[Show abstract][Hide abstract] ABSTRACT: The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
[Show abstract][Hide abstract] ABSTRACT: Background: The reprogramming of somatic cells into iPSCs provides potential to model human diseases in vitro, as has been demonstrated in the cardiac and neuronal fields. Nephrology has not yet benefited from these advancements primarily due to lack of a robust kidney differentiation protocol.
Methods: We aimed to generate and characterize iPSCs from patients with different GRDs. Families with clinically diagnosed GRD were recruited from a Renal Genetics Clinic for concurrent research-based massively parallel sequencing (MPS) and iPSC generation. One affected and unaffected member of each family was recruited. To establish transgene-free iPSC lines, fibroblasts were isolated via skin biopsy and reprogrammed using non-integrating Sendai virus (21day protocol).
Results: 7 families (14 participants) were recruited with a variety of GRD diagnoses. Fibroblast culture was successful in 6 families. Established iPSC lines have typical hESC-like morphology and express pluripotency markers after 4 (TRA1-60) and 15 passages (NANOG). Moreover, iPCS lines have cleared the Sendai virus vectors, as confirmed by RT PCR after only 7 passages. G-band analysis of 2 lines from each isolation confirmed that each of the derived lines had maintained the normal karyotype after reprogramming. Paired patient and control iPSC are being redifferentiated towards kidney employing an established protocol. Renal organoids will be analyzed using IF, FACS and transcriptional profiling.
Conclusions: Patient derived iPSC have been generated and renal redifferentiation commenced. We hope to uncover the biological consequences of novel genetic variants causing GRD as identified via MPS, thereby beginning to explain patient phenotypes and disease pathogenesis.
American Society of Nephrology 2015 Kidney Week; 10/2015
[Show abstract][Hide abstract] ABSTRACT: Background: Genetic Renal Disease (GRD) accounts for 10% of adults and 50% of children with end stage kidney disease. Advances in molecular genetic diagnostic testing and multidisciplinary renal clinic models suggest opportunities for clinical redesign. Here we describe the initial characteristics, experience and outcomes of the first Australian multidisciplinary RGC.
Methods: A retrospective audit was undertaken of the RBWH Adult Conjoint RGC (1st year of operation; Aug’13-Jul’14). Each encounter involved a nephrologist, clinical geneticist and genetic counselor.
Results: 27/48 patients (56%) encountered had a known GRD, 9/48 (19%) had a suspected GRD and 12/48 (25%) had an unknown GRD with positive family history (FHx). Patients were most commonly referred by nephrologists (66%).The most common GRD category & diagnosis were cystic kidney disease (49%) and Autosomal Dominant Tubulointerstitial Kidney Disease (25%). Extra-renal features were associated with GRD in 26/48 (54%). During consultations differential diagnoses were explored (54%), management advice provided (83%) and genetic counseling undertaken (79%). A genetic test was requested in 58.3%, most commonly being indicated for combined diagnostic and genetic counseling reasons (78.6%). Of 12 returned genetic test results, 7 were positive and 1 was a variant of uncertain significance. Two negative results have prompted additional genetic testing. Of 27 patients referred with a known GRD, 6 now have a new suspected GRD. The majority of those referred with either a suspected (6/9) or unknown GRD diagnosis (10/12) now have a new suspected or confirmed GRD.
Conclusions: These findings demonstrate the early successful operation of this RGC model including diagnostic and genetic counseling benefit. Future assessment of clinical outcomes and expansion to telehealth and paediatric settings is anticipated.
American Society of Nephrology 2015 Kidney Week; 10/2015
[Show abstract][Hide abstract] ABSTRACT: Background: GRD accounts for 10% of adults and 50% of children with end stage kidney disease. An unknown proportion of cases remain refractory to current clinical genetic tests. Advances in MPS enable attempts to address this in a research setting. We aimed to utilize MPS with pedigree analysis in unresolved cases of GRD.
Methods: Families with clinically diagnosed GRD were recruited in whom clinical genetic testing was either negative or not available. Whole exome sequencing was undertaken and analyzed with custom in house bioinformatics assessment tools accounting for all inheritance patterns. Identified variants of interest were confirmed using Sanger sequencing in clinical laboratories.
