Detyrosinated tubulin is decreased in fetal vessels of preeclampsia placentas.

INTRODUCTION: Preeclampsia is a hypertensive, gestational disease, which is still the leading cause of pregnancy related morbidity and mortality. The impairment of placental angiogenesis and vascularization is discussed to be of etiopathologic relevance. Deytrosination and tyrosination of α-tubulin is important for the stability and dynamics of microtubules. An increase of α-tubulin detyrosination leads to microtubule stabilization, which is an essential prerequisite for physiologic vascular tube morphogenesis during angiogenesis. So far, little is known about the specific localization of detyrosinated (detyr) and tyrosinated (tyr) tubulin in the placenta and its relevance for preeclampsia. METHODS: Placental expression of detyr- and tyr-tubulin was analyzed by immunohistochemistry, immunofluorescence and western blot. For western blot quantification we used biopsies from healthy placentas (n = 21) and placentas from pregnancies complicated with small for gestational age (n = 5), preeclampsia (n = 5) or both (n = 5). RESULTS: Specific placental localization of detyr-tubulin was detected in the fetal endothelial cells of the placenta. Villous and extravillous trophoblasts as well as villous stroma cells were tyr-tubulin positive. Detyr-tubulin protein expression was significantly decreased in placentas complicated by preeclampsia. CONCLUSIONS: In summary, we report an accumulation of detyr-tubulin in villous vessels of the placenta and a significantly reduced level of detyr-tubulin in placental biopsies of preeclampsia cases. The reduction of placental detyr-tubulin in preeclampsia could suggest a deficit in villous vascular plasticity and might be associated with the impaired arborization of the disease.

Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma.

BACKGROUND: Human placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases. METHODS: Three choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2'-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry. RESULTS: In the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2'-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots. CONCLUSIONS: Our findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

Searching the web: a survey on the quality of advice on postnatal sequelae of intrauterine growth restriction and the implication of developmental origins of health and disease.

Intrauterine growth restriction (IUGR) and fetal growth restriction (FGR) are pregnancy complications associated with morbidity in later life. Despite a growing body of evidence from current research on developmental origins of health and disease (DOHaD), little information is currently provided to parents on long-term metabolic, cardiovascular and neurologic consequences. As parents strongly rely on internet-based health-related information, we examined the quality of information on IUGR/FGR sequelae and DOHaD in webpages used by laypersons. Simulating non-clinicians experience, we entered the terms 'IUGR consequences' and 'FGR consequences' into Google and Yahoo search engines. The quality of the top search-hits was analyzed with regard to the certification through the Health On the Net Foundation (HON), currentness of cited references, while reliability of information and DOHaD-related consequences were assessed via the DISCERN Plus score (DPS). Overall the citation status was not up-to-date and only a few websites were HON-certified. The results of our analysis showed a dichotomy between the growing body of evidence regarding IUGR/FGR-related sequelae and lack of current guidelines, leaving parents without clear directions. Furthermore, detailed information on the concept of DOHaD is not provided. These findings emphasize the responsibility of the individual physician for providing advice on IUGR/FGR-related sequelae, monitoring and follow-up.

The placental mTOR-pathway: correlation with early growth trajectories following intrauterine growth restriction?

Idiopathic intrauterine growth restriction (IUGR) is a result of impaired placental nutrient supply. Newborns with IUGR exhibiting postnatal catch-up growth are of higher risk for cardiovascular and metabolic co-morbidities in adult life. Mammalian target of rapamycin (mTOR) was recently shown to function as a placental nutrient sensor. Thus, we determined possible correlations of members of the placental mTOR signaling cascade with auxologic parameters of postnatal growth. The protein expression and activity of mTOR-pathway signaling components, Akt, AMP-activated protein kinase α, mTOR, p70S6kinase1 and insulin receptor substrate-1 were analysed via western blotting in IUGR v. matched appropriate-for-gestational age (AGA) placentas. Moreover, mTOR was immunohistochemically stained in placental sections. Data from western blot analyses were correlated with retrospective auxological follow-up data at 1 year of age. We found significant catch-up growth in the 1st year of life in the IUGR group. MTOR and its activated form are immunohistochemically detected in multiple placental compartments. We identified correlations of placental mTOR-pathway signaling components to auxological data at birth and at 1 year of life in IUGR. Analysis of the protein expression and phosphorylation level of mTOR-pathway components in IUGR and AGA placentas postpartum, however, did not reveal pathognomonic changes. Our findings suggest that the level of activated mTOR correlates with early catch-up growth following IUGR. However, the complexity of signals converging at the mTOR nexus and its cellular distribution pattern seem to limit its potential as biomarker in this setting.

