of PC1 through a mammalian-2-hybrid screen. SH3BP1 is a RhoGAP with selectivity for the Rho-GTPases Cdc42 and Rac1, converting them to their inactive GDP-bound states. SH3BP1 can modulate cell-cell junction formation, directional cell migration and endocytosis. In ADPKD cyst development, a number of these processes are known to be defective, suggesting that a functional SH3BP1-PC1 interaction could be a major regulator of actin cytoskeletal dynamics.

Methods:
Analysis of protein-protein interactions was performed using immunoprecipitation techniques and recombinant tagged protein expression. Renal epithelial mutant cell lines were generated using the CRISPR-CAS9 system. Endocytosis was evaluated through incubation in 3kDa Fluorescein-conjugated dextran before analysis via fluorescence-activated cell sorting (FACS).

Results:
An interaction between SH3BP1 and the C terminus of PC1 has been confirmed in PC1 and SH3BP1. Knockout SH3BP1 renal epithelial cell lines have been generated and are currently undergoing phenotypic analysis alongside PKD1 knockout cells and wild type controls.

Conclusion:
This work identifies a novel interaction between PC1 and SH3BP1 which may have a functional relevance in cyst development. The potential link between SH3BP1 and cyst development will be explored through analysis of SH3BP1 and PKD1 mutant cell lines.

Lay summary

Lay title: Investigating how cell structure and behavior might contribute to cyst growth in autosomal dominant polycystic kidney disease

Background:  
Autosomal dominant polycystic kidney disease (ADPKD) is the most common kidney disease which is passed down through families. Patients with ADPKD develop fluid-filled cysts in their kidneys leading to kidney failure in some patients. ADPKD is usually caused by a fault in the proteins Polycystin-1 (PC1) or Polycystin-2 (PC2). We do not know why problems with these genes cause cysts, but recent work shows that PC1 can affect the internal scaffolding of cells causing changes in their shape and behaviour.

Your methodology - what have done in your study? 
We have used kidney cells in the lab to look at how PC1 interacts with other proteins that might affect cyst growth. We have also been able to look at how PKD cells take in material from its surrounding environment.

Results:  What have you discovered?  
We have discovered that PC1 can interact with another protein, SH3BP1, which also regulates how cells behave. By making kidney cells without SH3BP1 we can explore if it affects cyst growth. We have also seen changes in how PKD cells take up material from around the cell which may relate to how cysts develop.

What are the potential benefits that this research could bring to patients? 
By exploring these processes further, we hope to better understand how cysts develop and identify new drug targets that can be used for future treatments.

Authors and affiliations:  
John P Stone^1,2, William R Cowey^1,2, Corban JT Bowers^1,2, Amy F Stewart^1,2, Erin R Armstrong^1,2, Marc Clancy⁵ Timothy R Entwistle^1,2, Kavit Amin^1,2,3,4 and James E Fildes

¹The Ex-Vivo Research Centre, Alderley Park, Macclesfield, UK
²Pebble Biotechnology Laboratories, Building 3, Alderley Park, Macclesfield, UK
³ Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.
⁴ Department of Plastic Surgery & Burns, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK.
⁵Department of Renal Transplantation, NHS Greater Glasgow and Clyde-Queen Elizabeth University Hospital, Glasgow, UK.
⁶The Healthcare Technologies Institute, School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.

Background: 
Normothermic machine perfusion (NMP) offers a superior alternative to existing hypothermic preservation strategies but is currently limited to 1-3 hours. Extending the time a kidney can be sustained using this technology could electivise transplantation, and enable physiological assessments of renal function. We aimed to develop a protocol that allows the safe preservation of donor kidneys for 12 hours using this technique.

Methods:
Porcine kidneys (n=20) were retrieved and flushed with 1L preservation solution before being stored on ice. Following a cold ischaemic time of 3.5 hours, kidneys were placed onto a NMP circuit and perfused for 12 hours. Renal haemodynamics, biochemistry and urine output were recorded and analysed. At the end of perfusion, kidneys were scored based on the clinical assessment score and their suitability for transplant determined. Biopsies were collected at the end for histological assessment.

Results: 
All kidneys were successfully reperfused with immediate recordable renal blood flow (RBF). RBF continually improved over the course of the perfusions, peaking at 12 hours, and negatively correlated with intra-renal resistance. Perfusate sodium concentrations remained stable and within physiological parameters. Sodium bicarbonate increased over time with a corresponding decrease in lactate concentrations, demonstrating active renal gluconeogenesis. Urine production began immediately in all kidneys and was sustained, indicating a maintenance of functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury-free with a REMUZZI score of 0.

