Expression of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) Candidate Genes EDA2R, PCDH9, and TRAF7 in Normal Human Kidney Development and CAKUT.

Approximately half of the cases of chronic kidney disease (CKD) in childhood are caused by congenital anomalies of the kidney and urinary tract (CAKUT). Specific genes were identified as having significant importance in regard to the underlying genetic factors responsible for the CAKUT phenotype, and in our research, we focused on analyzing and comparing the expression levels of ectodysplasin A2 receptor (EDA2R), protocadherin9 (PCDH9), and TNF receptor-associated factor 7 (TRAF7) proteins in the cortex and medulla of healthy control kidneys during developmental phases 2, 3, and 4. We also performed an analysis of the area percentages of the mentioned proteins in the cortical and medullary sections of healthy embryonic and fetal kidneys compared to those affected by CAKUT, including duplex kidneys (DK), horseshoe kidneys (HK), hypoplastic kidneys (HYP), and dysplastic kidneys (DYS). We found that the CAKUT candidate gene proteins EDA2R, PCDH9, and TRAF7 are all expressed during normal human kidney development stages. In DYS, the expression of EDA2R was higher than in normal kidneys, likely due to EDA2R's role in apoptosis, which was upregulated in specific cases and could possibly contribute to the formation of DYS. The expression of PCDH9 was lower in HK, which can be attributed to the possible role of PCDH9 in cell migration suppression. Decreased PCDH9 expression is linked to increased cell migration, potentially contributing to the development of HK. The level of TRAF7 expression was reduced in all examined kidney disorders compared to normal kidneys, suggesting that this reduction might be attributed to the crucial role of TRAF7 in the formation of endothelium and ciliogenesis, both of which are essential for normal kidney development. Further research is required to ascertain the function of these proteins in both the typical development of the kidney and in CAKUT.

Prenatal prednisone exposure disturbs fetal kidney development and its characteristics.

Prednisone is a synthetic glucocorticoid that is commonly used in both human and veterinary medication. Now, it is also recognized as an emerging environmental contaminant. Pregnant women may be exposed to prednisone actively or passively through multiple pathways and cause developmental toxicity to the fetus. However, the impact of prenatal prednisone exposure (PPE) on fetal kidney development remains unclear. In this study, pregnant mice were administered prednisone intragastrically during full-term pregnancy with different doses (0.25, 0.5, or 1 mg/(kg·day)), or at the dose of 1 mg/(kg·day) in different gestational days (GD) (GD0-9, GD10-18, or GD0-18). The pregnant mice were euthanized on GD18. HE staining revealed fetal kidney dysplasia, with an enlarged glomerular Bowman's capsule space and a reduced capillary network in the PPE groups. The expression of the podocyte and the mesangial cell marker genes was significantly reduced in the PPE groups. However, overall gene expression in renal tubules and collecting ducts were markedly increased. All of the above effects were more pronounced in high-dose, full-term pregnancy, and female fetuses. Studies on the mechanism of the female fetal kidney have revealed that PPE reduced the expression of Six2, increased the expression of Hnf1ß, Hnf4α, and Wnt9b, and inhibited the expression of glial cell line-derived neurotrophic factor (GDNF) and Notch signaling pathways. In conclusion, this study demonstrated that there is a sex difference in the developmental toxicity of PPE to the fetal kidney, and the time effect is manifested as full-term pregnancy > early pregnancy > mid-late pregnancy.

Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development.

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.

Gestational diabetes mellitus induces congenital anomalies of the kidney and urinary tract in mice by altering RET/MAPK/ERK pathway.

Gestational diabetes mellitus (GDM) presents a substantial population health concern. Previous studies have revealed that GDM can ultimately influence nephron endowment. In this study, we established a GDM mouse model to investigate the embryological alterations and molecular mechanisms underlying the development of congenital anomalies of the kidney and urinary tract (CAKUT) affected by GDM. Our study highlights that GDM could contribute to the manifestation of CAKUT, with prevalent phenotypes characterized by isolated hydronephrosis and duplex kidney complicated with hydronephrosis in mice. Ectopic ureteric buds (UBs) and extended length of common nephric ducts (CNDs) were noted in the metanephric development stage. The expression of Ret and downstream p-ERK activity were enhanced in UBs, which indicated the alteration of RET/MAPK/ERK pathway may be one of the mechanisms contributing to the increased occurrence of CAKUT associated with GDM.

The extracellular matrix protein fibronectin promotes metanephric kidney development.

