A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron.

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.

Premature differentiation of nephron progenitor cell and dysregulation of gene pathways critical to kidney development in a model of preterm birth.

Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

Molecular detection of maturation stages in the developing kidney.

Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.

Developmental Renal Glomerular Defects at the Origin of Glomerulocystic Disease.

The architecture of renal glomeruli is acquired through intricate and still poorly understood developmental steps. In our study we identify a crucial glomerular morphogenetic event in nephrogenesis that drives the remodeling/separation of the prospective vascular pole (the future entrance of the glomerular arterioles) and the urinary pole (the tubular outflow). We demonstrate that this remodeling is genetically programmed. In fact, in mouse and human, the absence of HNF1B impairs the remodeling/separation of the two poles, leading to trapping and constriction of the tubular outflow inside the glomerulus. This aberration gives rise to obstructive glomerular dilations upon the initiation of primary urine production. In this context, we show that pharmacological decrease of glomerular filtration significantly contains cystic expansion. From a developmental point of view, our study discloses a crucial event on glomerular patterning affecting the "inside-outside" fate of the epithelia in the renal glomerulus.

Detailed process analysis for glomerular capillary formation by immunofluorescence on ultra-thick sections.

Glomerular capillary formation is one of the fundamental mysteries in renal developmental biology. However, there are still debates on this issue, and its detailed formation process has not been clarified. To resolve this problem, we performed antibody staining with ultra-thick section on embryonic and postnatal mouse kidneys. We obtained the expression patterns of several genes that play an important role in the development of glomerular capillaries. We found that blood vessel of the fetal kidneys expanded through proliferation and sprouting. During the comma-stage and S-shaped stage, 3-4 capillaries began to bud and migrate into the glomerular cleft, forming a capillary bed in the Bowman's capsule. Then, the capillary bed expanded into mature glomerular capillary by intussusceptive angiogenesis. The afferent and efferent arterioles were formed through pruning. The distribution of VEGFA in the nephron epithelial cells but not only in podocytes, induced multiple capillaries sprouted into the glomerular cleft. And CXCR4 played an important role in the differentiation and expansion of capillary bed into glomerular capillary. Immunofluorescence performed with ultra-thick section allowed us to investigate the development of complex structure tissues systematically and comprehensively.

A Novel Role for GATA3 in Mesangial Cells in Glomerular Development and Injury.

BACKGROUND: GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury. METHODS: To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/-) knockout mice, as well as injured human and rodent kidneys. RESULTS: We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis. CONCLUSIONS: These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.

Systemic Arterial Hypertension in Childhood: A Challenge Related to the Increasing Survival of Very Low Birth Weight Preterm Infants.

OBJECTIVE: To verify the prevalence of systemic arterial hypertension (SAH) and to identify possible early predictors of SAH at ages 2 and 4 years in very low birth weight (VLBW) infants. STUDY DESIGN: This is a prospective cohort study including inborn children with birth weight (BW) <1,500 g. Arterial blood pressure measurements were performed at 2 and 4 years. Model 1 compared children with and those without SAH at age 4. Model 2 compared children who had SAH at ages 2 and 4 with the others. SAH was diagnosed if the systolic or/and diastolic pressures were above the 95th percentile. RESULTS: A total of 198 patients were included during the 5-year study period, of whom 56% had SAH at age 4. In model 1, white matter injury (WMI) and catch-up growth at age 2 were predictors of SAH at age 4. In model 2, bronchopulmonary dysplasia, WMI, catch-up growth at age 2, and BW were predictors of SAH at 2 and 4 years. SAH at age 2 was an independent risk factor for SAH at age 4. After a multivariate analysis of model 2, BW and catch-up growth were associated with SAH. CONCLUSION: Prevalence of SAH was high in VLBW infants; it was associated with low BW and catch-up growth at age 2.

Development of urinary organs in domestic cat during the embryonic and fetal periods.

The embryonic origin of the urogenital system came from the intermediate mesoderm. Kidney development involves three successive renal systems with a fast chronological overlap: the pronephro, the mesonephro, and the metanephro. Due to the lack of specific knowledge about this system in cats the present work aimed to describe their urinary organs development, focusing on the structures seen in pronephro, mesonephro, and metanephro during the embryonic and fetal stages of development. The techniques used in this study were: light microscopy, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. For that, embryos and fetuses from 12 pregnant mixed-breed domestic cats in different gestational stages were used to describe the proposed organs. The pronephro is present at early stages of embryonary development in embryos from 15 to 19 days with the presence of pronephro's corpuscles, ducts and tubules. The mesonephro is found, in general, between days 17 and 37, and contains mesonephric ducts, mesonephric tubules, and glomeruli. The metanephro is seen since 21 days of pregnancy with the presence of glomeruli, proximal and distal contorted tubules and at day 37, the cortex-medullary region is already differentiated. The evaluation of these structures enhances the knowledge about embryology of the urinary system in cats, aiding a better anatomical understanding of the system in the specie allowing the correlation with other species.

