Expression of transforming growth factor-β type II receptor mRNA in embryonic and adult rat kidney

Summary: The transforming growth factor-β (TGF-β) family of growth factors regulates cell proliferation, differentiation, extracellular matrix synthesis and angiogenesis in many developing tissues. Transforming growth factor-β1 was recently shown to affect the branching of ureteric epithelium and nephron formation in cultured rat metanephroi. As the TGF-β type II receptor is specific for the TGF-β family, the present study used in situ hybridization to localize mRNA for this receptor in metanephroi from Sprague-Dawley rat embryos. Transforming growth factor-β type II receptor mRNA was located in ureteric duct epithelium, undifferentiated mesenchymal cells in the nephrogenic zone, vesicles, comma-shaped bodies and S-shaped bodies. In some S-shaped bodies, TGF-β type II receptor mRNA was not expressed in the lower limb, which subsequently forms the renal corpuscle. Expression was not observed in capillary loop stage glomeruli and maturing glomeruli, or in proximal tubules and interstitial cells. In adult rat kidney, TGF-β type II receptor mRNA was expressed in cortical collecting ducts and distal tubules but not in glomeruli or proximal tubules. These findings demonstrate that the prominent expression of TGF-β type II receptor mRNA decreases as glomeruli and tubules develop. Expression then remains undetectable in adult glomeruli and proximal tubules. the developmentally-regulated expression of this receptor suggests a key role in glomerular and nephron development.     

Localization of fibroblast growth factor-2 (basic FGF) and FGF receptor-1 in adult human kidney.

BACKGROUND: The expression pattern of fibroblast growth factor-2 (FGF-2; basic FGF), a pleiotrophic growth factor, as well as one of its receptors (FGFR1), in the kidney is highly controversial. METHODS: Using an approach that combines multiple antibodies for immunohistochemistry and correlative in situ hybridization, we assessed the intrarenal expression of both FGF-2 and FGFR1 in 13 specimens of adult kidney removed during tumor nephrectomy. RESULTS: The FGF-2 expression pattern in the kidneys as detected by immunohistochemistry was variable and depended on the antibody used. The most consistent expression of FGF-2 protein was demonstrated in glomerular parietal epithelial cells, tubular cells (mainly of the distal nephron), as well as arterial endothelial cells. These locations also corresponded to areas of FGF-2 mRNA expression. Additionally, by immunohistochemistry, FGF-2 protein was detected in arterial smooth muscle cells and occasional podocytes. The expression of FGFR1 protein and mRNA was most consistently present in tubular cells of the distal nephron and in vascular smooth muscle cells. In situ hybridization, but not immunohistochemistry, also suggested FGFR1 expression in cells that could not be precisely identified within the glomerular tuft as well as some interstitial cells. CONCLUSION: These data suggest potential autocrine and paracrine pathways within the FGF-2 system, particularly within the vascular walls and in the distal nephron, and thereby provide information for further mechanistic understanding of the role of the FGF-2 system in human renal disease.     

Fibroblast growth factor receptor signaling in kidney and lower urinary tract development

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.     

Differential expression of fibroblast growth factor receptor-1, -2, and -3 and syndecan-1, -2, and -4 in neonatal rat mandibular condyle and calvaria during osteogenic differentiation in vitro

Fibroblast growth factors (FGFs) play important roles in the control of skeletal cell growth and differentiation. To identify the mechanisms of regulation of FGF actions during chondrogenesis and osteogenesis, we investigated, by immunohistochemistry, the spatiotemporal expression of the high-affinity FGF receptors (FGFR-1, -2, and -3) and coreceptors (syndecans-1, -2, and -4) in newborn rat condyle and calvaria during chondrogenesis and osteogenesis in vitro. During chondrogenesis at 4 days of culture, condyle chondrocytes showed weak FGFR-1, FGFR-2, and syndecan-1 immunoreactivity; stronger syndecan-2 expression; and marked FGFR-3 and syndecan-4 immunolabeling. At a later stage (i.e., 9 days of culture), FGFR-1, -2, and -3 were coexpressed with syndecan-4 in chondrocytes. Condyle progenitor cells located in the condyle perichondrium initially expressed strong syndecan-2 and -4 and weak syndecan-1 labeling, whereas no FGFR was detectable. When these cells differentiated into osteoblasts, they expressed syndecan-2 and -4 coincidently with FGFR-1, -2, and -3 at 9 days of culture. In newborn rat calvaria, syndecan-1, -2, and -4 were coexpressed mainly with FGFR-1 and -2 in osteoblasts. In the two models, treatment with FGF-2 (100 ng/mL) at 4-9 days of culture increased cell growth and decreased glycosaminoglycan or collagen synthesis, respectively, suggesting interactions of FGF-2 with distinct FGFRs and syndecans during chondrogenesis and osteogenesis. The coincident or distinct spatiotemporal expression pattern of FGFRs and syndecans in chondrocytes, progenitor cells, and osteoblasts represents a dynamic mechanism by which FGF effects on skeletal cells may be controlled in a coordinate manner during cartilage and bone formation in vitro.     

Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.     

In vitro effects of growth factors on lung hypoplasia in a model of congenital diaphragmatic hernia

BACKGROUND/PURPOSE: Pulmonary hypoplasia, a leading contributor to the lethality of congenital diaphragmatic hernia (CDH), precedes diaphragmatic malformation in the nitrofen model and persists to allow experimental manipulations in organ culture. Fibroblast growth factors (FGFs) are crucial to early lung development. Acidic FGF (FGF-1) binds to all FGF receptors and enhances in vitro branching morphogenesis. Basic FGF (FGF-2) is localized to developing airway epithelium, basement membrane, and extracellular matrix. Heparin (HEP) modulates FGF kinetics and inhibits smooth muscle proliferation in lung primordia. The aim of this study was to examine the morphological effects of fibroblast growth factors and heparin on lung hypoplasia in an organ culture model. METHODS: Sprague-Dawley rats were fed nitrofen on day 9.5 of pregnancy to induce lung hypoplasia and CDH in newborns. Control rats received olive oil. Normal and hypoplastic lung primordia were microdissected on day 13.5 of gestation and cultured up to 78 hours in plain media with or without FGF-1 or FGF-2, with or without HEP. In vitro morphological development was studied by serial measurements of terminal bud count, lung area, and lung perimeter. RESULTS: Over 120 fetal lung specimens were studied (n > or = 4 per group). Significant increases in area, perimeter, and bud count were seen in normal lungs cultured with FGF-1 plus HEP compared with control media (P < .05). In the nitrofen lungs, FGF1 plus HEP yielded reductions in all parameters compared with those in control media (P < .05), whereas FGF-2 produced significant expansion in lung area but marked reductions in bud count and lung perimeter divided by square root of area (P < .05). Heparin did not produce substantial or sustained alteration of morphology in normal or hypoplastic lungs. CONCLUSIONS: These observations may indicate an intrinsic abnormality of FGF processing in the hypoplastic nitrofen lung before diaphragmatic malformation. Heparin did not rescue abnormal lung development. Mechanisms underlying the differential effects of these agents now need to be explored to target fetal lung growth and improve the dismal prognosis of human CDH.     

Development and functional capacity of transplanted rat metanephroi.

BACKGROUND: Transplantation of embryonic kidneys (metanephroi) offers a potential solution to the problem of kidney donor shortage. The aim of this study was to characterise the haemodynamic capacity of transplanted rat metanephroi and to determine the number and maturity of the tubules. METHODS: Metanephroi from E15 Lewis rat embryos were transplanted adjacent to the abdominal aorta of uninephrectomised adult female syngeneic Lewis rats. Twenty-one days later, a single metanephros ureter was anastomosed to the host's urinary system. Three months later animals were prepared for standard clearance measurements. RESULTS: Effective renal blood flow (149 +/- 33 microl min(-1) per g kidney weight) and glomerular filtration rate (17 +/- 9 microl min(-1) per g kidney weight), standardised to kidney weight, were significantly lower in transplanted metanephroi compared with control adult kidneys (P < 0.001); renal vascular resistance (934 +/- 209 mmHg ml min(-1) per g kidney weight) was significantly higher (P < 0.001). Nephron number in transplanted metanephroi was significantly greater than that of E21 kidneys (P < 0.01) but lower than that of postnatal day (PND) 1 kidneys (P < 0.001). Angiotensin II type 2 receptor mRNA expression, a marker of nephrogenesis, was markedly reduced in metanephroi. Aquaporins 1 and 2, epithelial Na channel and Na-K-2Cl cotransporter type 2 mRNA and protein were expressed in transplanted metanephroi; the urea transporters-A1, 2 and 3 were absent. Vascular markers (alpha-smooth muscle actin and CD31) were identified in metanephroi but their expression did not differ from that of E21 and PND 1 kidneys. CONCLUSIONS: This study shows that metanephroi continue to develop post-transplantation but only reach a stage of development equivalent to that of a normal rat kidney at birth.     

Mesenchymal hamartoma of the liver--new insight into histogenesis.

