Towards Computer Aided Management of Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the fourth most common cause of kidney transplant worldwide accounting for 7-10% of all cases. Although ADPKD usually progresses over many decades, accurate risk prediction is an important task. Identifying patients with progressive disease is vital to providing new treatments being developed and enable them to enter clinical trials for new therapy. Among other factors, total kidney volume (TKV) is a major biomarker predicting the progression of ADPKD. Consortium for Radiologic Imaging Studies in Polycystic Kidney Disease (CRISP) have shown that TKV is an early, and accurate measure of cystic burden and likely growth rate. It is strongly associated with loss of renal function. While ultrasound (US) has proven as an excellent tool for diagnosing the disease; monitoring short-term changes using ultrasound has been shown to not be accurate. This is attributed to high operator variability and reproducibility as compared to tomographic modalities such as CT and MR (Gold standard). Ultrasound has emerged as one of the standout modalities for intra-procedural imaging and with methods for spatial localization has afforded us the ability to track 2D ultrasound in the physical space in which it is being used. In addition to this, the vast amount of recorded tomographic data can be used to generate statistical shape models that allow us to extract clinical value from archived image sets. Renal volumetry is of great interest in the management of chronic kidney diseases (CKD). In this work, we have implemented a tracked ultrasound system and developed a statistical shape model of the kidney. We utilize the tracked ultrasound to acquire a stack of slices that are able to capture the region of interest, in our case kidney phantoms, and reconstruct 3D volume from spatially localized 2D slices. Approximate shape data is then extracted from this 3D volume using manual segmentation of the organ and a shape model is fit to this data. This generates an instance from the shape model that best represents the scanned phantom and volume calculation is done on this instance. We observe that we can calculate the volume to within 10% error in estimation when compared to the gold standard volume of the phantom

In Vivo Characterisation of ANKS3- A New Candidate for Ciliopathic Disease

ANKS3 is an Ank and SAM domain containing protein. It interacts with ciliopathy associated NPHP proteins as well as ANKS6 and the RNA binding protein BICC1 which cause polycystic kidney disease (PKD) when mutated in rodents. The ANKS3 SAM domain is capable of assembling homopolymers and forms large complexes with BICC1. This ANKS3 homopolymerisation is prevented by ANKS6-ANKS3-SAM domain binding. The PKD causing Anks6p.R823W mutation prevents ANKS6 from binding to ANKS3 which might result in increased ANKS3-SAM domain homopolymerisation. The function of ANKS3 in mammals is quite unresolved. This thesis aimed to clarify the role of Anks3 in rats and the pathophysiological role of ANKS3-SAM domain homopolymerisation in PKD. To this end, we generated novel rat models, using CRISPR/Cas9, carrying either an Anks3 knockout (Anks3KO), an ANKS3-SAM domain deletion (Anks3ΔSAM) or a missense mutation in the SAM domain (Anks3KI) which prevents ANKS3-SAM domain homopolymerisation but not ANKS3-ANKS6 binding. Furthermore, we crossed the Anks3KI rat into the Anks6p.R823W PKD rat (TGRAnks6) to study the effect of defective ANKS3-SAM domain homopolymerisation in PKD (TGRAnks6-Anks3KI/KI). We provide the following crucial results: 1. The spatial expression pattern of ANKS3 and ANKS6 in the kidney is tightly regulated during development and is disturbed by the Anks6p.R823W mutation in PKD rats. The Anks6p.R823W mutation decreases aquaporin 2 (Aqp2) expression and urinary concentration ability while the defective ANKS3 homopolymerisation in the Anks3KI/KI rats increases them, supporting a role of ANKS3 homopolymerisation in water regulation in the kidney. 2. Defective ANKS3 polymerisation in TGRAnks6-Anks3KI/KI rats, retards cyst growth and reverses most pathways altered in TGRAnks6 PKD rats, including the key signalling pathways Hippo, Wnt and cAMP as well as metabolic pathways. In addition, all DNA replication and repair pathways were upregulated in TGRAnks6-Anks3KI/KI rats vs. TGRAnks6 and vs. wildtype rats. 3. We provide evidence that the ANKS3-SAM domain, but not ANKS3-SAM domain homopolymerisation, is required for proper morphogenesis in embryos. Both, Anks3 knockout and ANKS3-SAM domain deletion, results in an embryonically lethal, ciliopathic phenotype including disturbances in organ morphogenesis and laterality defects. Cilia formation did not appear to be disturbed. Expression profiling in Anks3KO/KO embryos revealed a significant downregulation of DNA replication and repair pathways vs. wildtype embryos, indicating a significant role of ANKS3 in DNA damage response and repair, which becomes crucial during periods of high proliferative stress, including embryogenesis and PKD. Unlike Anks6, the Anks3 mutations did not cause a PKD phenotype. Altogether, in the course of this thesis we provided three novel mutated Anks3 rat models which, for the first time, allow the in vivo study of Anks3 function in mammals and will contribute to further elucidate the molecular pathways of PKD and other ciliopathies, and their interactions