Intussusceptive Pillar Formation in Developing Porcine Glomeruli.

BACKGROUND/AIMS: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated. METHODS: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney. RESULTS: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation. CONCLUSION: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries.

Glomerulogenesis and the role of endothelium.

PURPOSE OF REVIEW: Earlier works of the glomerulogenesis described morphological steps and protein expression during in-vivo and in-vitro kidney development. Recent technologies using cell-specific or conditional knock-out mice for several factors provide important knowledge about cross-talk signaling among resident cells as local events. Based on the recent advancement, this review revisits comprehensive morphological development of the glomerulus. RECENT FINDINGS: Interactions of presumptive podocyte vascular endothelial growth factor with vascular endothelial growth factor-2 on angioblasts initiate glomerular vascularization. In induced pluripotent stem cells or organoid-derived nephron formation, the lack of endothelium and mesangial cells under differentiated podocytes suggests the presence of another unknown mechanism for glomerular neovascularization. Mesangial cell migration is prerequisite for glomerular looping by interaction of endothelial platelet-derived grothe factor beta and mesangial platelet-derived growth factor receptor beta and requires the coreceptor neuropilin1. Development of the filtration barrier is promoted by cross-talk among resident cells and may need shear stress. The components of the glomerular basement membrane change during glomerulogenesis, and endothelium and podocytes produce laminin and type IV collagen α1 and α2, whereas type IV collagen α3, α4, α5 is derived only from podocytes. SUMMARY: Glomerulogenesis progresses by dynamic cellular migration/differentiation induced by cross-talk signaling in resident cells. Glomerular vasculogenesis and subsequent capillary development provide insight into glomerular regeneration and remodeling for medical application.

Fryl deficiency is associated with defective kidney development and function in mice.

FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl-/- mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl-/- mice died soon after birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl-/- neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl-/- neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.

Development of the Human Fetal Kidney from Mid to Late Gestation in Male and Female Infants.

BACKGROUND: During normal human kidney development, nephrogenesis (the formation of nephrons) is complete by term birth, with the majority of nephrons formed late in gestation. The aim of this study was to morphologically examine nephrogenesis in fetal human kidneys from 20 to 41weeks of gestation. METHODS: Kidney samples were obtained at autopsy from 71 infants that died acutely in utero or within 24h after birth. Using image analysis, nephrogenic zone width, the number of glomerular generations, renal corpuscle cross-sectional area and the cellular composition of glomeruli were examined. Kidneys from female and male infants were analysed separately. FINDINGS: The number of glomerular generations formed within the fetal kidneys was directly proportional to gestational age, body weight and kidney weight, with variability between individuals in the ultimate number of generations (8 to 12) and in the timing of the cessation of nephrogenesis (still ongoing at 37weeks gestation in one infant). There was a slight but significant (r2=0.30, P=0.001) increase in renal corpuscle cross-sectional area from mid gestation to term in females, but this was not evident in males. The proportions of podocytes, endothelial and non-epithelial cells within mature glomeruli were stable throughout gestation. INTERPRETATION: These findings highlight spatial and temporal variability in nephrogenesis in the developing human kidney, whereas the relative cellular composition of glomeruli does not appear to be influenced by gestational age.

USP40 gene knockdown disrupts glomerular permeability in zebrafish.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Nephron, Wilms' tumor-1 (WT1), and synaptopodin expression in developing podocytes of mice.

Newborn mouse glomeruli are still immature with a morphological feature of an early capillary loop stage, but infant mice do not manifest proteinuria. Little is known about the molecular mechanism whereby infant mice are resistant to proteinuria. Nephrin and synaptopodin are crucial for slit diaphragm and foot process (FP) formation for avoiding proteinuria. Nephrin tyrosine phosphorylation means a transient biological signaling required for FP repair or extension during nephrotic disease. Using an immunohistochemical technique, we examined the natural course of nephrin, Wilms' tumor-1 (WT1) and synaptopodin at 16.5 days of embryonic age (E16.5d) and E19.5d, 7 days of post-neonatal age (P7d) and P42d during renal development of mice. As a result, nephrin and synaptopodin were detected at E19.5d in S-shaped bodies. WT1, a transcriptional factor for nephrin, was detected in nucleus in podocyte-like cells in all stages. Nephrin tyrosine phosphorylation was evident in glomeruli at P7d, and this was associated with an early-stage of FP extension. Inversely, nephrin phosphorylation became faint at P42d, along with maturated FP. Based on the present results, we suggest the sequential molecular mechanism to protect growing mice from proteinuria: (i) WT1-induced nephrin production by podocytes in S-shaped bodies at E19.5d; (ii) Synchronized induction of synaptopodin at the same period; and (iii) FP extension is initiated at a milk-suckling stage under a nephrin tyrosine-phosphorylated condition, while it is arrested at an adult stage, associated with a loss of nephrin-based signaling.

