3D kidney organoids for bench-to-bedside translation.

The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

Evaluation of the Intravesical Ureters after Failed Endoscopic Treatment of Vesicoureteral Reflux with Dextranomer/hyaluronic Acid in Children via Light and Transmission Electron Microscopic Analysis. A Matched Case-Control Study.

INTRODUCTION: The cytokine profile and the ultrastructural changes of refluxing ureterovesical junctions(UVJs) of children treated with failed dextranomer/hyaluronic-acid (Dx/HA) injections were investigated using immunohis-tochemical methods and transmission electron microscopy(TEM). PATIENTS AND METHODS: Eighteen children who had undergone injection for reflux were included the study. The smooth muscle arrangement of the ureteral wall, transforming growth factor-? (TGF-?1),vascular-endotheli-al-growth factor (VEGF) and CD34 were evaluated immunohistochemically, and the results were compared with 10 age-matched autopsy specimens as controls. The ultrastructural evaluation and morphological description was made semi-quantitatively and compared with published data. RESULT: Four of the patients (22%) were male, and 14 (78%) were female. The mean age of the patients was 105.4 ± 44.5(48-184) months. There was no correlation between the vesicoureteral reflux (VUR) grade and age (P = 0.85). The mean VEGF and CD34 scores were 16.2 ± 9.6 (0-90) cells per HPF and 10.2 ± 3.5 (4-16) vessels per HPF in ureters with reflux; these values were 60.6±16.4 (32-84) cells per HPF and 17.8 ± 4.1 (12-24) vessels per HPF in the control group. The amount of VEGF and CD34 were significantly decreased in patients compared with the control group (P < 0.001, P < 0.001).The TGF-?1 levels were significantly higher in patients with VUR compared with the control group (34.2 ± 19.9 vs 5.0±1.9; P=0.001).The amount of VEGF, CD34, and TGF-?1 were not correlated with the grade of reflux (P = 0.26, P = 0.94, and P = 0.42, respectively). Ultrastructural changes in the muscle cells were observed in all the VUR specimens (Grade II-IV). CONCLUSION: Refluxing ureters exhibited immune-histopathological abnormalities and ultrastructural changes of the muscle cells in all VUR specimens in the ureterovesical junctions of children treated with failed Dx/HA injec-tions for reflux.

Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy.

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited. The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na+-Cl- cotransporter, H+-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development. Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

Hydronephrosis with ureteritis developed in C57BL/6N mice carrying the congenic region derived from MRL/MpJ-type chromosome 11.

Inbred MRL/MpJ mice show several unique phenotypes in tissue regeneration processes and the urogenital and immune systems. Clarifying the genetic and molecular bases of these phenotypes requires the analysis of their genetic susceptibility locus. Herein, hydronephrosis development was incidentally observed in MRL/MpJ-derived chromosome 11 (D11Mit21-212)-carrying C57BL/6N-based congenic mice, which developed bilateral or unilateral hydronephrosis in both males and females with 23.5% and 12.5% prevalence, respectively. Histopathologically, papillary malformations of the transitional epithelium in the pelvic-ureteric junction seemed to constrict the ureter luminal entrance. Characteristically, eosinophilic crystals were observed in the lumen of diseased ureters. These ureters were surrounded by infiltrating cells mainly composed of numerous CD3+ T-cells and B220+ B-cells. Furthermore, several Iba-1+ macrophages, Gr-1+ granulocytes, mast cells and chitinase 3-like 3/Ym1 (an important inflammatory lectin)-positive cells were detected. Eosinophils also accumulated to these lesions in diseased ureters. Some B6.MRL-(D11Mit21-D11Mit212) mice had duplicated ureters. We determined >100 single nucleotide variants between C57BL/6N- and MRL/MpJ-type chromosome 11 congenic regions, which were associated with nonsynonymous substitution, frameshift or stopgain of coding proteins. In conclusion, B6.MRL-(D11Mit21-D11Mit212) mice spontaneously developed hydronephrosis due to obstructive uropathy with inflammation. Thus, this mouse line would be useful for molecular pathological analysis of obstructive uropathy in experimental medicine.

Interkinetic nuclear migration in the mouse embryonic ureteric epithelium: Possible implication for congenital anomalies of the kidney and urinary tract.