The tumor suppressor gastrokine-1 is expressed in placenta and contributes to the regulation of trophoblast migration.

INTRODUCTION: Gastrokine-1 (GKN1) is a secreted auto-/paracrine protein, described to be expressed in the gastric mucosa. In gastric cancers GKN1 expression is commonly down-regulated. While current research focusses on the exploration of tumor-suppressive properties of GKN1 with regard to its potential clinical use in the treatment of gastroenterologic tumor disease, nothing is known about GKN1 expression and function in other organ systems. We investigated GKN1 expression in placental tissue and cells. MATERIALS AND METHODS: GKN1 was localized using immunohistochemistry in first and third trimester placental tissue, hydatidiform moles and various gestational trophoblastic neoplasias. We determined the expression of GKN1 in immunomagnetic bead-separated term placental cells and in choriocarcinoma cell lines. The role of GKN1 for JEG-3 migration was studied using live cell imaging. E-cadherin, MMP-2 and -9, TIMP-1 and -2, as well as urokinase (uPA) expression levels were determined. RESULTS: GKN1 is expressed in healthy third trimester placentas. Its expression is specifically limited to the extravillous trophoblast (EVT). GKN1 expression is significantly reduced in choriocarcinoma cell lines and gestational trophoblastic neoplasias. GKN1 attenuates the migration of JEG-3 choriocarcinoma cells in vitro, possibly via AKT-mediated induction of E-cadherin. GKN1 treatment reduced MMP-9 expression in JEG-3. DISCUSSION: Besides its role in gastric physiology our results clearly indicate regulatory functions of GKN1 in the EVT at the feto-maternal interface during pregnancy. Based on our findings in the JEG-3 choriocarcinoma cell line, an auto-/paracrine role of GKN1 for EVT motility and villous anchorage at the basal plate is conceivable. Thus, the tumor suppressor GKN1 is expressed in placental EVT and might contribute to the regulation of EVT migration/invasion.

Sympathetic blockade prevents the decrease in cardiac VEGF expression and capillary supply in experimental renal failure.

Uremic cardiomyopathy of men and rodents is characterized by lower myocardial capillary supply that in rats could be prevented by central and peripheral blockade of the sympathetic nervous system. The underlying pathomechanisms remain largely unknown. We investigated whether alterations of cardiac vascular endothelial growth factor (VEGF) gene and protein expression were involved. In our long-term experiment, we analyzed whether VEGF gene and protein expression was altered in the heart of male Sprague-Dawley rats with either sham operation (sham, n=10) or subtotal nephrectomy (SNX, n=10). In our short-term experiment (17 sham, 24 SNX), the effect of a putative downregulation of sympathetic nervous activity by surgical renal denervation (interruption of renal afferent pathways) on cardiac gene expression of VEGF, flt-1, and flk-1 and on myocardial capillary supply was analyzed. In the long-term study, cardiac capillary supply and vascular endothelial growth factor gene and protein expression were significantly lower in SNX than in sham. In the short-term experiment, cardiac VEGF mRNA expression was significantly lower in untreated SNX (4,258±2,078 units) than in both sham groups (11,709±4,169 and 8,998±4,823 units); this decrease was significantly prevented by renal denervation (8,190±3,889, P<0.05). We conclude that cardiac VEGF gene and protein expression is reduced in experimental renal failure, and this may be considered as one potential reason for impaired myocardial adaptation under the situation of cardiac hypertrophy. The beneficial effect of sympathetic downregulation on cardiac structure and function in renal failure may be at least in part explained by increased cardiac VEGF gene expression.

Lack of nidogen-2 increases blood pressure, glomerular and tubulointerstitial damage in DOCA-salt hypertension.

BACKGROUND: Nidogen-2, an extracellular matrix protein, is ubiquitous in renal basement membranes linking the laminin and collagen IV networks. Nidogen-2-deficient (nidogen-2(-/-)) mice do not exhibit a phenotype, and renal basement membranes appear normal. The functional role of nidogen-2 in the adult kidney under pathological conditions however remains unclear. We tested the hypothesis that nidogen-2 mediated cell-matrix interactions are important to maintain glomerular integrity and structure in renal hyperperfusion and hypertension. MATERIALS AND METHODS: Two weeks after unilateral nephrectomy (UNX), desoxycorticosterone (DOCA)-salt hypertension was induced in nidogen-2(-/-) mice and their wild type littermates for 6 weeks. Renal damage was assessed by means of semiquantitative scoring, morphometric analysis, immunohistochemistry and measurement of serum creatinine and albumin excretion. RESULTS: UNX alone resulted in a very mild increase in renal damage in nidogen-2(-/-) mice compared to wild type animals. Following DOCA-salt treatment, blood pressure, serum creatinine and albumin excretion were significantly higher in nidogen-2(-/-) than in wild type mice. In addition, nidogen-2(-/-) mice showed increased mesangial cell hyperplasia and matrix expansion with higher expression of fibronectin and its receptor alpha8 integrin. Glomerular capillaries were significantly reduced in size and number. CONCLUSIONS: We demonstrate that in both mild and severe glomerular damage, lack of nidogen-2 is associated with: (i) increased mesangioproliferation; (ii) higher mesangial matrix expansion; and (iii) reduction in glomerular capillary supply. These findings suggest a critical role for nidogen-2 in the maintenance of glomerular structure in the diseased kidney.