Results: 14 families (62 participants) were recruited with a variety of GRD diagnoses and modes of inheritance. A molecular genetic diagnosis has been resolved in 5 families. These include mutations in RMND1 (compound heterozygous), IFT140 (compound heterozygous), HNF4A (heterozygous), COL4A5 (hemizygous) and tRNA(Phe) (mtDNA) genes. All have been confirmed in a clinical diagnostic laboratory and reported back to the participants with further genetic counseling. Research continues in the remaining 9 families, including application of whole genome sequencing. No reportable incidental genetic findings were identified.
Conclusions: MPS confirmed and clarified a clinical genetic diagnosis in 5/14 families with previously and diagnostically refractory GRD. These results were integrated into clinical practice and demonstrate an emerging role for MPS in the delivery of clinical care. Further studies are required to resolve the remaining families.
American Society of Nephrology 2015 Kidney Week; 10/2015
[Show abstract][Hide abstract] ABSTRACT: Aim: To investigate the genetic aetiology of deafness, lactic acidosis (LA) and nephropathy in Australian patients.
Background: RMND1 is an integral inner membrane mitochondrial protein. Mutations in RMND1 cause an autosomal recessive mitochondrial oxidative phosphorylation deficiency disorder recently phenotypically expanded to encompass syndromic nephropathy.
Methods: Two Australian patients with global developmental delay (GDD), deafness, LA and nephropathy underwent whole exomic sequencing (WES).
Results: Patient 1 presented in the neonatal period with seizures and sensorineural hearing loss (SNHL) having been born at 24 weeks gestation. The child had tubulopathy with polyuria, hyponatraemia, hyperkalaemia, metabolic acidosis (LA), positive sweat test (negative CFTR analysis) and progressive renal impairment. Renal ultrasound showed normal sized echogenic kidneys. Renal biopsy demonstrated chronic tubulointerstitial nephritis and nephronophthisis. WES identified compound heterozygous RMND1 mutations (c.[713A>G][829_830del];p.[ Asn238Ser][ Glu277Glyfs*20]). At 4years, GDD continues to improve, bilateral cochlear implants have been successful and renal impairment continues to progress (eGFR 33 ml/min/1.73 m2).
Patient 2 was born at term by normal delivery and had unremarkable neonatal course except for bilateral SNHL. At 5 months of age, he was noted to have generalised hypotonia, poor weight gain, GDD, LA and 3-methylglutaconic aciduria in the context of a viral illness. A homozygous RMND1 mutation (c.[1349G>C];p.[*450Serext*32]) was identified by WES (Melbourne Genomics Health Alliance). Subsequent screening for renal disease showed hyperkalemia and renal hypodysplasia. At 18 months, he is globally delayed and hypotonic.
Conclusions: These cases reinforce the relationship between RMND1 mutations and autosomal recessive nephropathy associated with SNHL and LA, whilst demonstrating the utility of WES in diagnosis. Tubulointersitital changes on renal biopsy suggest a potential relationship with other forms of ciliopathy. This and how oxidative phosphorylation deficiencies mediate renal disease require further investigation.
[Show abstract][Hide abstract] ABSTRACT: Aim: To identify the genetic cause of genetic renal disease (GRD) in a large Australian family whose pedigree is consistent with autosomal dominant or mitochondrial inheritance.
Background: Mutations in tRNA(Phe) are rarely reported to cause renal disease, usually with syndromic features such as epilepsy and myopathy.
Methods: A large Australian family with GRD underwent whole exomic sequencing (WES) with mtGenome capture, and mtDNA sequencing.
Results: The family has extensive kidney disease historically identified as “MedullaryCystic Kidney Disease”. Six surviving members receive renal replacement therapy or have a functioning renal transplant. Seven further deceased family members had Chronic or End-Stage Kidney Disease. No clear history of an extrarenal phenotype is apparent, though one affected family member has an undiagnosed oromotor dysphagia parkinsonian syndrome and another died of apparent herpetic encephalitis. Renal histopathology in three affected family members >30 years ago demonstrated tubulointerstitial disease with mild tubular dilatation and some corticomedullary cyst formation. WES did not reveal any candidate variants in the nuclear genome. mtDNA sequencing identified the T616C mutation in the anticodon loop of tRNA(Phe). This mutation was homoplasmic in blood and present in all affected family members including 4th degree cousins. Northern blot demonstrated decreased phenylalanine tRNA in affected fibroblasts compared to wild-type control fibroblasts. A respiratory chain defect was present in affected fibroblasts and cybrids compared to wild-type, confirming the mitochondrial origin.
