Marijan Saraga

Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

Mutations in DSTYK and Dominant Urinary Tract Malformations

BACKGROUND Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

Role of mitotic, pro-apoptotic and anti-apoptotic factors in human kidney development

The expression pattern of mitotic Ki-67 and anti-apoptotic bcl-2 proteins, as well as apoptotic caspase-3 and p53 proteins, were investigated in the human mesonephros and metanephros of 5–9 week-old human conceptuses. Apoptotic cells were additionally detected using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) method. Between the 5th and 7th developmental weeks Ki-67, caspase-3 and TUNEL-positive cells characterized all mesonephric structures, indicating importance of cell proliferation in the growth of the mesonephros and role of apoptosis in nephrogenesis. From the 7th week on, p53 and bcl-2 positive cells appeared in the mesonephros as well. Regressive changes in the mesonephros could be regulated by activation of p53, while bcl-2 could contribute to selective survival of some tubules giving rise to adult structures. In the early human metanephros (5–7 weeks), Ki-67 positive cells characterized all metanephric structures, indicating a role of cell proliferation in branching of the ureteric bud and in nephron formation. During the same period bcl-2, caspase-3 and TUNEL-positive cells were found only in the metanephric mesenchyme and nephrons. Bcl-2 protein probably protected nephrons from apoptosis, while caspase-3 protein controlled cell death in the mesenchyme. At later stages (7–9-weeks), appearance of p53-expressing cells could participate in further morphogenesis of the metanephric collecting system. The factors investigated had a spatially and temporally restricted pattern of appearance in developing kidneys. Changes in that pattern might lead to serious disturbances of kidney formation and function in early childhood.

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10‐14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370‐kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein‐altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370‐kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Expression of intermediate filaments, EGF and TGF-α in early human kidney development

The spatial and temporal expression patterns of cytokeratins, vimentin, epithelial growth factor (EGF) and transforming growth factor alpha (TGF-α), were investigated in the 5–9-week old human mesonephros and metanephros. Vimentin was found in all mesonephric structures, while cytokeratins were seen only in the mesonephric tubules. EGF and TGF-α were detected early in all mesonephric structures, and immunoreactivity to both factors decreased in later stages. In the 5–6-week metanephros, vimentin immunoreactivity was found in all structures and later increased in the collecting system and interstitium. In the 5th week, cytokeratins 8 and 19 appeared in the ureteric bud and ampullae, and later showed increasing immunoreactivity in the collecting system and nephrons. The coexpression of intermediate filament proteins in metanephric development is a temporary feature and might be associated with mesenchymal to epithelial transformation of developing nephrons. In adult kidneys, such coexpression is associated with fibrosis or carcinomatous changes. At early stages, immunoreactivity to EGF and TGF-α was detected in all metanephric structures and from the 7th week onward, it decreased in differentiating nephrons. EGF and TGF-α patterns of appearance indicate their role in induction, proliferation and growth of metanephric structures. Disturbances in that pattern might cause reduction in kidney growth.

Changes in bacterial resistance patterns in children with urinary tract infections on antimicrobial prophylaxis at University Hospital in Split

Summary Background We assessed prevalence and resistance of uropathogens on antimicrobial agents (AA) from urine cultures (UC) in children hospitalized with urinary tract infections (UTI) at University Hospital in Split. Material/Methods During the 7-year period, children hospitalized only once with UTI alone were compared to those repeatedly hospitalized, and who received long-term antimicrobial prophylaxis (LTAP), as well as those with associated anomalies of the urinary system (US). Results E. coli was the most frequent isolate (67.7%) with resistance to ampicillin by 69.5%, amoxicillin/clavulonic acid by 3.5%, cephalexin by 6.6%, trimethoprim/sulfamethoxazole (TMP-SMX) by 27.5%, and nitrofurantoin by 0.4%. For other uropathogens, AA resistance rates were the following: 64.3%, 5.8%, 10.5%, 21.3%, and 7.9%. The high or increasing resistance to TMP-SMX is characterized by all uropathogens. Patients with anomalies of US showed a lower prevalence of E. coli and Enterococcus sp., but a higher prevalence of Pseudomonas sp., ESBL-producing E. coli and Klebsiella sp. than those without US anomalies. Repeatedly hospitalized patients showed a lower prevalence of E. coli, but a higher prevalence of Pseudomonas sp. and Klebsiella sp. than patients hospitalized only once. Both groups displayed significantly less resistance of Enterococcus sp. In patients receiving LTAP before hospitalization, E. coli was significantly more resistant to ampicillin, amoxicillin/clavulonic acid and TMP/SMX than in those without LTAP. Conclusions Based on our results, we recommend excluding ampicillin altogether, and reconsideration of further use of TMP-SMX, as well as use of nitrofurantoin, cephalexin and amoxicillin/clavulonic acid for LTAP in our region.