Intrauterine growth retardation aggravates the course of acute mesangioproliferative glomerulonephritis in the rat.

Intrauterine growth retardation (IUGR) aggravates the course of acute mesangioproliferative glomerulonephritis (GN) in the rat. Observational studies in children suggest that IUGR may be associated with a severe course of kidney diseases such as IgA nephropathy. We tested the hypothesis that IUGR leads to aggravation of acute mesangioproliferative GN in former IUGR rats. IUGR was induced in Wistar rats by isocaloric protein restriction in pregnant dams. Litter size was reduced to six male neonates in low protein animals (LP) and normal protein animals (NP). At 8 weeks GN was induced by injection of an anti-Thy-1.1 antibody. Rats were killed on days 4 and 14 after induction of GN and kidneys were investigated for inflammation and sclerosis using real-time polymerase chain reaction and histological methods. On day 4 after induction of GN, LP animals showed more glomerulosclerosis and tubulointerstitial lesions. On day 14, inflammatory markers (expression of monocyte chemoattractant protein 1, osteopontin, tumor necrosis factor and interleukin-6), extracellular matrix accumulation and markers of sclerosis (plasminogen activator inhibitor-1 expression, transforming growth factor-beta1 expression, score for glomerulosclerosis, glomerular deposition of collagen I and collagen IV) were more severe in LP animals. Some degree of induction of inflammatory and profibrotic markers was also present in non-nephritic LP animals. However, these rats did not display marked glomerulosclerosis or interstitial fibrosis. We conclude that after IUGR inflammatory damage is aggravated and the reparation of the kidney is impaired during the course of acute mesangioproliferative GN, leading to more sclerotic lesions.

Fibrillin-1 regulates mesangial cell attachment, spreading, migration and proliferation.

The microfibrillar protein fibrillin-1 is present in many organs, including the vasculature, eye, and dermis, and is thought to convey structural anchorage and elastic strength. Fibrillin-1 is also a component of the mesangial matrix. To assess the functional relevance of fibrillin-1 for cell-matrix interactions in the glomerulus, we studied the attachment, spreading, migration and proliferation of mesangial cells on fibrillin-1 and the regulation of fibrillin-1 in experimental anti-Thy1.1 nephritis displaying mesangial cell migration and proliferation in vivo. During the acute phase of experimental Thy1.1 nephritis, glomerular fibrillin-1 messenger ribonucleic acid expression and protein immunoreactivity were significantly induced as compared to controls. In a hexosaminidase-based adhesion assay, mesangial cells showed concentration-dependent attachment to fibrillin-1, similar to what was observed for fibronectin. The cell attachment was Arg-Gly-Asp dependent. Further, fibrillin-1 significantly promoted spreading and focal contact formation detected by immunostaining for vinculin. Mesangial cell migration, assessed by a transmigration assay, and proliferation, measured by a 5-bromo-2'-deoxy-uridine incorporation assay, were augmented by fibrillin-1. In diabetic mice underexpressing fibrillin-1, glomerular cell proliferation, determined by counting proliferating cell nuclear antigen-positive cells in renal sections, was significantly lower than in diabetic control mice. We conclude that fibrillin-1 promotes mesangial cell attachment, spreading, migration, and proliferation. We speculate that fibrillin-1 may thus contribute to mesangial hypercellularity during glomerular disease.

Angiotensin II type 1 receptor blockade prevents lethal malignant hypertension: relation to kidney inflammation.