Interkinetic nuclear migration (INM) is a phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium, which is characterized by the presence of apical primary cilia, in synchrony with the cell cycle in a manner of apical mitosis. INM is suggested to regulate not only stem/progenitor cell proliferation/differentiation but also organ size and shape. INM has been reported in epithelia of both ectoderm and endoderm origin. We examined whether INM exists in the mesoderm-derived ureteric epithelium. At embryonic day (E) 11.5, E12.5 and E13.5, C57BL/6J mouse dams were injected with 5-bromo-2'-deoxyuridine (BrdU) and embryos were killed 1, 2, 4, 6, 8, 10 and 12 h later. We immunostained transverse sections of the ureter for BrdU, and measured the position of BrdU (+) nuclei in the ureteric epithelia along the apico-basal axis at each time point. We analyzed the distribution patterns of BrdU (+) nuclei in histograms using the multidimensional scaling. Changes in the nucleus distribution patterns suggested nucleus movement characteristic of INM in the ureteric epithelia, and the mode of INM varied throughout the ureter development. While apical primary cilia are related with INM by providing a centrosome for the apical mitosis, congenital anomalies of the kidney and urinary tract (CAKUT) include syndromes linked to primary ciliary dysfunction affecting epithelial tubular organs such as kidney, ureter, and brain. The present study showed that INM exists in the ureteric epithelium and suggests that INM may be related with the CAKUT etiology via primary ciliary protein function.

Aberrant differentiation of urothelial cells in patients with ureteropelvic junction obstruction.

AIM: To investigate the urothelial changes in the pathogenesis of ureteropelvic junction obstruction (UPJ-O). METHODS: A total of 12 patients of UPJ-O were respectively studied. The expression of Annexin A7, Annexin A11, EGFR, Keratin 5, uroplakin III, and SMA in the urothelium of obstructed UPJ segment and of the normal ureter below the obstructed segment were determined by immunofluorescence. Transmission electron microscopy was used to determine the morphological changes in UPJ epithelium in compared to normal ureteral epithelium. RESULTS: We found that Annexin A7, Annexin A11, EGFR, Keratin 5, and SMA were upregulated, while uroplakin III was downregulated in the urothelium of UPJ-O patients. Furthermore, ultrastructural analyses showed that intercellular spaces between urothelial cells were dilated and the number of microvilli on superficial cells was increased in UPJ-O patients. CONCLUSIONS: We propose that a disrupted urothelial barrier in UPJ-O may results in urothelial inflammatory response and truncated differentiated urothelial cells, which may play an important role in the development and pathogenesis of UPJO.

[Scanning electron microscopy of resected ureters in children with ureterohydronephrosis].

Using scanning electron microscopy, various portions of the ureter in reflux and obstructive ureterohydronephrosis in children were evaluated. In the first case, architectonics of the distal portions is preserved, while in the second case connective tissue is proliferated. Differences in the structure of proximal and distal portions in both forms of ureterohydronephrosis consist in inflammatory changes and violation of the integrity of the epithelial lining, edema and infiltration of the underlying layers, especially the inner muscle layer of the distal portion. The wall of the ureter in proximal part is much thinner, especially the muscular and mucous layers.

Ultrastructural and immunohistopathological evaluation of intravesical ureters via electron and light microscopy in children with vesicoureteral reflux.

PURPOSE: We used immunohistochemical methods and transmission electron microscopy to investigate the cytokine profiles and ultrastructural changes in the ureterovesical junction of children with primary vesicoureteral reflux. MATERIALS AND METHODS: A total of 39 distal intravesical ureters were obtained from 23 children who underwent ureteroneocystostomy for primary vesicoureteral reflux. Ureteral wall smooth muscle organization and transforming growth factor-ß1, vascular endothelial growth factor and CD34 were evaluated immunohistochemically and compared to controls, which consisted of 10 age matched autopsy specimens. Ultrastructural evaluations and morphological descriptions were performed semiquantitatively and compared to the published data. RESULTS: Of the patients 6 (26%) were male and 17 (74%) were female, and mean ± SD age was 73.2 ± 34.3 months (range 12 to 168). There was no correlation between reflux grade and age (p = 0.39). Smooth muscle disorganization score differed significantly between patients with intravesical ureters and controls (p = 0.01). Transforming growth factor-ß1 levels were significantly higher (p = 0.001) and vascular endothelial growth factor levels and microvessel densities were significantly lower in the patients with reflux compared to controls (both p <0.001). Vascular endothelial growth factor, CD34 and transforming growth factor-ß1 levels did not correlate with reflux grades (p = 0.84, p = 0.76 and p = 0.10, respectively). Urothelium, lamina propria and tunica adventitia appeared normal in the specimens for all grades of vesicoureteral reflux using transmission electron microscopy. Damage was observed in the muscular layers of the ureterovesical junction, especially in patients with grade IV or V reflux. CONCLUSIONS: Primary refluxing ureters exhibit immunohistopathological abnormalities compared to normal ureters irrespective of reflux grade, and ultrastructural changes are especially severe in cases of high grade reflux. These abnormalities can hinder the normal ureteral valve mechanism, and may lead to reflux due to smooth muscle dysfunction and microvascular alterations.