Conclusion:  
We have developed a NMP protocol that safely preserves donor kidneys for 15.5 hours. Successful perfusion was achieved with stable haemodynamics, blood-perfusate biochemistry, and maintained urine output. Importantly, kidneys remained in optimal health, with no evidence of injury. This protocol may enable the electivisation of transplantation, while reducing ischaemic injury associated with static cold storage.

Lay summary 

Lay title: Keeping Kidneys Healthy: Safely Preserving Donor Kidneys for 12 Hours with New Technique

Background: 
Maintaining donor kidney health before transplantation is crucial. Presently, kidneys are preserved on ice which can harm them. An alternative technique, "normothermic machine perfusion," involves circulating blood through kidneys to warm them and provide nutrients. However, it's limited to 1-3 hours. Our goal was to extend this to 12 hours, offering flexibility for transplants and enhancing post-transplant organ performance.

Your methodology - what have done in your study?
Kidneys were collected from abattoir pigs and stored on ice for a short period. They were then connected to a machine that copies the body's conditions, keeping them healthy and functioning for 12 hours. Blood flow, chemicals, and urine production were monitored continuously to see how well the kidneys were doing. Kidney tissue was collected after 12 hours to look at how healthy it was.

Results:  What have you discovered? 
Kidneys quickly received sufficient blood flow upon connection, improving over 12 hours, indicating better function. Blood chemistry remained stable. All kidneys consistently produced urine, showing maintained function. After 12 hours, kidneys were suitable for transplant.

What are the potential benefits that this research could bring to patients? 
A 12-hour normothermic machine perfusion protocol was developed, preserving kidney health. This promising technique could improve organ transplant safety and success by extending the time kidneys remain viable before surgery.

Katie Mylonas

Authors and affiliations:   
Ross A Campbell¹, Marie-Helena Docherty¹, David P. Baird¹, David A Ferenbach¹, Jeremy Hughes¹ and Katie J Mylonas¹.

¹Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, 4-5 Little France Drive, Edinburgh EH16 4UU, Scotland, United Kingdom.
 
Scientific summary 

Background:  
Senescent cells (SCs) accumulate in the kidney with age/injury. They are metabolically active, promoting inflammation/fibrosis via release of senescence associated secretory phenotype (SASP) cytokines. We have shown ageing induces renal epithelial senescence, with administration of the senolytic drug ABT- 263 prior to renal ischaemia reperfusion injury (IRI) being protective; reducing fibrosis and inflammation, whilst promoting preserved function. ABT-263 also reduces macrophage numbers. I hypothesise that SCs compromise renal repair/regeneration after IRI in mice by driving excessive monocyte/macrophage recruitment and negatively influencing macrophage phenotype.
Methods:

In vitro: human proximal tubular epithelial cells (PTECs) were irradiated with 10 gy radiation, to induce senescence. Chemokine CCL2, phospatidylserine (PS), a pro-phagocytic ‘eat me’ signal, and CD47, an anti-phagocytic ‘don’t eat me’ signal, were measured by various methods.

Macrophages were cultured from healthy human volunteer blood and exposed to the supernatant (conditioned medium; CM) of senescent or control PTECs. The phagocytic receptor, MerTK, was measured by flow cytometry (FC).

In vivo: macrophages were interrogated in old, young and young mice with inducible renal senescence (TG) by FC and immunofluorescence. IRI was carried out on WT and CD47-/- mice, with nephrectomy of the CLK at 5 weeks. Kidney function was measured via serum cystatin c 6 weeks post-IRI.

Results:   
In vitro: senescent PTECs produced significantly increased levels of the chemokine CCL2 (p<0.01) and PS (p<0.01), but also CD47 (p<0.001). Macrophage MerTK was reduced after exposure to senescent CM vs control (p<0.05).

In vivo: old and TG mice had more renal inflammatory macrophages than young WTs (p<0.05).
CD47-/- mice had better kidney function after 6 weeks of IRI than WTs(p<0.01) indicating that SC clearance by macrophage can be enhanced with positive results in the kidney.

Conclusion:  
SC negatively affect macrophage phenotype/function. These studies will establish the foundation for immune-enhancing therapies in disease to reduce renal fibrosis.

Lay summary  

Lay title: Exploring how harmful senescent cells interact with the immune system in order to promote kidney repair.