BACKGROUND/PURPOSE: Mesenchymal hamartoma (MH) of the liver is thought to develop from the ductal plates of the prenatal liver. This immunohistochemical study was performed to gain insight into the pathophysiology of its development. METHODS: Specimens from four MHs with adjacent liver, in one case from a biopsy and from the resected lesion after 6 years follow-up, were investigated with immunostaining on cryostatsectionswith antibodies against cytokeratins, vimentin, desmin and alpha-actin, as well as von Willebrand factor (factor VIII), fibroblast growth factor (FGF) receptors, FGF-1 (acidic FGF), FGF-2 (basic FGF), and the proliferation-associated Ki67 antigen. RESULTS: Fibrous tissue of MH stained positive not only for vimentin, but also for desmin and alpha-actin, whereas cytokeratins and factor VIII showed specific staining in biliary cysts and endothelial cells, respectively. All mesenchymal cells expressed proteins of the FGF receptor family. Although FGF-1 was only scarcely detectable, there was an accumulation of FGF-2 in borderline areas of liver to MH. Multiple Ki67-positive mesenchymal cells could be identified in these regions in all three MHs. However, we could not detect any proliferative activity in the MHs after follow-up. CONCLUSIONS: The proliferative process in MH is still active during early childhood. FGF-2 may have a role in promoting this process. The positivity for desmin and alpha-actin of the lesions suggests that fat-storing (Ito) cells of the immature liver may be involved in the development of MH.     

ERK and p38 MAP kinase are required for rat renal development

BACKGROUND: We previously demonstrated that extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase (p38) are strongly expressed in the embryonic kidney. In the present study, we investigated the role of ERK and p38 during kidney development. METHODS: Rat metanephroi were cultured from 15-day-old embryos, and exposed to inhibitors of MEK, an activator of ERK, PD98059 (300 micromol/L), U0126 (10 micromol/L), or a p38 inhibitor SB203580 (30 micromol/L) 24 to 120 hours after the start of culture. Growth of metanephroi was measured by surface area and thymidine incorporation. Ureteric buds and glomeruli were identified by labeling with Dolichos biflorus lectin and peanut agglutinin, respectively. PCNA staining and TUNEL assay were performed on kidney sections. The level of apoptosis was evaluated by examining DNA ladder formation. RESULTS: Growth of metanephroi was significantly inhibited by SB203580 but not by PD98059 or U0126. Ureteric bud branching was not affected by SB203580 or MEK inhibitors. Glomerular number was markedly reduced by SB203580 and to a lesser extent by U0126 (14 +/- 2 and 48 +/- 10% of controls, respectively). On histological examination, the number of tubuloglomerular structures was reduced in MEK inhibitor-treated metanephroi compared to controls. Very few mesenchymal condensates were observed in kidneys incubated with SB203580. PCNA-positive cells were reduced in SB203580-treated metanephroi compared to control and PD98059-treated kidneys. Apoptosis was increased in SB203580-treated kidneys and to a lesser extent in PD98059-treated cultures. CONCLUSIONS: Both ERK and p38 are required for renal development. ERK appears to play a role in nephrogenesis and p38 for kidney growth and nephrogenesis.     

One day exposure to FGF-2 was sufficient for the regenerative repair of full-thickness defects of articular cartilage in rabbits

OBJECTIVES: Administration of fibroblast growth factor (FGF)-2 for 2 weeks induces a successful cartilaginous repair response in 5-mm full-thickness articular cartilage defects in rabbits. The purpose of this study was to investigate the effects of a short time exposure to FGF-2 on the repair of the defects. METHODS: Five-mm-diameter cylindrical defects, which do not repair spontaneously, were created in the femoral trochlea of the rabbit knees. The defects were administered sterile saline or FGF-2 (150pg/h) via an osmotic pump for the initial 1 day, 3 days, or 2 weeks, and we assessed the FGF-2 action on the proliferation and migration of mesenchymal cells in the reparative tissue. Using a total of 126 rabbits, we performed three sets of experiments. We also studied the effect of FGF-2 on migration of marrow-derived mesenchymal cells in vitro. RESULTS: FGF-2 treatment for 1 day or 3 days induced the sequential chondrogenic repair responses that led to successful cartilaginous resurfacing of defects within 8 weeks as well as the 2-week treatment did. We confirmed by a radioisotope study that FGF-2 injected was rapidly eliminated from the defects (a residual ratio of 50% within 30min). The effect of FGF-2 on cultured marrow-derived cells suggested that FGF-2 facilitated the mobilization and migration of replicating mesenchymal cells from bone marrow. CONCLUSIONS: Only 1 day exposure to FGF-2 is sufficient for induction of the chondrogenic repair response in 5-mm-diameter full-thickness defects of articular cartilage in rabbits. FGF-2 stimulated the recruitment of mesenchymal cells into the defects, which was a limiting step for the induction of cartilage.