Complex interactions of the branching ureteric bud (UB) and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Impaired nephron endowment predisposes to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Since only little is known about their physiological role in kidney development, we aimed for analyzing the expression and role of fibronectin. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.5 and E13.5, fibronectin lined the UB epithelium, which became less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes and impaired glomerulogenesis following reduced cell proliferation and branching of the UB epithelium. Fibronectin colocalized with alpha 8 integrin and fibronectin loss caused a reduction in alpha 8 integrin expression, release of glial-derived neurotrophic factor and expression of Wnt11, both of which are promoters of UB branching. In conclusion, the ECM protein fibronectin acts as a regulator of kidney development and is a determinant of the final nephron number.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Engineering physiological environments to advance kidney organoid models from human pluripotent stem cells.

During embryogenesis, the mammalian kidney arises because of reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), driving UB branching and nephron induction. These morphogenetic processes involve a series of cellular rearrangements that are tightly controlled by gene regulatory networks and signaling cascades. Here, we discuss how kidney developmental studies have informed the definition of procedures to obtain kidney organoids from human pluripotent stem cells (hPSCs). Moreover, bioengineering techniques have emerged as potential solutions to externally impose controlled microenvironments for organoid generation from hPSCs. Next, we summarize some of these advances with major focus On recent works merging hPSC-derived kidney organoids (hPSC-kidney organoids) with organ-on-chip to develop robust models for drug discovery and disease modeling applications. We foresee that, in the near future, coupling of different organoid models through bioengineering approaches will help advancing to recreate organ-to-organ crosstalk to increase our understanding on kidney disease progression in the human context and search for new therapeutics.

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development.

We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad-mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

Differential expression of epithelial and smooth muscle lineage-specific markers of metanephros in one-humped camel foetuses.

The development of the metanephros in one-humped camels involves a complex series of interactions between epithelial and mesenchymal cells. As a result, there is a synchronized differentiation process of stromal, vascular and epithelial cell types during glomerulogenesis, angiogenesis and tubulogenesis. In the current work, the metanephros of camel foetuses were divided into four stages where kidneys from each stage were processed and immunoassayed, followed by quantitative analysis to determine target protein intensities throughout metanephrogenesis in the camel. This study demonstrated robust expression of α-smooth muscle actin (α-SMA) in the glomerular mesangium, as well as in interlobular and glomerular arterioles during the earlier stages of development. However, in the late stages, α-SMA expression became more localized around the blood capillaries in both the cortex and medulla. Strong expression of CD34 was observed in the immature glomerular and peritubular endothelial cells within the subcapsular zone, as well as in the glomerular, proximal tubular and distal tubular epithelium of stage one foetuses, although its expression gradually diminished with foetal maturation. The expression pattern of osteopontin was prominently observed in the distal convoluted tubules throughout all stages, however, no expression was detected in the proximal tubules, glomeruli and arterioles. E-cadherin was detected in the developing renal tubular epithelial cells but not in the glomeruli. In conclusion, this study reveals the spatiotemporal distribution of key proteins, including α-SMA, CD34, Osteopontin and E-cadherin, which play a crucial role in metanephrogenesis in camel foetuses.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Ultrasound Measurement of Fetal Kidney Length in Normal Pregnancy and its Correlation with Gestational Age.

BACKGROUND: To establish a nomogram of fetal kidney length for a normal pregnancy from 20 to 41 weeks of gestational age. METHODS: A prospective cross-sectional study was carried out in the Department of radio-diagnosis and imaging BPKIHS, Dharan. 400 kidneys of 200 fetuses between the gestational age 20 to 41 weeks were scanned. Renal measurement was performed by ultrasonography. RESULTS: Analysis was performed on data obtained from 200 normal fetuses. Size charts for right, left and mean fetal kidney length with standard deviation were presented for each weeks of gestation from 20 weeks to 41 weeks. The difference in renal length between right and left side was statistically not significant.There was significant correlation between gestational age and fetal kidney length (r=0.947, p=0.001). CONCLUSIONS: Fetal kidney length can be used as an adjunct parameter for estimation of gestation age.

Temporally and spatially regulated collagen XVIII isoforms are involved in ureteric tree development via the TSP1-like domain.

Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development.

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.

Acyl-CoA binding protein (ACBP) is highly expressed in the developing human kidney.