Imaging tools for analysis of the ureteric tree in the developing mouse kidney.

The structure of the ureteric tree in developing mouse and rat kidneys has previously been quantified in two dimensions. While this type of analysis may provide evidence of changes in ureteric growth, these measurements are effectively inaccurate, as the ureteric tree is a three-dimensional (3D) object. Here we describe a method for measuring the ureteric tree in three dimensions. This technique involves (1) culture of the metanephric kidney at embryonic day 12 (mouse) or 14 (rat), (2) whole-mount immunofluorescence to selectively stain ureteric tree epithelium, (3) confocal microscopy to obtain a complete Z series through the ureteric tree, and (4) image analysis algorithms to binarize, skeletonize, and measure individual branch lengths in 3D. This method has been extended to analysis of the same ureteric tree over time (4D). The results obtained provide accurate and precise quantitation of ureteric tree growth in the developing mouse or rat kidney.

Study of the ureter structure in anencephalic fetuses

Purpose The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. Materials and Methods We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). Results The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. Conclusions Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate ...

Study of the ureter structure in anencephalic fetuses.

PURPOSE: The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. MATERIALS AND METHODS: We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). RESULTS: The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. CONCLUSIONS: Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate that anencephalic fetal ureters tend to have significant structural alterations, probably due to cerebral lesions with consequent brain control damage of ureter nerves.

Analysis of 3D branching pattern: hematoxylin and eosin method.

Accurate analysis of the three-dimensional (3D) architecture of developing organs is critical to understanding how developmental defects can be linked with structural abnormalities. Here, we describe a 3D reconstruction technique of the developing kidney including the outer kidney capsule, ureteric epithelium, and developing nephrons. This 3D reconstructive process involves generating serial sections of the developing kidney, followed by histological staining. Each serial image is projected on the monitor and each tissue lineage or structure is traced. The kidney tracings are aligned and a 3D image is rendered. Each reconstructed tissue/lineage can then be subjected to quantitative analysis (e.g., surface area or volume). The reconstructed ureteric epithelium can be skeletonized to determine the branching architecture.

Tissue engineering of ureteral grafts by seeding urothelial differentiated hADSCs onto biodegradable ureteral scaffolds.

The study is aimed to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into urothelial lineage, and to assess possibility of constructing ureteral grafts using the differentiated hADSCs and a novel polylactic acid (PLA)/collagen scaffolds. HADSCs were indirectly cocultured with urothelial cells in a transwell coculture system for urothelial differentiation. After 14 days coculturing, differentiation was evaluated by detecting urothelial lineage markers (cytokeratin-18 and uroplakin 2) in mRNA and protein level. Then the differentiated hADSCs were seeded onto PLA/collagen ureteral scaffolds and cultured in vitro for 1 week. The biocompatibility of the scaffolds was tested by scanning electron microscopy (SEM) and MTT analysis. At last, the cell/scafflod grafts were subcutaneously implanted into 4-week-old female athymic mice for 14 days. The results demonstrated that the hADSCs could be efficiently induced into urothelial lineage by indirect coculture. The differentiated cells seeded onto the PLA/collagen ureteral scaffolds survived up to 7 days and maintained proliferation in vitro, which indicated that the scaffolds displayed good biocompatibility. In vivo study showed that the differentiated cells in the grafts survived, formed multiple layers on the scaffolds and expressed urothelial lineage markers. In conclusion, hADSCs may serve as an alternative cell resource in cell-based tissue engineering for ureteral reconstruction. These cells could be employed to construct a model of ureteral engineering grafts and be effectively applied in vivo, which could be a new strategy on ureteral replacement with applicable potential in clinical research.