Regulated ion transport in mouse liver cyst epithelial cells

AbstractDerived from bile duct epithelia (BDE), secretion by liver cyst-lining epithelia is positioned to drive cyst expansion but the responsible ion flux pathways have not been characterized. Cyst-lining epithelia were isolated and cultured into high resistance monolayers to assess the ion secretory pathways. Electrophysiologic studies showed a marked rate of constitutive transepithelial ion transport, including Cl− secretion and Na+ absorption. Na+ absorption was amiloride-sensitive, suggesting the activation of epithelial sodium channels (ENaC). Further, both cAMPi and extracellular ATP induced robust secretory responses. Western blotting and immunohistologic analysis of liver cyst epithelia demonstrated expression of P2X4, a potent purinergic receptor in normal BDE. Luminometry and bioassaying measured physiologically relevant levels of ATP in a subset of liver cyst fluid samples. Liver cyst epithelia also displayed a significant capacity to degrade extracellular ATP. In conclusion, regulated ion transport pathways are present in liver cyst epithelia and are positioned to direct fluid secretion into the lumen of liver cysts and promote increases in liver cyst expansion and growth

Molecular genetic studies of inherited cystic kidney disease in Oman

Ph. D. ThesisInherited kidney diseases are fundamental causes of chronic kidney disease (CKD) and end stage kidney disease (ESKD); accounting for approximately 20% of all CKD cases and up to 10% of adults and over 70% of children reaching ESKD. Oman is the second largest country in the South East of Arabian Peninsula. Omani population is characterized by large family size, presence of tribal and geographical settlements and higher rates of consanguineous marriages, which facilitate the study of autosomal recessive disorders. Rare genetic disorders create considerable burden on healthcare system in Oman and are major causes of congenital abnormalities and perinatal deaths in hospitals. The prevalence of inherited kidney disease was estimated to be high, but there is a lack for a comprehensive data. Therefore, this study aimed to evaluate the magnitude of inherited kidney disease in this population and identify the molecular genetic causes of inherited cystic kidney diseases in Omani patients. First, I performed a population-based retrospective analysis of ESKD patients commencing RRT from 2001 to 2015 using the national renal replacement therapy (RRT) registry and evaluated the epidemiological and etiological causes of ESKD with focused attention on inherited kidney diseases. Second, I designed a targeted gene panel (49 genes) and used massive parallel sequencing technologies for the molecular genetic diagnosis of cystic kidney disease in 53 patients. An overall molecular genetic diagnostic yield of 75% was achieved; with 46% of detected causative variants were novel genetic findings. Third, I evaluated the utility of molecular genetic testing in patients with autosomal recessive polycystic kidney disease (ARPKD) and described the clinical and genetic profile of this cohort. Finally, whole exome sequencing (WES) was used to determine the genetic causes of CKD in 11 unrelated children suspected with recessively inherited kidney diseases. Definite genetic diagnosis was achieved in 54.5% of cases, reflecting the importance of genomic implications in those with uncertain aetiology causing CKD. This study creates a solid basis reflecting the genotype-phenotype of some inherited kidney diseases in Omani population and reveals the enormous diagnostic power of genomic technologies.The Research Council (TRC), Ministry of Health (MOH), Ministry of Higher Education (MOHE