Background:  
Harmful senescent cells accumulate with age/injury. Removing them increases healthy lifespan in mice. Ageing mice had increased kidney cell senescence which was reversed with the novel drug ABT-263 that destroys senescent cells. ABT-263-treatment protected kidneys from scarring after injury.
White blood cells of the immune system called macrophages appear in the kidney after injury to aid healing. However, excessive macrophages can also be damaging.
I propose that the ability of these macrophages to promote kidney repair is negatively affected by the presence of senescent cells. I will test whether I can enhance kidney repair by changing how these cells interact with each other.

Your methodology: what have you done in your study?  
We have measured:
- factors produced by senescent cells, and macrophages that have interacted with senescent cells
- levels of macrophages in the kidneys of mice that have lots of senescent cells
- kidney function in mice where macrophages can clear senescent cells more easily.

Results: what have you discovered?  
Senescent cells change the way macrophages work, affecting how they help heal injured kidneys.

What are the potential benefits that this research could bring to patients? 
I hope that these studies will establish the foundation for therapies to improve how macrophages help heal the injured kidney.

Winnie Chan

Authors and affiliations:   
Winnie Chan^1,2, Carolyn Greig¹, Richard Borrows^2,3
1School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham
²Department of Nephrology, University Hospitals Birmingham NHS Foundation Trust
³School of Immunity and Infection, University of Birmingham

Scientific summary  

Background:  Kidney transplantation is the treatment of choice for patients reaching end-stage renal disease.  In addition to rectifying uraemia and metabolic complications contributing to an overall sense of well-being, kidney transplantation improves mortality, morbidity and quality of life.  After successful transplantation, many patients regain some physical capacity, but frailty represents a prevalent phenomenon, affecting >50% of this population.

Frailty is a state of increased vulnerability to physical stressors.  It is characterised by progressive and sustained degeneration in multiple physiological systems, and is associated with worsened mortality, morbidity and quality of life in kidney transplant recipients. To date, no studies have evaluated the effects of resistance exercise training (RET) combined with protein supplementation (RETPS) in ameliorating frailty in these patients.  This randomised controlled feasibility study explores 1] the feasibility of implementing a novel low-cost home-based RET programme with/without protein supplementation; 2] the effects of RET with/without protein supplementation on frailty and functional status, muscle strength, and quality of life; 3] the cost-effectiveness of RETPS compared to RET alone; and 4] the potential efficacy measures.

Methods and Results:  A total of 40 clinically stable kidney transplant recipients will be enrolled and randomly assigned to either a RET group (n=20) or a RETPS group (n=20), each with a 12-week intervention period.  Measurements will be undertaken pre- and post- intervention, including assessments of study feasibility, frailty and functional status, muscle strength, muscle thickness, body composition, kidney function, inflammation, cardiometabolic status and patient-reported outcome measures.

Conclusion:  This study sets out to introduce a holistic approach, incorporating optimum and safe level of protein supplementation with achievable and effective home-based exercises, thereby improving strength and combating frailty in kidney transplant recipients.  Ultimately, this study will inform the design of a full-scale randomised controlled trial to investigate the impact of RET and RETPS in kidney transplantation.

Lay summary  
  
Lay Title:  Can home-based exercise and protein supplements improve frailty in kidney transplant recipients?

Background:  
Kidney transplantation is the preferred treatment for advanced kidney disease. It restores the ability to eliminate body toxins, prolongs longevity and improves quality of life.  However, frailty remains a common problem after transplantation, accounting for recurrent hospitalisation and death.

No studies have evaluated the effects of resistance exercise training with or without protein supplementation on frailty status in kidney transplant patients. This study aims to answer 4 research questions:  1. Is it feasible to implement a home-based resistance exercise training programme?  2. What is the impact of resistance exercise training with or without protein supplementation on frailty status?  3. Is combining protein supplementation with exercise training cost-effective?  4. What are the potential efficacy measurements?

Your methodology: what have you done in your study? 
Forty kidney transplant patients will be randomly assigned to either resistance exercise training only, or resistance exercise training in combination with protein supplementation for 12 weeks.  Measurements will be undertaken before and after the intervention including assessments of feasibility, frailty status, functional status, muscle strength, muscle thickness, body composition, kidney function, inflammation, cardiometabolic status, and patient-reported outcome measures.
  
Potential Benefits:  This study has the potential to introduce a holistic approach, combining the correct and safe level of protein supplementation with achievable home-based exercises to combat frailty and inform future larger-scale studies.