OBJECTIVE: Acyl-CoA-binding protein (ACBP), also known as diazepam binding inhibitor (DBI), is a small phylogenetically conserved protein. This ancestral peptide is multifunctional, performing intracellular activities as ACBP protein or extracellular roles as DBI. Several studies showed its endless facets, including a relevant activity as appetite stimulator and as anabolic factor. High levels of ACBP have been described in erythrocytes, liver, kidney, and gut cells. The aim of this study was to analyze, at immunohistochemical level, the expression of ACBP in fetal human tissues during development, focusing on the developing kidney. MATERIALS AND METHODS: Immunohistochemistry for ACBP was performed on 30 human fetal kidneys, from 15 fetuses of gestational age ranging from 13 to 19 weeks. At autopsy, all kidney samples were 10% formalin-fixed, routinely processed and paraffin-embedded. Five micron-thick paraffin sections were stained with Hematoxylin and Eosin and PAS stain for a morphological examination. RESULTS: ACBP was detected in all 30 kidneys analyzed in this study. No significant changes in ACBP expression were observed at different gestational ages. Immunostaining for ACBP was restricted to the epithelium covering the renal pelvis, the papillae, the collecting tubules, and the proximal and distal tubules. On the other hand, medullary regions and in the metanephric mesenchymal stem/progenitor cells did not show any reactivity for ACBP. CONCLUSIONS: According to our findings, ACBP should be considered as a new player in the complex field of human nephrogenesis, given that it was detected in all fetal kidneys immunostained. Its preferential localization in the renal structures derived from the Wolf duct, such as pelvis epithelium and collecting ducts, suggests a major role for ACBP in the induction of the metanephric mesenchymal cells toward the differentiation into glomerular structures. ACBP expression in proximal and distal tubules, two structures originating from the metanephric mesenchyme, indicates a further role of this protein in nephron development. In conclusion, ACBP should be added to the multiple molecules involved in human nephrogenesis.

Disruption of mitochondrial complex III in cap mesenchyme but not in ureteric progenitors results in defective nephrogenesis associated with amino acid deficiency.

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.

Disruption of Gen1 causes ectopic budding and kidney hypoplasia in mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a family of often-concurrent diseases with various anatomical spectra. Null-mutant Gen1 mice frequently develop multiple urinary phenotypes, most commonly duplex kidneys, and are ideal subjects for research on ectopic budding in CAKUT development. The upper and lower kidney poles of the Gen1PB/PB mouse were examined by histology, immunofluorescence, and immunohistochemistry. The newborn Gen1PB/PB mouse lower poles were significantly more hypoplastic than the corresponding upper poles, with significantly fewer glomeruli. On embryonic day 14.5, immediately before first urine formation, the upper pole kidney was already larger than the lower pole kidney. In vivo and in vitro, embryonic kidney upper poles had more ureteric buds than lower poles. Gen1PB/PB embryos exhibited ectopic ureteric buds, usually near the original budding site, occasionally far away, or, rarely, derived from the primary budding site. Therefore, ectopia of the ureteric buds is the core of CAKUT formation. Further studies will be needed to investigate the regulatory roles of these genes in initial ureteric budding and subsequent ontogenesis during metanephros development.

Growth Hormone and IGF1 Actions in Kidney Development and Function.

Growth hormone (GH) exerts multiple effects on different organs including the kidneys, either directly or via its main mediator, insulin-like-growth factor-1 (IGF-1). The GH/IGF1 system plays a key role in normal kidney development, glomerular hemodynamic regulation, as well as tubular water, sodium, phosphate, and calcium handling. Transgenic animal models demonstrated that GH excess (and not IGF1) may lead to hyperfiltration, albuminuria, and glomerulosclerosis. GH and IGF-1 play a significant role in the early development of diabetic nephropathy, as well as in compensatory kidney hypertrophy after unilateral nephrectomy. Chronic kidney disease (CKD) and its complications in children are associated with alterations in the GH/IGF1 axis, including growth retardation, related to a GH-resistant state, attributed to impaired kidney postreceptor GH-signaling and chronic inflammation. This may explain the safety of prolonged rhGH-treatment of short stature in CKD.

Adverse Impact of Environmental Chemicals on Developmental Origins of Kidney Disease and Hypertension.

Chronic kidney disease (CKD) and hypertension are becoming a global health challenge, despite developments in pharmacotherapy. Both diseases can begin in early life by so-called "developmental origins of health and disease" (DOHaD). Environmental chemical exposure during pregnancy can affect kidney development, resulting in renal programming. Here, we focus on environmental chemicals that pregnant mothers are likely to be exposed, including dioxins, bisphenol A (BPA), phthalates, per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAH), heavy metals, and air pollution. We summarize current human evidence and animal models that supports the link between prenatal exposure to environmental chemicals and developmental origins of kidney disease and hypertension, with an emphasis on common mechanisms. These include oxidative stress, renin-angiotensin system, reduced nephron numbers, and aryl hydrocarbon receptor signaling pathway. Urgent action is required to identify toxic chemicals in the environment, avoid harmful chemicals exposure during pregnancy and lactation, and continue to discover other potentially harmful chemicals. Innovation is also needed to identify kidney disease and hypertension in the earliest stage, as well as translating effective reprogramming interventions from animal studies into clinical practice. Toward DOHaD approach, prohibiting toxic chemical exposure and better understanding of underlying mechanisms, we have the potential to reduce global burden of kidney disease and hypertension.