Intussusceptive angiogenesis and expression of Tie receptors during porcine metanephric kidney development.

Intussusceptive angiogenesis (IA) is required for normal embryonic vascular development. The Tie family of receptors and their ligands, the angiopoietins, play an important role in the growth or regression of blood vessels which are important not only during development but also throughout an organism's life. The presence of IA was investigated in glomerular capillaries of the fetal porcine metanephros using Mercox II resin casts. The first signs of IA were observed in stage III glomeruli. Stage IV and V glomeruli showed numerous signs of aligned pillar formation and their successive merging to delineate the vascular entities. Furthermore, immunohistochemistry was used to determine the exact locations of the Tie receptors in the developing porcine metanephric kidneys. Tie1 and Tie2 were found in endothelial cells of all glomeruli. Strong expression of the receptors was found in podocytes of stage V glomeruli whereas a weaker expression was observed in the cuboidal epithelial cells of stage III and IV glomeruli. Remarkably, the receptors were also found in the parietal epithelium of Bowman's capsule. These findings indicate that there might be an association between the Tie receptors and the IA during porcine metanephric development and during glomerulogenesis in particular.

Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis.

BACKGROUND: Wnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system. RESULTS: The Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11 (-/-) mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11 (-/-) mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency. CONCLUSIONS: The Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11 (-/-) mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.

Ultrastructural characterization of the pronephric glomerulus development in zebrafish.

The zebrafish pronephros is a valuable model for studying kidney development and diseases. Ultrastructural studies have revealed that zebrafish and mammals share similarities in nephron structures such as podocytes, slit diaphragms, glomerular basement membrane, and endothelium. However, the basic ultrastructural features of the pronephric glomerulus during glomerulogenesis have not been characterized. To understand these features, it is instructive to consider the developmental process of the pronephros glomerulus. Here, we describe the ultrastructural features of pronephric glomerulus in detail from 24 h hours post-fertilization (hpf) to 144 hpf, the period during which the pronephric glomerulus develops from initiation to its mature morphology. The pronephric glomerulus underwent progressive morphogenesis from 24 to 72 hpf, and presumptive glomerular cells were observed ventral to the aorta region at 24 hpf. The nascent glomerular basement membrane and initial lumen were formed at 36 hpf. A lumen was clearly visible in the region of the pronephros at 48 hpf. At 60 hpf, the pronephric glomerulus contained more patches of capillaries. After these transformations, the complex capillary vessel networks had formed inside the glomerulus, which was surrounded by podocyte bodies with elaborate foot processes as well as well-formed glomerular basement membrane by 72 hpf. The number of renal glomerular cells rapidly increased, and the glomerulus presented its delicate structural features by 96 hpf. Morphogenesis was completed at 120 hpf with the final formation of the pronephric glomerulus. J. Morphol. 277:1104-1112, 2016. © 2016 Wiley Periodicals, Inc.

Administration of Cyclosporine A in Pregnant Rats - the Effect on Blood Pressure and on the Glomerular Number in Their Offspring.

BACKGROUND/AIMS: Cyclosporine A (CsA) is a commonly used immunosuppressive agent. In some patients treatment with CsA has to be continued during pregnancy. The aim of the study was to assess in an experimental model whether the exposure to CsA during fetal life influences the number and volume of glomeruli, kidney function and blood pressure in the offspring. METHODS: Eight pregnant female Sprague-Dawley rats were allocated to 2 treatment regimens: with CsA or solvent. Blood pressure was measured in the offspring at 7 and 11 weeks of age and albuminuria was determined at 11 weeks of age. In the kidney the number and mean volume of glomeruli was assessed using stereological methods. RESULTS: In the offspring of pregnant rats treated with CsA the number of glomeruli was significantly lower and the mean volume of glomeruli was higher when compared to the offspring of pregnant rats receiving solvent. Systolic and diastolic blood pressures as well as albuminuria were significantly higher in the offspring of mothers treated with CsA during gestation compared to the offspring from the control group. CONCLUSIONS: Exposure of rats to CsA during fetal life impairs kidney development, thus potentially predisposing to chronic kidney disease and hypertension in the adult life.