Characterisierung der embryonalen Nierenentwicklung in Danio rerio und Eine Funktionelle Analyse von Arl13b/ Scorpion waehrend der Embryonalentwicklung in Danio rerio

Joubert syndrome and Joubert syndrome and related disorders are human diseases that are characterized by brain stem and cerebellar defects but can also be accompanied by polycystic kidney disease, retinitis pigmentosa, situs inversus among other defects. It is crucial to understand the normal kidney development in order to understand the cyst pathogenesis, however not much is known about embryonic kidney development in danio rerio. The present study aimed to elucidate the earliest events during embryonic kidney formation in Danio rerio. We show for the first time that cells of the intermediate mesoderm aquire apico-basolateral polarity, undergo cavitatation and exhibit oriented cilia concurrently around the 12 somite stage and are completed at the 14 somite stage. Furthermore the intermediate mesoderm undergoes extensive elongation and narrowing processes which are not due to proliferation and apoptosis but might be governed by the Planar cell polarity pathway. Arl13b/ Scorpion, a small GTPase, localizes to the cilium in various organs and plays a crucial role for ciliogenesis in the KV, retinal photoreceptor layer and the pronephric duct. As a conseqeunce, the Arl13b mutant or morphant embryos show laterality defects, photoreceptor degeneration due to apoptosis in the outer segment of the photoreceptor layer, duct dilation in the pronephros and kidney cyst formation. Performing careful histological analysis in scorpionhi459 mutant embryos, we could show that hyperproliferation and/ or defects in cell rearrangements occur as a consequence of dilation, and are not causative. In addition, scorpionhi459 mutant embryos extend cilia tufts from multicilliated cells which present a defective basal body alignment that is goverened by PCP but cilia on single ciliated cells can not be detected. Remaning cilia have a normal axonemal ultrastructure as shown by electron microscopy, but exhibit a defective beating pattern. The apicao-basolaterael polarity is not perturbed in mutant pronephric ducts. A deletion analysis of Arl13b/ Sco protein allowed us to study the roles of the truncation proteins in regards to cilia localization and the capability to rescue the mutant phenotype. The GTPase domain, coiled coil region and part of the C-terminus (C-terminal to amino acid 308) were necessary for proper ciliary targeting. We found a close correlation for cilia localization being necessary for the protein being able to rescue the mutant phenotype. In addition, despite the GTPase activity playing no role in trafficking the protein to the cilium, it was crucial for the rescue capability of the protein

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%), p\u3c0.01. Among 218 non-smokers, 130 (59%) were male, 142 (65%) were Caucasian; 60 (28%) reported SHS exposure compared to 158 (72%) with no exposure. Non-smoker adolescents with SHS exposure were compared to those without SHS exposure. There was no racial, age, or gender differences between both groups. Baseline creatinine, diastolic hypertension, C reactive protein, lipid profile, GFR and hemoglobin were not statistically different. Significantly higher protein to creatinine ratio (0.90 vs. 0.53, p\u3c0.01) was observed in those exposed to SHS compared to those not exposed. Exposed adolescents were heavier than non-exposed adolescents (85th percentile vs. 55th percentile for BMI, p\u3c 0.01). Uncontrolled casual systolic hypertension was twice as prevalent among those exposed to SHS (16%) compared to those not exposed to SHS (7%), though the difference was not statistically significant (p= 0.07). Adjusted multivariate regression analysis [OR (95% CI)] showed that increased protein to creatinine ratio [1.34 (1.03, 1.75)] and higher BMI [1.14 (1.02, 1.29)] were independently associated with exposure to SHS among non-smoker adolescents. These results reveal that among adolescents with CKD, cigarette use is low and SHS is highly prevalent. The association of smoking with hypertension and SHS with increased proteinuria suggests a possible role of these factors in CKD progression and cardiovascular outcomes

