Galectin-3 modulates ureteric bud branching in organ culture of the developing mouse kidney

Galectin-3 is a mammalian beta-galactoside-specific lectin with functions in cell growth, adhesion, and neoplastic transformation. On the basis of expression patterns in humans, it is proposed that galectin-3 modulates fetal collecting duct growth. This article provides evidence that galectin-3 can modulate branching morphogenesis of the mouse ureteric bud/collecting duct lineage. With the use of immunohistochemistry, galectin-3 was not detected in early metanephrogenesis but was upregulated later in fetal kidney maturation when the protein was prominent in basal domains of medullary collecting ducts. Addition of galectin-3 to embryonic days 11 and 12 whole metanephric cultures inhibited ureteric bud branching, whereas galectin-1 did not perturb morphogenesis, nor did a galectin-3 mutant lacking wild-type high-affinity binding to extended oligosaccharides. Exogenous galectin-3 retarded conversion of renal mesenchyme to nephrons in whole metanephric explants but did not affect nephron induction by spinal cord in isolated renal mesenchymes. Finally, addition of a blocking antiserum to galectin-3 caused dilation and distortion of developing epithelia in embryonic day 12 metanephroi cultured for 1 wk. The upregulation of galectin-3 protein during kidney maturation, predominantly at sites where it could mediate cell/matrix interactions, seems to modulate growth of the ureteric tree.     

PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases

PDGF-C is a new member of the PDGF-family and has recently been identified as a rat mesangial cell mitogen. Its expression and function in human kidneys is unknown. Localization of PDGF-C protein was analyzed by immunohistochemistry using a rabbit polyclonal antibody directed against the core-domain of PDGF-C in human fetal kidneys (n = 8), normal adult human kidneys (n = 9), and in renal biopsies of patients with IgA nephropathy (IgAN, n = 31), membranous nephropathy (MGN, n = 8), minimal change disease (MC, n = 7), and transplant glomerulopathy (TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissected glomeruli by real-time RT-PCR in cases of normal adult kidneys (n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In the fetal kidney, PDGF-C localized to the developing mesangium, ureteric bud epithelium, and the undifferentiated mesenchyme. In the adult kidney, PDGF-C was constitutively expressed in parietal epithelial cells of Bowman's capsule, tubular epithelial cells (loops of Henle, distal tubules, collecting ducts), and in arterial endothelial cells. A marked upregulation of glomerular PDGF-C protein was seen in MGN and TxG with a prominent positivity of virtually all podocytes. In MC, PDGF-C localized to podocytes in a more focal distribution. In MGN, increased glomerular PDGF-C protein expression was due to increased mRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detected in microdissected glomeruli from MGN compared with normal. PDGF-C protein was additionally expressed in individual mesangial cells in TxG. Finally, upregulated PDGF-C protein expression was detected within sclerosing glomerular and fibrosing tubulointerstitial lesions in individual cases from all analyzed groups. We conclude that PDGF-C is constitutively expressed in the human kidney and is upregulated in podocytes and interstitial cells after injury/activation of these cells.     

Immunolocalization of the four prostaglandin E2 receptor proteins EP1, EP2, EP3, and EP4 in human kidney.

Four prostaglandin E2 receptor subtypes designated EP1, EP2, EP3, and EP4 have been shown to mediate a variety of effects of prostaglandin E2 (PGE2) on glomerular hemodynamics, tubular salt and water reabsorption, and on blood vessels in the human kidney. Despite the important role of renal PGE2, the localization of PGE2 receptor proteins in the human kidney is unknown. The present study used antipeptide antibodies to the EP1 to EP4 receptor proteins for immunolocalization in human kidney tissue. Immunoblot studies using these antibodies demonstrated distinct bands in membrane fraction from human kidney. By means of immunohistochemistry, expression of the human EP1 receptor subtype protein in renal tissue was detected mainly in connecting segments, cortical and medullary collecting ducts, and in the media of arteries and afferent and efferent arterioles. The human EP2 receptor subtype protein was detectable only in the media of arteries and arterioles. The human EP3 receptor subtype protein was strongly expressed in glomeruli, Tamm-Horsfall negative late distal convoluted tubules, connecting segments, cortical and medullary collecting ducts, as well as in the media and the endothelial cells of arteries and arterioles. Staining of the human EP4 receptor subtype protein was observed in glomeruli and in the media of arteries. However, no signal of either receptor subtype was detected in the thick ascending limb, the macula densa, or in adjacent juxtaglomerular cells. These results support the concept that PGE2 modulates specific functions in different anatomical structures of the human kidney.     

