Search

Connexins (Cxs) are membrane-spanning proteins which enable flow of information important for kidney homeostasis. Changes in their spatiotemporal patterning characterize blood vessel abnormalities and chronic kidney diseases (CKD). We analysed spatiotemporal expression of Cx37, Cx40, Cx43 and Cx45 in nephron and glomerular cells of developing, postnatal kidneys, and nephrotic syndrome of the Finnish type (CNF) by using immunohistochemistry, statistical methods and electron microscopy. During kidney development, strong Cx45 expression in proximal tubules and decreasing expression in glomeruli was observed. In developing distal nephron, Cx37 and Cx40 showed moderate-to-strong expression, while weak Cx43 expression gradually increased. Cx45/Cx40 co-localized in mesangial and granular cells. Cx43 /Cx45 co-localized in podocytes, mesangial and parietal epithelial cells, and with podocyte markers (synaptopodin, nephrin). Different Cxs co-expressed with endothelial (CD31) and VSMC (α –SMA) markers in vascular walls. Peak signalling of Cx37, Cx43 and Cx40 accompanied kidney nephrogenesis, while strongest Cx45 signalling paralleled nephron maturation. Spatiotemporal Cxs patterning indicate participation of Cx45 in differentiation of proximal tubules, and Cx43, Cx37 and Cx40 in distal tubules differentiation. CNF characterized disorganized Cx45 expression in proximal tubules, increased Cx43 expression in distal tubules and overall elevation of Cx40 and Cx37, while Cx40 co-localized with increased number of interstitial myofibroblasts.

The spatiotemporal expression of α-tubulin, inversin and dishevelled-1 (DVL-1) proteins associated with the Wnt-signaling pathway, and primary cilia morphology were analyzed in developing kidneys (14th–38th developmental weeks), healthy postnatal (1.5- and 7-years old) and pathologically changed human kidneys, including multicystic dysplastic kidneys (MCDK), focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). The analysis was performed by double immunofluorescence, electron microscopy, semiquantitative and statistical methods. Cytoplasmic co-expression of α-tubulin, inversin and DVL-1 was observed in the proximal convoluted tubules (pct), distal convoluted tubules (dct) and glomeruli (g) of analyzed tissues. During kidney development, the overall expression of α-tubulin, inversin and DVL-1 decreased, while in the postnatal period slightly increased. The highest expressions of α-tubulin and inversin characterized dct and g, while high DVL-1 characterized pct. α-tubulin, inversin and DVL-1 expression pattern in MCDK, FSGS and CNF kidneys significantly differed from the healthy control. Compared to healthy kidneys, pathologically changed kidneys had dysmorphic primary cilia. Different expression dynamics of α-tubulin, inversin and DVL-1 during kidney development could indicate that switch between the canonical and noncanonical Wnt-signaling is essential for normal kidney morphogenesis. In contrast, their disturbed expression in pathological kidneys might be associated with abnormal primary cilia, leading to chronic kidney diseases.

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 (MET), 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, citokeratin10-CK10, vimentin and α –SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advances glycation 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 (CNF), 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.

The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein ( VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7(th) week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5(th) week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP- 7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

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.