Dr Jessica Kepple

Authors and affiliations:   
Jessica D. Kepple¹², Phalguni Rath¹, Katherine R Bull¹²³

Wellcome Centre for Human Genetics1, Nuffield Department of Medicine², MRC Human Immunology Unit, Radcliffe Department of Medicine³; University of Oxford, Oxford, UK.

Scientific summary  
Background: SHDRA or “structural heart defects and renal anomalies syndrome” is a rare genetic disorder arising from mutations in the TMEM260 gene, encoding a broadly expressed endoplasmic reticulum-bound O-mannosyltransferase protein. Infants with biallelic mutations have severe multiorgan abnormalities, including early-onset renal impairments and defects, with high paediatric mortality rates. Given the patient renal phenotypes, we hypothesize that TMEM260 is a novel gene required for early renal development.

Methods: To examine the renal function of human TMEM260 variants and isoform-specific deletions, we are generating TMEM260 deficient renal organoids from induced pluripotent stem cells (iPSC) using CRISPR-Cas9 gene editing. We will assess morphology, gene expression, and proteomic changes in control, full deletion, and TMEM260 variant organoids. Immunofluorescent imaging will elucidate cellular changes in tubular and glomeruli structures at serial developmental timepoints. Single-cell RNA Sequencing (ScRNA-Seq) will reveal cell type specific transcriptional changes, supported by mass spectrometry for 4000+ proteins.

Results: We have generated RNA sequencing data from human renal transplant donors demonstrating broad renal TMEM260 expression. We performed analysis mapping known human mutations to the protein structure, revealing most mutations map to the ER luminal domain involved in substrate binding. We have established a complete TMEM260 deficient renal organoid model, with the deletion confirmed by Sanger sequencing and qPCR. Assessment of gross organoid morphology revealed variable structures and sizes between mutants and controls. Organoids lacking TMEM260 have reduced expression in developmental genes including DAPL1, SIX1, and GATA3. Further assessment is being done to determine cell specific changes upon TMEM260 loss in the developing organoid.

Conclusion: Overall, this project will provide novel insights into renal TMEM260 function, with possible implications for alternatively impacted organ systems and gene discovery. Having established an organoid model for complete TMEM260 loss, we seek to develop human variants and isoform-specific deletion models to measure cellular interactions and identify altered regulatory pathways.
  
Lay summary  
  
Lay Title:   
Exploring TMEM260 as a novel protein required for kidney development.

Background:
The rare disease SHDRA or “structural heart defects and renal anomalies syndrome” is caused by loss of the gene TMEM260, leading to severe developmental defects in the heart, brain, and kidney and death in early childhood. Currently, we know very little about how TMEM260 loss causes disease. We predict that TMEM260 is important for normal kidney development, with its loss leading to significant structural and functional changes.

Your methodology: what have you done in your study?  
To determine TMEM260 function, we have deleted it in human cells that we then programmed into kidney organoids, miniaturised kidneys in a dish. We are measuring how TMEM260 loss affects kidney structures, genes, and proteins in the different cell types.

Results:  what have you discovered?  
We found that TMEM260 is present in many human kidney cells. Organoids that lack TMEM260 grow visibly different structures and sizes to controls and have lower amounts of important developmental genes. We are working to further explore cell changes in these kidney organoids.

What are the potential benefits that this research could bring to patients?   
Given the unknown function of TMEM260 in human kidneys, this project will advance our understanding of its role in kidney development, expand the use of organoids as a tool to investigate kidney disease, and may lead to uncovering new targets for SHDRA treatments.

Louise Oni

Authors and affiliations:   
Alexandra Weightman¹, Andrew J. Chetwynd¹, Louise Oni^1
1University of Liverpool & Alder Hey Children’s Hospital, Liverpool

Scientific summary  

Background:   
A limitation to understanding rare diseases is capturing every patient. In high throughput discovery science (‘omics’) large amounts of data can be collected from small biosample volumes (<100µL). Micro-sampling is therefore an interesting approach to mechanistic studies. Dried blood spots (DBS) are collected via a finger prick. This technique allows patients to collect samples from home and return by post, saving time and money. A growing theme is the expansion to other biofluids such as urine and saliva. This work aimed to evaluate the perception of the use of at home dried biofluid collection.

Methods: We devised a 23-item questionnaire distributed to members of Kidney Research UK’s`s ‘Kidney Voices for Research’.