Conclusions: Here we confirm the T616C tRNA(Phe) mutation as causative for Mitochondrially Inherited Tubulointerstitial Kidney Disease, in the apparent absence of extrarenal syndromic features. This has implications for genetic counselling and demonstrates the utility of collaborative genetic diagnostic approaches. The pathobiology of how such mitochondrial mutations cause renal disease requires further research.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND:
Genetic renal diseases (GRD) are a heterogeneous and incompletely understood group of disorders accounting for approximately 10 % of those diagnosed with kidney disease. The advent of Next Generation sequencing and new approaches to disease modelling may allow the identification and validation of novel genetic variants in patients with previously incompletely explained or understood GRD.
This study will recruit participants in families/trios from a multidisciplinary sub-specialty Renal Genetics Clinic where known genetic causes of GRD have been excluded or where genetic testing is not available. After informed patient consent, whole exome and/or genome sequencing will be performed with bioinformatics analysis undertaken using a customised variant assessment tool. A rigorous process for participant data management will be undertaken. Novel genetic findings will be validated using patient-derived induced pluripotent stem cells via differentiation to renal and relevant extra-renal tissue phenotypes in vitro. A process for managing the risk of incidental findings and the return of study results to participants has been developed.
This investigator-initiated approach brings together experts in nephrology, clinical and molecular genetics, pathology and developmental biology to discover and validate novel genetic causes for patients in Australia affected by GRD without a known genetic aetiology or pathobiology.
[Show abstract][Hide abstract] ABSTRACT: Aim: To identify a rare genetic cause for atypical autosomal dominant Fanconi renotubulopathy mimicking Dent’s Disease amongst Australian patients.
Background: The HNF4A gene is associated with “Maturity Onset Diabetes of the Young Type 1” however a specific variant has recently been identified causing distinctive autosomal dominant renal tubulopathy. Translation to clinical diagnostic sequencing is desirable.
Methods: Two Australian patients with atypical Fanconi renotubulopathy respectively underwent small-pedigree whole exomic sequencing (WES) and diagnostic clinical exome sequencing via the Australian Renal Genetic Disorders Panels (ARGP) at Westmead.
Results: Patient 1 presented at 10 years with rickets (requiring multi-level orthopaedic procedures), short stature, asymptomatic low-molecular-weight proteinuria with Fanconi renotubulopathy and progressive chronic kidney disease (CKD). Development was otherwise normal with past history of surgically repaired ventricular septal defect at 5 years. Growth hormone therapy precipitated diabetes mellitus, which resolved upon its cessation. Renal biopsy, imaging and CLCN5/OCRL were normal. Small-pedigree WES identified the de novo p.R76W (NM_000457.4): c.[187C>T];[=]) variant in HNF4A.
Patient 2 presented in early childhood with asymptomatic low-molecular-weight proteinuria. She progressively developed Fanconi renotubulopathy, progressive CKD, recurrent morning ketotic hypoglycaemia, hyperaldosteronism, minor osteopenia, subclinical rickets, intermittently raised intraocular pressure, hypermetropia, mild intellectual impairment, generalised joint hypermobility, and pancreatic hyperechogenicity of unknown cause. Renal biopsy, imaging and CLCN5 were normal. ARGP analysis was initially negative, however reanalysis based on recent reports of patients with the p.R76W variant in HNF4A, identified this variant (NM_000457.4):c.[187C>T][=]).
Conclusions: These cases reconfirm the association of this heterozygous variant in HNF4A with atypical Fanconi renotubulopathy whilst demonstrating diagnostic translation and integration into the clinical ARGP service for identification of further cases. Further research into the pathogenic mechanism of this variant is required.