Genetic and molecular studies in Hyperinsulinemic Hypoglycemia and congenital Polycystic Kidney Disease (HIPKD)

Background: Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence (HIPKD) in 17 children from 11 unrelated families suggested a shared cause. // Methods: We ascertained the clinical phenotype and performed genetic studies. The effect of the identified shared mutation was assessed in vitro. // Results: All patients exhibited HI and enlarged polycystic kidneys. Whole genome linkage analysis in 5 informative families identified a single significant (LOD 6.5) locus on chromosome 16p13.2. A promoter mutation (c.-167G>T) in PMM2 was found in all patients, either homozygous or in trans with PMM2 coding mutations. Yet, typical systemic features of congenital disorder of glycosylation type 1a were absent and the diagnostic test of transferrin isoelectric focusing was normal. The promoter mutation showed decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggests an important role for ZNF143 for the formation of a chromatin loop including PMM2 that could affect tissue-specific transcription. In order to investigate this further in a chromatin conformation study a HIPKD cell model homozygous for the promoter mutation was generated with CRISPR-Cas9. // Conclusions: We report a rare disease characterized by the combination of hyperinsulinemic hypoglycemia and polycystic kidney disease. Our findings extend the spectrum of genetic causes for both disorders, provide insights into gene regulation and implicate glycosylation in the disease etiology. The identified promoter mutation appears critical for tissue-specific regulation of PMM2 transcription, leading to an organ-specific phenotype and explaining PMM2 pleiotropy

Discovery and analysis of genes important in kidney development and disease

Abnormal kidney development is a relatively prevalent health issue; however, the genetic basis is mostly unknown. The aim of this thesis is to identify genes important in kidney development and disease and to study their molecular functions. We hypothesized that human diseases associated with kidney anomalies can uncover novel genes important in kidney development and disease. The thesis is divided into three independent projects that examined three genes (i.e. Zeb2, Ilk, Robo2) at three stages of mouse kidney development: nephrogenesis, glomerular podocyte, and early ureteric bud outgrowth. In the first project, we identified Zeb2, a gene encoding the zinc finger E-box binding homeobox 2 transcription factor that is mutated in the Mowat Wilson syndrome, as a novel gene important in nephrogenesis. Zeb2 conditional knockout mice (Zeb2 cKO) develop glomerulocystic kidney disease with many atubular glomeruli and decreased expression of proximal tubular markers before cyst formation. These data suggest that abnormal nephrogenesis leads to the congenital atubular glomeruli and primary glomerular cysts in the Zeb2 cKO mice. This study implies that ZEB2 is a novel candidate gene for glomerular cystic disease in patients. Additionally we found that Pkd1, the gene mutated in autosomal dominant polycystic kidney disease, is upregulated in non-cystic glomeruli and knockout of one copy of the Pkd1 gene exacerbates the cystic phenotype of the Zeb2 cKO mice. These findings suggest a genetic interaction between Zeb2 and Pkd1 and that Zeb2 might be a novel PKD1 modifier. In the second project, we studied the roles of integrin-linked kinase (ILK) and roundabout 2 (ROBO2) in glomerular podocytes. We found that ILK and ROBO2 form a protein complex, and that loss of Robo2 improves survival and alleviates the podocyte and basement membrane abnormalities seen in Ilk knockout mice. In the third project, using microarray gene expression analysis, we found lower gene expression levels of extracellular matrix proteins during early ureteric bud outgrowth in the Robo2 homozygous knockout embryos as compared to wild type controls. These findings suggest that ROBO2 may regulate extracellular matrix components in the kidney. In conclusion, we found a new role for Zeb2 in nephrogenesis, and identified a novel function of Robo2 in regulating extracellular matrix gene expression in podocytes and during early kidney development.2017-11-03T00:00:00