Identification of telocytes in the upper lamina propria of the human urinary tract.

The upper lamina propria (ULP) area of interstitial cells (IC) has been studied extensively in bladder, but is rather unexplored in the rest of the urinary tract. This cell layer is intriguing because of the localization directly underneath the urothelium, the intercellular contacts and the close relationship with nerve endings and capillaries. In this study, we examine the ULP layer of IC in human renal pelvis, ureter and urethra, and we make a comparison with ULP IC in bladder. Tissue was obtained from normal areas in nephrectomy, cystectomy and prostatectomy specimens, and processed for morphology, immunohistochemistry and electron microscopy. A morphological and immunohistochemical phenotype for the ULP IC was assessed and region-dependent differences were looked for. The ULP IC in renal pelvis, ureter and urethra had a similar ultrastructural phenotype, which differed somehow from that of bladder IC, that is, thinner and longer cytoplasmic processes, no peripheral actin filaments and presence of dense core granules and microtubules. Together with their immunohistochemical profile, these features are most compatible with the phenotype of telocytes, a recently discovered group of stromal cells. Based on their global ultrastructural and immunohistochemical phenotype, ULP IC in human bladder should also be classified as telocytes. The most striking immunohistochemical finding was the variable expression of oestrogen receptor (ER) and progesterone receptor (PR). The functional relevance of ULP telocytes in the urinary tract remains to be elucidated, and ER and PR might therefore be promising pharmacological research targets.

Aberrant planar cell polarity induced by urinary tract obstruction.

Flow sensing by primary cilia of the epithelial cells is involved in cystogenesis in polycystic kidney disease. We investigate whether a similar mechanism applies to the pathogenesis of cyst-like tubular dilatation induced by ureteral obstruction in mice. Robust proliferation occurs in the obstructed tubules when urine flow is interrupted as well as in the repairing tubules when urine flow is reestablished after relief of the obstruction, suggesting a urine flow-independent mechanism of proliferation. In the urothelium, proliferation is only detected above the obstruction, although urine flow ceased both above and below the obstruction. Our results support mechanical strain- rather than flow-mediated proliferation in obstructive uropathy. To understand the mechanism of cell proliferation leading to increased tubular diameter in cyst-like tubular dilatation, we examine planar cell polarity (PCP), which is necessary for oriented cell division and maintenance of tubular diameter. In dilated tubules, the orientation of cell division is randomized, atypical PKC (aPKC) is mislocalized, and the pattern of the expression of a core PCP protein, Frizzled3 (Fz3), is altered. In addition, the level of Fz3 expression is increased. These results indicate that aberrant PCP may contribute to cyst-like tubular dilatation in obstructive uropathy. Interestingly, the orientation of cell division, localization of aPKC, and Fz3 expression return to normal when obstruction is relieved, which suggest a role of normal PCP signaling in tubular repair.

Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat.

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

Relation between radionuclide imaging and pathologic findings of ureteropelvic junction obstruction in neonatal hydronephrosis.

OBJECTIVE: An excessive amount of collagen fibers around the muscle cells in the ureteropelvic junction could be responsible for obstruction in patients with hydronephrosis. We aimed to elucidate the ultrastructure of the ureters and correlate this finding with the prognostic outcome and to correlate the histopathological findings with diuretic radionuclide renography findings. MATERIAL AND METHODS: Biopsy specimens of 20 children who underwent dismembered pyeloplasty for ureteropelvic junction obstruction were analyzed. The patients were grouped according to their age: infants (<12 months) and others (>12 months). Diuretic radionuclide imaging was performed using (99m)Tc mercaptylacetyltriglycine in the pre- and postoperative periods. Changes in differential renal function and excretion patterns on diuretic renography were evaluated in relation to the findings noted on histopathological examination of the biopsy specimens. Excretion patterns were classified as follows: A, normal; B, responsive to diuretic; C, minimal response to diuretic with some excretion after postural change; and D, very poor/no drainage despite diuretics. Biopsy materials were analyzed for the presence and extent of inflammation, fibrosis and changes in the smooth muscle layer using Masson's trichrome stain and immunohistochemical staining. Histopathological findings were graded from zero to three, depending on severity. RESULTS: In patients aged <12 months, preoperative differential renal function (DRF) was associated with fibrosis (F) and smooth muscle hypertrophy (SMH) [mean (SD) DRF for both F and SMH were Grade 0-1, 47.8% (6.4%); Grade 2-3, 36.2% (11.3%); p<0.05]; and change in DRF was associated with inflammation [Grade 0-1, -0.1% (4.0%); Grade 2-3, 5.8% (3.0%); p<0.05]. Excretion patterns or improvement in excretion were not associated with any of the histopathological features. Change in DRF was significantly associated with inflammation Grade 2-3 (beta coefficient, 5.8; 95% CI 1.4-10.3). CONCLUSIONS: Histopathological evaluation of renal parenchymal biopsy specimens obtained during pyeloplasty may be useful to provide an objective method for predicting the recovery of renal function. In addition, this will allow comparison of the types of histopathological alterations with the changes in differential renal function in order to predict the potential final improvement.