Role of the notochord in the development of cephalic structures in normal and anencephalic human fetuses

Normal and anencephalic human conceptuses were analysed histologically to investigate the role of differentiation of the intracranial notochord and its relation to the formation of the basichondrocranium. We have examined 16 normal embryos and fetuses and 4 anencephalic fetuses. Each developmental stage of formation of the normal basichondrocranium presented specific morphological changes during the course of notochord depletion. In contrast with normal specimens, anencephalic fetuses presented malformations of the basichondrocranium which were always related to an abnormal position of the notochord. Macroscopical differences between craniorachischisis and cranioschisis in fetuses with anencephaly correlated with the existence of two histologically different degrees of malformation. In fetuses with craniorachischisis we found severe disturbances in the shape, position and ossification of the basichondrocranium and in the course of the intracranial notochord. In fetuses with cranioschisis the described disturbances of the basichondrocranium and intracranial notochord were mild. In addition, marked differences in affection of the central nervous system and the hypophysis were observed. These findings suggest different periods of dysmorphogenesis. Our results underline the importance of the chordal mesoderm in the differentiation for the formation of cephalic structures in Man.

Expression of intermediate filaments and desmosomal proteins during differentiation of the human spinal cord

Differentiation of the human spinal cord and involution of its caudal end were investigated in 4-9-week human conceptuses using immunofluorescence and electron microscopy. In the spinal cord, several types of intermediate filament proteins and desmoglein were expressed in parallel: in early stages (4 to 6 weeks), neurofilaments were expressed in low amounts only in the neuroblast processes of the marginal layer. At 6 weeks, differences in staining intensity and distribution patterns of neurofilaments became apparent between lumbar and sacrococcygeal (tail) parts of the spinal cord. Neurofilament expression increased in the mantle and marginal layers of the lumbar spinal cord coinciding with advancing neurogenesis. In contrast, neurofilament expression decreased in the sacrococcygeal spinal cord in association with regression of all tail organs. Regression was characterized by the appearance of large amounts of dead cells and macrophages. Strong vimentin expression was found in neuroepithelial (ependymal) cells and in the radial glia of the spinal cord throughout all stages examined. Coexpression of vimentin and glial fibrillary acidic protein was found only in the radial glia in the earliest developmental stage. Desmoglein was expressed in low amounts around the central canal which was probably associated with the immature junctional complexes that were present between ependymal cells. In conclusion, temporal and spatial distribution patterns of intermediate filament proteins in specific cell populations characterizes differentiation and caudal involution of the human spinal cord.

The interstitial expression of alpha-smooth muscle actin in glomerulonephritis is associated with renal function

Summary Background In a healthy kidney, contractile protein alpha-smooth muscle actin (ASMA) is immunohistochemically strongly expressed only in the blood vessels, while in pathological conditions it can be visualized in glomerular mesangial cells and interstitial myofibroblasts. The aim of this study was to explore the possible correlation between expression of ASMA in glomerulonephritis (GN) and indicators of renal function. Material/Methods We analyzed expression of ASMA in percutaneous renal biopsy of 142 adult and pediatric patients with GN and its correlation with blood pressure, serum creatinine, creatinine clearance and 24-hour urine protein at the time of biopsy. Immunoexpression of ASMA was analyzed quantitatively using computer-assisted morphometric analysis. Relative surface of ASMA expression in all glomeruli and interstitium was calculated for each patient. Results In adults and children, greater expression of ASMA in interstitium was associated with higher serum creatinine and reduced creatinine clearance. Conversely, greater ASMA expression in glomeruli was associated with normal or decreased serum creatinine in adults and increased creatinine clearance in children. In children, correlation was found between high blood pressure and ASMA expression in interstitium. Conclusions We confirmed that interstitial expression of ASMA is associated with reduced renal function at time of biopsy. The connection of ASMA expression in glomeruli with lower serum creatinine and normal or increased creatinine clearance suggests a favorable role of this phenotypic change in glomerular filtration rate; further investigation is needed.

Immunohistochemical and electronmicroscopic features of mesenchymal-to-epithelial transition in human developing, postnatal and nephrotic podocytes

Differentiation of human podocytes starts with mesenchymal-to-epithelial transition (MET) of the metanephric mesenchyme into the S-shaped nephrons. During further development, differentiating podocytes regain mesenchyme-like cell characteristics by epithelial-to-mesenchymal transition (EMT), leading to formation of the terminally differentiated, non-dividing cell. Both MET and EMT processes involve changes in content and organization of cytoskeletal and actin filaments, accompanied by the increased glomerular vascularization. Here, we analyze and compare normal human developing, postnatal and nephrotic podocytes and glomeruli, using immunohistochemical and double immunofluorescent methods for detection of markers of cytoskeletal filaments (nestin, cytokeratin 10—CK10, vimentin and α-SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advanced glycation end products, RAGE). In addition, electron microscopy is used to detect ultrastructural changes of the podocytes. Early metanephric cup mesenchyme expresses all investigated markers except α-SMA, which characterizes only surface mesenchymal cells. In differentiating podocytes and cells of Bowman’s capsule (parietal podocytes) nestin decreases, vimentin increases, while CK10 gradually disappears. Increase in α-SMA is associated with blood vessels development, appearance of podocyte pedicles and slit diaphragm and loss of intercellular connections (zonulae adherentes). Increase in CD31 characterizes vascular glomerular tufts development, while decrease in RAGE expression accompanies normal podocyte differentiation. In congenital nephrotic syndrome of the Finnish type, dedifferentiated podocytes display changes in cytoskeletal filaments and depletion of podocyte pedicles, while glomerular vascular supply is diminished. Our data also suggest high potential of metanephric mesenchyme and parietal podocytes in possible regeneration of the damaged podocytes.