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Significance Statement Congenital obstructive uropathy (COU) is a prevalent human developmental defect with highly heterogeneous clinical presentations and outcomes. Genetics may refine diagnosis, prognosis, and treatment, but the genomic architecture of COU is largely unknown. Comprehensive genomic screening study of 733 cases with three distinct COU subphenotypes revealed disease etiology in 10.0% of them. We detected no significant differences in the overall diagnostic yield among COU subphenotypes, with characteristic variable expressivity of several mutant genes. Our findings therefore may legitimize a genetic first diagnostic approach for COU, especially when burdening clinical and imaging characterization is not complete or available. Background Congenital obstructive uropathy (COU) is a common cause of developmental defects of the urinary tract, with heterogeneous clinical presentation and outcome. Genetic analysis has the potential to elucidate the underlying diagnosis and help risk stratification. Methods We performed a comprehensive genomic screen of 733 independent COU cases, which consisted of individuals with ureteropelvic junction obstruction (n=321), ureterovesical junction obstruction/congenital megaureter (n=178), and COU not otherwise specified (COU-NOS ; n=234). Results We identified pathogenic single nucleotide variants (SNVs) in 53 (7.2%) cases and genomic disorders (GDs) in 23 (3.1%) cases. We detected no significant differences in the overall diagnostic yield between COU sub-phenotypes, and pathogenic SNVs in several genes were associated to any of the three categories. Hence, although COU may appear phenotypically heterogeneous, COU phenotypes are likely to share common molecular bases. On the other hand, mutations in TNXB were more often identified in COU-NOS cases, demonstrating the diagnostic challenge in discriminating COU from hydronephrosis secondary to vesicoureteral reflux, particularly when diagnostic imaging is incomplete. Pathogenic SNVs in only six genes were found in more than one individual, supporting high genetic heterogeneity. Finally, convergence between data on SNVs and GDs suggest MYH11 as a dosage-sensitive gene possibly correlating with severity of COU. Conclusions We established a genomic diagnosis in 10.0% of COU individuals. The findings underscore the urgent need to identify novel genetic susceptibility factors to COU to better define the natural history of the remaining 90% of cases without a molecular diagnosis.

Introduction: Blood pressure is controlled by kallilkrein-kinin system (KKS) and renin- angiotenzin-aldosteron system (RAAS). In developing kidneys, renin progenitor cells appear in the kidney arterial tree and juxtaglomerular apparatus (JGA), while kallikrein expression characterizes distal nephron. Both renin and kallikrein seem to influence kidney development and maturation. Data on kallikrein expression are conflicting, especially during human kidney development. In this study, we analyze the spatiotemporal and functional relationship between kidney renin and kallikrein during human development, in the postnatal period, and in the nephrotic syndrome of the Finnish type (CNF). Material and methods: The spatiotemporal expression of renin and kallikrein was analyzed in human kidneys by double immunofluorescence techniques in paraffin sections of 10 human conceptuses 8 to 38 weeks old, in 1, 5-year-old healthy kidney tissues and in the CNF. Results: In the embryonic period, renin - kallikrein expression characterized metanephric cup cells, S- form nephrons, immature glomeruli, and collecting ducts (CD). In fetal and postnatal kidneys, kallikrein was partly expressed in distal tubules (DT) and CD, and strongly along connecting tubules (CT), while renin expression characterized DT and CD, and partly CT. Their co-expression was observed in small areas of DT and CT. In CNF kidneys, renin and kallikrein co-expression was stronger and with longer overlapping areas than in the healthy kidneys. Conclusions: Our data suggested the interaction of renin and kallikrein during kidney maturation and in the blood pressure control. The two proteins were predominantly expressed in neighboring structures, while their co-expression characterized CT region. In CNF, their changed expression indicated increased efforts of affected kidneys in the blood pressure control.

Introduction: Primary cilia are non-motile sensory organelles involved in the control of different cellular processes, including cell differentiation. The role of primary cilia has been implicated in numerous cystic kidney diseases. By now, primary cilia have been detected on the surfaces of tubular cells and collecting ducts in developing and postnatal human kidneys. In the present study we analyze appearance and percentage of primary cilia on the surfaces of developing human healthy and nephrotic podocytes. Material and methods: Kidney tissue were dissected from human conceptuses 8th-38th postovulatory week old, postnatal healthy and nephrotic syndrome of the Finnish type (CNF) kidneys. They were stained by double immunofluorescent technique, using α- tubulin as primary cilia marker and DAPI as nuclear marker. Cilia were counted on cell surfaces and their percentage was calculated using Kruskal–Wallis test, followed by Dunns post-hoc test. Ultrastructural characteristics of podocytes were analyzed by transmission electron microscopy. Results: Between the 8th and 10th developmental week, population of metanephric cup cells contained 24% of ciliated cells, immature glomeruli 47% and Bownam’s capsule cells (parietal podocytes) 26%. Percentage of ciliated podocytes decreased to 10% in the 38th week and then to 3% in postnatal period. Number of ciliated cells in Bowman’s capsule first increased to 32% by the 22nd developmental week, and postnatally decreased to 16%. Compared to healthy kidneys, CNF podocytes showed increased number of ciliated podocytes (12%) and Bowman’s capsule cells (19%). Conclusions: Prospective human podocytes contain primary cilia already at the metanephric cup stage. During development, number of ciliated podocytes decreases in accordance with podocyte differentiation, while Bowman’s capsule cells retain higher percentage of ciliated cell, thus indicating their potential in podocyte regeneration. Compared to healthy kidneys, both cell populations of CNF kidneys display higher percentage of primary cilia, pointing to their incomplete maturation.