An electron microscopic examination of the intravesical ureter in children with primary vesico-ureteric reflux.

OBJECTIVE: To determine the structure of the intravesical distal ureteric wall of patients with primary vesico-ureteric reflux (VUR), and to compare the findings with previous reports. MATERIALS AND METHODS: Specimens of the distal intravesical ureteric segments were taken surgically from children undergoing ureteric reimplantation surgery for primary VUR. There were 24 distal intravesical ureteric specimens from 15 children (nine female and six male). Ultra-thin sections were cut from the specimens and examined with a transmission electron microscope. RESULTS: The appearance of the muscular layers of the specimens of different grades differed markedly. There were intercellular oedematous areas in the muscular layer in specimens from patients with grade 2 and 3 VUR. In specimens from grade 4 VUR there were also intracytoplasmic vacuoles in the smooth muscle cells. The most marked and striking changes were in the specimens from children with grade 5 VUR, in which there were large intercellular oedematous areas and prominent large intracytoplasmic vacuoles. CONCLUSION: Refluxing ureters differ from normal ureters in having disorganized smooth muscle fibres and altered smooth muscle cell structure, leading to incompetence of the valve mechanism. Although we cannot confirm that these pathological changes in the smooth muscle layer of the intravesical ureteric wall are caused by VUR we conclude that, with increasing degrees of reflux, the degree of smooth muscle damage increases, and that the rate of spontaneous resolution decreases.

Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Ureteric branching morphogenesis in BMP4 heterozygous mutant mice.

Exogenous bone morphogenetic protein 4 (BMP4) inhibits ureteric branching morphogenesis and amplifies the already existing branching asymmetry in the developing mouse kidney in vitro. In the present study we examined ureteric branching morphogenesis in BMP4/lacZ heterozygous (BMP4(+/-)) mice in vitro under control conditions and in the presence of exogenous BMP4 using three-dimensional image analysis software. The relative expression of BMP4 mRNA was determined in BMP4(+/-) and wildtype urogenital ridges using real-time PCR. Embryonic day 12.5 (E12.5) BMP4(+/-) and wildtype mouse metanephroi were cultured for 48 h with or without 260 ng mL(-1) recombinant human BMP4 (rhBMP4) and were then wholemount immunostained in order to identify the ureteric epithelium, which was quantified in three dimensions. Despite a significant reduction in BMP4 mRNA in BMP4(+/-) mice, qualitative and quantitative studies identified no differences in ureteric branching morphogenesis between phenotypically normal BMP4(+/-) and wildtype metanephroi in either BMP4-treated or control cultures. Both BMP4(+/-) and wildtype metanephroi cultured in the presence of BMP4 showed a decrease in total ureteric length, branch number and ureteric volume, and increased average branch length compared with control cultures. A marked anterior-posterior asymmetry in both ureteric length, branch number and average branch length was observed in BMP4-treated metanephroi from both genotypes. A similar asymmetry was revealed in control metanephroi from both genotypes. This asymmetry is the result of reduced ureteric branching morphogenesis but not elongation in the posterior region of the kidney. These results suggest that despite reduced endogenous BMP4 mRNA levels, most BMP4(+/-) embryos can still facilitate normal ureteric branching morphogenesis during development. In addition, reduced endogenous levels of BMP4 do not alter the inhibitory effects of exogenous BMP4 on ureteric branching or amplification of normal renal asymmetry.