[Show abstract][Hide abstract] ABSTRACT: Modelling of disease via the directed differentiation of human pluripotent stem cells is now becoming feasible for a number of tissue types. We recently reported the stepwise differentiation of human embryonic stem cells and human induced pluripotent stem cells through posterior primitive streak and intermediate mesoderm to simultaneously form both ureteric epithelium and nephron-forming metanephric mesenchymei. We also demonstrated that these component cell types would contribute to multiple renal structures when re-aggregated and cultured in three dimensions. This approach was in contrast to other protocols in which pluripotent stem cells had given rise to either ureteric epitheliumii or nephron forming mesenchymeiii. We have now investigated the growth factor conditions able to optimise ureteric bud versus metanephric mesenchyme derivatives and show that the resulting 3D organoids contain evidence of derivatives of all four progenitor populations required for the formation of the mammalian kidney, including the ureteric, nephron, stromal and vascular lineages. We also show the application of this protocol to iPSC derived from patients carrying identified mutations in kidney disease genes, illustrating the potential utility of such lines for disease modelling.
2015 International Workshop on Developmental Nephrology, Snowbird, Utah, USA; 07/2015
[Show abstract][Hide abstract] ABSTRACT: The reprogramming of somatic cells into induced pluripotent stem cells (iPSC) provides the potential to model human diseases in the laboratory, as has been applied extensively in the cardiac and neuronal fields. Nephrology has not yet benefited from these advancements primarily due to the lack of a robust kidney differentiation protocol. We have recently demonstrated a capacity to differentiate human iPSC to cells which self-organize into kidney organoids containing appropriate mesenchymal and epithelial components, including nephrons.1 Here we report the generation and characterisation of iPSCs from patients with clinical diagnoses of either Nephronophthisis (NPHP) or Medullary Cystic Kidney Disease (MCKD), both cystic kidney disorders with diverse co-morbidities. While many genes have now been associated with cystic kidney disease, the genetic aetiology remains unclear for many patients, including all cases reported here. iPSC have been derived from 5 families (5 probands and matched unaffected relative) in which the proband lacks mutations in any known disease-associated gene. In order to establish transgene-free iPSC lines, fibroblasts were isolated via skin biopsy and reprogrammed using non-integrating Sendai virus in a 21-day protocol. Established iPSC lines have a typical hESC-like morphology and express pluripotency markers after 4 (TRA1-60) and 15 passages (NANOG). Moreover, iPCS lines have cleared the Sendai virus vectors, as confirmed by RT PCR after only 7 passages. G-band analysis of 2 lines from each isolation confirmed that each of the derived lines had maintained the normal karyotype after reprogramming. We now aim to redifferentiate patient and control iPSCs lines towards kidney by employing our previously established protocol (1). Kidney organoids will be analysed using IF and FACS and subjected to transcriptional profiling. We hope that these studies will uncover the biological consequences of novel genetic variants identified via NextGeneration sequencing, thereby beginning to explain the patient pathogenic phenotype.
International Society for Stem Cell Research 2015 Annual Meeting, Stockholm, Sweden; 06/2015
[Show abstract][Hide abstract] ABSTRACT: There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and human macrophage colony stimulating factor (M-CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug-induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK-2) and primary (commercially available and freshly in-house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM-1, NGAL, and M-CSF assessed using ELISA and real-time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK-2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM-1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M-CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug-induced nephrotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Recent studies on the directed differentiation of human pluripotent stem cells report tissue self-organization in vitro such that multiple component cell types arise in concert and arrange with respect to each, thereby recapitulating the morphogenetic events typical for that organ. Such self-organization has generated pituitary, optic cup, liver, brain, intestine, stomach and now kidney. Here, we will describe the cell types present within the self-organizing kidney, how these signal to each other to form a kidney organoid and the potential applications of kidney organoids.
Protocols for the directed differentiation of human pluripotent cells focus on recapitulating the developmental steps required during embryogenesis. In the case of the kidney, this has involved mesodermal differentiation through posterior primitive streak and intermediate mesoderm. Recent studies have observed the simultaneous formation of both ureteric epithelium and nephron progenitors in vitro. These component cell types signal to each other to initiate nephron formation as would occur during development.
The generation of kidney organoids is a major advance in nephrology. Such organoids may be useful for disease modelling and drug screening. Ultimately, our capacity to generate organoids may extend to the development of tissues for transplantation.
Current Opinion in Organ Transplantation 04/2015; 20(2). DOI:10.1097/MOT.0000000000000174 · 2.88 Impact Factor