Results: In total 86 people responded, 95% expressed that they would provide a dried biofluid from home. In addition, 80% of patients replied stating that it would increase their likelihood to take part in medical research with the remaining 20% saying they would have volunteered regardless. Greater than 99% reported use of at home testing ranging from Covid-19 screening to DNA testing for ancestry research. The majority of patients reported willingness to provide dried blood (90.7%), urine (95.3%) and saliva (95.3%) with an overall preference in collecting samples monthly (35%-36%).

Patients were more willing to provide urine and saliva samples (weekly if needed) compared to blood sampling, this may be a result of the more invasive nature of a finger prick test. Patients cited that this method was ‘convenient’, ‘easy to do’ ‘no blood taken from vein’ hence ‘saving’ them for future.

Conclusion:  
Patients are supportive of at home biofluid micro-sampling. This enhances equity in involvement in clinical research and increases opportunities to monitor diseases.

Lay summary  

Lay title: What do people with kidney disease think of taking ‘microsamples’?

Background:  
A lot of people are unable to help with kidney research because they live a long way from a research hospital or cannot afford the time or money on travelling to give samples. This makes it hard to do research that is reflective of the true kidney patient populations.

Your methodology: what have you done in your study?  
To fix this we are interested in patients collecting small amounts of blood, urine and saliva from home and posting them back to us once they have dried. To see if this idea is popular with patients, we asked the Kidney Research UK Kidney Voices for Research members to complete a questionnaire looking at different aspects of this work.

Results: what have you discovered?  
Over 95% of patients are happy to provide samples this way with 80% saying they are more likely to give samples for research this way. They were happy to provide blood (91%), urine (95%) and saliva (95%) with around 80% of patients willing to provide a sample at least once a month.

What are the potential benefits that this research could bring to patients? 
These findings show that kidney patients are happy to provide samples from home and post them back. This makes it easier for patients and researchers to collect samples more frequently to track disease. It also reduces barriers to taking part in research and includes people from all locations and backgrounds.

Manjo Valluru

Authors and affiliations:
Valluru MK¹, Genomics England Research Consortium, and Ong ACM^1
1Division of Clinical Medicine, University of Sheffield

Scientific summary  

Background: ADPKD is generally an adult-onset disease caused by germline variants in PKD1 and PKD2.  However, ADPKD can rarely present in infancy or early childhood and occasionally in late childhood or early teenage years. We recently reported that 70% of cases with very-early onset ADPKD (age<18 months) had biallelic PKD1 variants, most commonly a pathogenic variant in combination with a hypomorphic variant (Durkie et al, 2021; 2023). To investigate the potential of other known or novel genes to contribute to an early-onset-ADPKD phenotype (2-16 Years), we hypothesised that an early-onset clinical presentation was more likely to be associated with a digenic or oligogenic inheritance. To address this question, whole genome sequencing data from young ADPKD patients (age<16yr) recruited to the UK 100,000 Genomes Project (100K GE), found to carry a PKD1 variant, was analysed alongside that of one or both parents.

Methodology: The inclusion and genotyping of participants in the 100K GE were managed by Genomics England Limited (GEL). Initially, affected PKD1 cystic kidney disease Trio or Duo probands were filtered based on AGE<16 and analysed in GEL Research Environment. Recorded data were subjected to downstream bioinformatics analysis.

Findings: 21 PKD1 positive cases under 16 years were identified in 100K GE. Ten cases had a negative family history of PKD, and no cases of very early-onset-ADPKD were found. Of these, 6 were monoallelic, 2 were biallelic and 13 were digenic or oligogenic. Unlike the very early-onset-ADPKD cohort, only two children with early-onset-ADPKD in the 100K GE cohort had biallelic PKD1 variants. We identified 24 PKD1 variants inherited in trans with a second gene variant derived from 21 other genes (REVEL >0.4). Of these, 10 were known to be associated with PKD or congenital anomalies of the kidney and urinary tract (CAKUT) phenotypes and 11 were associated with other renal syndromes. These digenic variants could be strong candidates as potential genetic modifiers of the human ADPKD phenotype.

Lay summary 

Lay title: Exploring Genetic Factors in Childhood-onset Autosomal Dominant Polycystic Kidney Disease

Background:  ADPKD most commonly presents in adults but can rarely occur in infancy, childhood, or teenage years. Our recent research found that ADPKD presenting in infancy (<18 months) is commonly due to two PKD1 gene faults, often a combination of a harmful and a milder change.