Cycloxygenase-2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henle's loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.     

Gene expression of prostanoid forming enzymes along the rat nephron

BACKGROUND: To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. METHODS: For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. RESULTS: Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. CONCLUSION: Given that the existence of cyclooxygenase in combination with the different PG-synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable.     

Preferential expression of insulin-like growth factor binding proteins-1, -3, and -5 during early diabetic renal hypertrophy in rats

The renal insulin-like growth factor-I (IGF-I) system has been implicated in the pathogenesis of renal hypertrophy, altered hemodynamics, and extracellular matrix expansion associated with early diabetes. The relative abundance of IGF binding proteins (IGFBPs) in the renal microenvironment may modulate IGF-I actions. However, the precise IGFBPs expressed in the glomerular and tubulointerstitial compartments during diabetic renal growth have not been characterized. In the present study, in situ hybridization studies were performed to examine the expression of IGFBP-1 to -6 messenger RNAs (mRNAs) 3, 7, and 14 days after streptozotocin (STZ) injection in rats. In control, nondiabetic kidneys, all six IGFBP mRNAs were differentially expressed with a predominance of IGFBP-5. The onset of renal hypertrophy in STZ-induced diabetes was associated with a rapid and site-specific induction of IGFBP-1, -3, and -5 mRNAs. In contrast, basal expression of IGFBP-2, -4, and -6 mRNAs was not altered in diabetic rats. IGFBP-5 mRNA expression increased in diabetic glomeruli, cortical, and inner medullary peritubular interstitial cells at days 3, 7, and 14. Although normal glomeruli failed to express IGFBP-3, it was induced concomitantly with IGFBP-5 in diabetic glomeruli and cortical peritubular interstitial cells. IGFBP-1 mRNA levels also increased in cortical tubular cells at each time point tested. Peak induction of IGFBP-3 and -5 was observed at day 3, whereas IGFBP-1 was delayed until day 7. IGFBP-1, -3, and -5 mRNA levels declined by day 14, but remained persistently elevated above control. By immunoperoxidase staining, similar alterations in the pattern of IGFBP-3 and -5 protein expression were observed at each time point. The preferential and site-specific increase in IGFBP-1, -3, and -5 suggest that these IGFBPs may regulate the local autocrine and/or paracrine actions of IGF-I and contribute to the pathogenesis of the early manifestations of diabetic nephropathy.     

Endothelin synthesis by rat inner medullary collecting duct cells

Endothelin has been shown to affect a broad range of renal functions, including rat inner medullary collecting duct Na/K ATPase activity, renin release, renal blood flow, and glomerular filtration rate. The source of endothelin in the kidney has been assumed to be endothelial cells. However, the inner medulla contains the highest concentration of immunoreactive endothelin in the kidney. Additionally, MDCK cells, a distal tubule-like cell line, synthesize endothelin. In order to determine if primary renal tubule cells release endothelin, supernatants collected from rat inner medullary collecting duct cells in culture were tested for endothelin-1 detected by specific radioimmunoassay. Inner medullary collecting duct cells produced endothelin-1 in a time-dependent manner, releasing 1,016.7 +/- 60.1 pg of endothelin-1 per mg/cell protein/24 h. Inner medullary collecting duct cells expressed a 2.2-kilobase mRNA on blot hybridization with rat prepro endothelin-1 cDNA. Vasopressin, thrombin, bradykinin, and epinephrine did not affect endothelin-1 release. These data demonstrate endothelin-1 production by inner medullary collecting duct cells and suggest a possible autocrine role for the peptide.     

The role of adrenomedullin and receptors in glomerular hyperfiltration in streptozotocin-induced diabetic rats