Up to 16.6 % of patients with congenital kidney defects have a molecular diagnosis attributable to a genomic disorder. A search for genomic structural variantsis indicated in this patient population to diagnose theirspecific genomic disorders, conduct adequate geneticcounseling, and individualize medical care.

Introduction: During early human kidney development, metanephric mesenchyme cells undergo mesenchymal–to–epithelial transition (MET). In the S-shape nephron, cells of the proximal nephron differentiate into podocytes and parietal epithelial cells (PECs). Near the vascular glomerular pole, PECs share podocyte markers and ultrastructure. While podocytes are unique terminally differentiated cells, PECs form a simple squamous epithelium. Pathologically changed kidneys are characterized by the proliferation and detachment of podocytes and PECs, and the appearance of cellular bridges between them. We analyzed the expression of Notch2, WNT4 and Snail in developing and postnatal human kidneys, in focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). Material and methods: Expression of Notch2, WNT4 and Snail was analyzed in sections of 10 human conceptuses 8 to 38 weeks old, in 3 healthy postnatal kidneys and in 7 CNF and FSGS samples, using immunohistochemistry and electron microscopy (EM). Results: In developing kidneys, WNT4 expression increased moderately in podocytes and strongly in PECs, while in CNF and FSGS kidneys decreased in both cell populations. Notch2 was widely expressed in the developing kidneys, moderately in podocytes and strongly in PECs. In CNF, expression of Notch2 increased, while it decreased in FSGS. Snail was expressed moderately in early nephrogenesis, while in later development Snail increased only in PECs. In CNF and FSGS kidneys Snail increased both in the podocytes and PECs. EM showed cytoplasmic processes between podocytes and PECs in developing and healthy kidneys, in CNF and FSGS. Conclusions: Developmental expression of WNT4, Notch2 and Snail indicate their role in MET and nephrogenesis of human kidneys, while changed expression pattern characterizes kidneys affected by CNF and FSGS. Cellular bridges characterize both developing podocytes and PECs, while they multiply in CNF and FSGS kidneys.

Introduction. Morphological and immunohistochemical characteristics of human podocytes were analyzed during normal kidney development, and during postnatal period in healthy and nephrotic kidneys. Materials and methods. Tissue pieces were dissected from embryos and fetuses (8th to 22nd postovulatory weeks), and from postnatal healthy and nephrotic kidneys, including nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS). Glomerules were analyzed by immunohistochemical and double immunofluorescent techniques, applying proliferation marker Ki-67, differentiation marker Oct-4 and α-tubulin marker for detection of primary cilia. Ultrastructural characteristics of podocytes were analyzed by transmission electron microscopy. Results. Between the 8th and 22nd developmental week, S- forms of immature nephrons were characterized by presence of the short primary cilia at the luminal surfaces of differentiating glomerules and strong Oct-4 expression. During further progression of development, in glomerules of fetal kidneys and in healthy postnatal kidneys, the primary cilia gradually disappeared from the surfaces of the podocytes, while they retained on the parietal layer of Bowman's capsule. Oct-4 expression gradually decreased during development as well. Ultrastructurally, healthy postnatal podocytes had characteristic foot processes, forming part of the glomerular filtration barier (GFB). In contrast, podocytes of postnatal nephrotic kidneys displayed numerous cilia on their surfaces, while their Oct-4 expression remained strong. At places, increased proliferation characterized parietal cells of Bowman's membrane. Ultrastructurally, podocytes of nephrotic kidneys showed absence of foot processes, but had numerous microvili on their surfaces. Conclusions. Differentiation of healthy human podocytes during development and postnatal life is characterized by disappearance of primary cilia from their surfaces, decrease of Oct-4 expression and appearance of podocyte foot processes. In nephrotic kidneys, podocytes are not completely differentiated as some of them retain primary cilia on their surfaces, showing also increased Oct-4 expression and depletion of foot processes.