To better understand whether this observation extended to children, we investigated whether faults in other genes might contribute to ADPKD presenting in childhood. We suspected that when ADPKD starts early in life, other genes could be involved.

Your methodology: what have you done in your study?   
We obtained genetic data from young ADPKD patients and their parents recruited in the UK's 100,000 Genomes Project (age<16years) and analysed children with a PKD1-related gene variation alongside their parents' genes.

Results:  what have you discovered?   
Among the participants, 21 with PKD1 gene changes were identified. Of significance, changes in 24 other kidney genes were present in the majority of these cases, often inherited from the unaffected parent. Of interest, these genes had been linked previously to other kidney diseases.

What are the potential benefits that this research could bring to patients? 
Our study uncovers a strong genetic basis for childhood-onset ADPKD. This insight could lead to better ways of identifying, managing and treating the disease in children.

Andrew Streets

Authors and affiliations: 
Streets AJ¹, Valluru MK¹, Chang L1, Simms RJ¹, Pei Y2 and Ong ACM^1
1Division of Clinical Medicine, University of Sheffield
²Division of Nephrology, University of Toronto

Scientific summary 

Background: ADPKD is the most common inherited cause of kidney failure, caused by germline mutations affecting PKD1 and PKD2 in >90% of patients. The approval of tolvaptan as a disease-modifying therapy in ADPKD patients at risk of rapid disease progression has highlighted the need to develop more accurate prognostic models. We recently showed that global urine extracellular vesicles (UEV) miRNA profiling was highly informative in this regard (Magayr TA, et al. Kidney Int. 2020). In combination, five miRNAs improved the diagnostic performance (AUC) of ultrasound mean kidney length (US-MKL) to discriminate patients with >3ml/min/yr and <3ml/min/yr annualised historical estimated glomerular filtration rate (eGFR) slopes over 5 years. In this study, we report the refinement of differentially expressed (DE) UEV miRNA between healthy controls and ADPKD patients in an independent validation cohort enriched for patients with early disease as the next step to validating its use in prognostic models.

Methodology: In silico bioinformatics analysis was performed to identify a panel of DE UEV miRNAs that discriminate between healthy controls and ADPKD patients. Panel miRNA screening by TaqMan qPCR was conducted in UEV microRNAs isolated from an independent validation cohort (n=70) comprising healthy volunteers (n=25), ADPKD patients with early (eGFR>60ml/min, n=35) or late (eGFR<60ml/min, n=10) disease. Based on this focused panel, bioinformatics analysis was performed to identify the major predicted miRNA-mRNA pathways altered in ADPKD datasets.

Findings: Of 21 DE miRNAs (Panel, AUC>0.8), seven miRNAs were found to be altered in the validation cohort. Furthermore, the combination of five of these improved the classification of early (AUC=0.76) and late (AUC=0.93) disease by ROC analysis. From miRNA-mRNA network analysis, we identified 90 targets altered in ADPKD. Over-representation analysis revealed enrichment for altered Cell-migration/adhesion/matrix and Rho-GTPase-signalling Genesets. Our results have validated a focused panel of five UEV miRNAs that can discriminate ADPKD patients from controls.

Lay summary 

Lay title: Spotting Kidney Disease Early: A Urine Test for Rapid ADPKD progression

Background:  
A common genetic kidney disease called ADPKD can lead to kidney failure. Scientists are working to develop better ways to predict the progression of this disease. One promising approach involves studying tiny particles called extracellular vesicles (UEVs) in urine. These UEVs contain molecules called microRNAs (miRNAs) that can provide important information about the disease. Our previous study found that analysing specific miRNAs in UEVs from urine samples could help predict the disease's severity.

Your methodology: what have you done in your study? 
To improve prediction, we tested urine samples from a new group using a selected panel of miRNAs.

Results: what have you discovered?  
We identified seven miRNAs that are altered between healthy people and ADPKD patients. By combining five of these miRNAs, it may be possible tobetter classify patients with early and late stages of the disease. We also looked at the connections between miRNAs and the genes they influence. We found that target genes are involved in cell movement, adhesion, and communication.

What are the potential benefits that this research could bring to patients, whether that be in the near or distant future?
We have found a way to analyse specific miRNAs in urine samples to predict the severity of ADPKD. This could help doctors identify patients at risk of rapid disease progression and provide them with more targeted care. The miRNAs we identified offer insights into the underlying processes involved in the disease.