BACKGROUND: Since adrenomedullin (AM) elicits vasodilatation by binding to specific AM receptors consisted of calcitonin-receptor-like receptor (CRLR)/receptor-activity-modifying protein 2 (RAMP2) or CRLR/receptor-activity-modifying protein 3 (RAMP3) on endothelial cells and stimulating nitric oxide production, AM possibly involves in glomerular capillary dilatation in early phase of diabetic nephropathy. METHODS: Streptozotocin (STZ)-induced diabetic Sprague-Dawley rats at 4 weeks after the injection were employed for expression studies of AM, RAPM2, and RAMP3. The measurement of AM peptide levels in kidney tissue, plasma, and urine was performed. Human aortic endothelial cells (HAEC) were used to investigate functional link between glucose-induced AM production and nitric oxide release. RESULTS: STZ rats showed glomerular hypertrophy and increased urinary NO2- and NO3- excretion. By Northern blot analyses, AM and RAPM2 mRNAs significantly increased in the kidneys of STZ rats, while RAMP3 mRNA was not altered. In STZ rats, AM peptide was actively secreted into urine (1280 +/- 360 fmol/day vs. control 110 +/- 36 fmol/day). AM peptide was mainly detected on cortical and medullary collecting duct cells in control rat kidneys and AM peptide and mRNA were up-regulated on afferent arterioles and glomeruli of STZ rats. RAMP2 expression was detected on afferent arterioles and not in glomeruli in control rats and it was up-regulated on glomerular endothelial cells in STZ rats. In HAEC culture, d-glucose stimulated AM and nitric oxide production and they were suppressed by addition of AM antisense oligodeoxynucleotides. CONCLUSION: Up-regulated expression of AM and RAMP2 in afferent arterioles and glomeruli may be related to selective dilatation of glomerular capillary in acute phase of type 1 diabetes.     

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.     

Immunolocalization of fibroblast growth factor-1 and -2 in the embryonic rat kidney

Summary: Fibroblast growth factors (FGF) regulate cell proliferation, migration, differentiation and angiogenesis during morphogenesis in many different tissues. Recent evidence indicates that exogenous FGF-2 stimulates mesenchymal condensation in cultured rat metanephroi, a crucial epithelial-mesenchymal induction event in the developing nephron. the aim of the present investigation was to determine the in vivo distribution of FGF-1 and FGF-2 in developing rat metanephroi at embryonic days 14, 15, 16, 18 and 20. Avidin-biotin enhanced indirect immunohistochemistry was used to demonstrate that both FGF-1 and FGF-2 were co-localized in metanephroi at all ages studied. High levels of FGF-1 and FGF-2 were present in ureteric bud branches and in developing distal tubules. Fibroblast growth factor-1 and FGF-2 were colocalized in developing nephron elements, from vesicles to S-shaped bodies, and in the mesangium of capillary loop and maturing stage glomeruli. Both growth factors were present in the mesenchyme of the nephrogenic zone and in the interstitium of the developing cortex. However, immunostaining for FGF was not evident in mesenchymal condensates, endothelial cells, medullary interstitial cells, or in the thin undifferentiated epithelium of the immature loop of Henle. These findings indicate that the expression of both FGF-1 and FGF-2 is tightly regulated in the embryonic kidney and suggest a role for these molecules in kidney development.     

Interacting molecule of AT1 receptor, ATRAP, is colocalized with AT1 receptor in the mouse renal tubules

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.     

Expression of transforming growth factor-beta 1 and beta 2 in rat glomeruli

The transforming growth factors-beta are potent modulators of cell growth and extracellular matrix metabolism in most types of cultured cells. The distribution and functions of TGF-beta in vivo are less well known. We utilized several different techniques including northern blots, a CCl-64 cell growth inhibition assay, and sandwich enzyme-linked immunosorbent assays (SELISA) to examine the expression of TGF-beta 1 and TGF-beta 2 in rat glomeruli. High levels of TGF-beta 1 mRNA and protein were found in glomeruli (56 +/- 22 ng TGF-beta 1/g tissue). These levels were several-fold higher than those present in whole kidney (10 +/- 5 ng/g). TGF-beta 2 mRNA was present in glomeruli but was not detected in whole kidney. TGF-beta 2 concentrations by SELISA were 19 +/- 8 ng TGF-beta 2/g in glomeruli and less than 5 ng/g in whole kidney. Since TGF-beta has such marked effects on cell growth, we also examined whether alterations in TGF-beta expression were associated with the renal hypertrophy which follows unilateral nephrectomy. Expression of TGF-beta 1 mRNA decreased in glomeruli following nephrectomy. However, this was not associated with a significant fall in glomerular TGF-beta 1 protein concentration. Whole kidney levels of TGF-beta 1 and its mRNA were unchanged following nephrectomy. Similar results were obtained for TGF-beta 2. Our data document the presence of high concentrations of TGF-beta 1 and beta 2 and their corresponding mRNAs in normal rat glomeruli. These results suggest that TGF-beta may play important regulatory roles in the normal glomerulus.