BACKGROUND: Angiotensin II is elevated in malignant hypertension. We tested the hypothesis that angiotensin II type 1 receptor blockade can prevent the development of malignant hypertension even in the absence of a blood pressure-lowering effect. METHODS AND RESULTS: Two-kidney, 1-clip rats were followed up for 28 days; blood pressure was measured by tail-cuff plethysmography and intra-arterially. After a 2-week run-in phase, rats received valsartan at a dose of 0.3 (n=14) or 3 (n=12) mg. kg(-1). d(-1) or solvent (n=27). Only the higher dose of valsartan, but not the lower dose, decreased blood pressure. Both doses of valsartan prevented the development of lethal malignant hypertension. Twenty of 27 solvent-treated renovascular hypertensive rats died, but only 3 of 14 rats treated with the low dose and 1 of 12 rats treated with the high dose of valsartan died. Histological signs of malignant nephrosclerosis were found in all rats examined that had died throughout the study and in 6 of 7 surviving solvent-treated renovascular hypertensive animals. Increased expression of monocyte chemoattractant protein-1 and prominent interstitial influx of macrophages occurred in the nonclipped kidneys exposed to high pressure in solvent-treated rats. These alterations were prevented by valsartan at both doses, irrespective of blood pressure effects. CONCLUSIONS: Angiotensin II type 1 receptor blockade by valsartan prevents lethal malignant hypertension independently of blood pressure. The results suggest that reduction of angiotensin-induced inflammation in the kidney may contribute to the protective effects of valsartan.

Renin uptake by the endothelium mediates vascular angiotensin formation.

We investigated the role of the vascular endothelium in the local production of angiotensin. Angiotensin release from isolated rat hindquarters perfused with an artificial medium was measured by high-performance liquid chromatography and radioimmunoassay. Perfused hindquarters with endothelium released angiotensin I spontaneously, indicating ongoing renin-angiotensinogen reaction. Endothelium denudation (by a detergent, validated by electron microscopy and by the absence of a vasodilator response to acetylcholine) reduced angiotensin I release by >90%, whereas bilateral nephrectomy 24 hours before perfusion abolished the release completely. Infusion of renin into perfused hindquarters induced sustained local angiotensin I release in the presence of an intact endothelium but not after endothelium denudation. The conversion of angiotensin I to angiotensin II was abrogated by endothelium denudation, whereas the disappearance of angiotensin II was unchanged. Endothelium denudation diminished the pressor response to angiotensin II but abolished the response to renin and angiotensin I. Expression of renin messenger RNA, investigated by reverse-transcription polymerase chain reaction using 4 different primer combinations, was not detected in up to 5 microg vascular RNA, whereas a renin signal was readily detected with 5 ng kidney RNA. The effects of endothelium destruction on Ang I formation support the notion that the endothelium mediates vascular angiotensin formation by taking up renin.

Inducible nitric oxide synthase and glomerular hemodynamics in rats with liver cirrhosis.

This study was designed to test the hypothesis that glomerular de novo expression of inducible nitric oxide synthase (iNOS) contributes to renal hemodynamic abnormalities in liver cirrhosis developed 3 wk after common bile duct ligature (CBDL). De novo expression of iNOS mRNA was detected by RT-PCR in RNA extracts from isolated CBDL rat glomeruli whereas no iNOS mRNA was found in control rat glomerular RNA. Immunohistochemical staining for iNOS was negative in control animals whereas, in CBDL rats, positive iNOS staining was detected in an apparently mesangial pattern in all glomeruli. Western blots of protein extracts from isolated glomeruli of CBDL rats, but not control animals, showed a prominent iNOS band of 130 kDa. Mean arterial pressure (MAP), renal plasma flow (RPF; p-aminohippurate clearance), and glomerular filtration rate (GFR; inulin clearance) were unaltered in CBDL rats, but the application of 4 mg/kg L-N(6)-(1-iminoethyl)lysine, a specific inhibitor of iNOS, reduced GFR and RPF significantly in CBDL rats, whereas control animals were not affected. Similar results were obtained with lipopolysaccharide (LPS)-pretreated animals, which were studied as a positive control for iNOS expression and as a model for recent iNOS induction. We conclude that de novo expression of iNOS occurs in glomeruli of rats with liver cirrhosis and that nitric oxide, generated by iNOS, contributes to the maintenance of glomerular filtration in the early state of this disease.

Glomerular osteopontin expression and macrophage infiltration in glomerulosclerosis of DOCA-salt rats.

Expression of the chemoattractant osteopontin (OPN) may contribute to macrophage infiltration in many types of tubulointerstitial kidney disease, but the role of OPN in chronic glomerulosclerosis is unknown. We hypothesized that glomerular OPN expression and macrophage infiltration occur in deoxycorticosterone acetate (DOCA)-salt glomerulosclerosis in rats. Uninephrectomized rats receiving DOCA pellets and 1% saline were compared with control rats. OPN mRNA was determined by Northern blot, and OPN protein was determined by Western blot. The localization of OPN was studied by in situ hybridization and double immunohistochemistry with glomerular cell markers. Macrophage infiltration was quantified by counting ED-1-positive cells, and semiquantitative glomerulosclerosis scores were obtained. In DOCA-salt rats, OPN mRNA in the kidney was increased 2-fold over control after 9 days and 3 weeks and 20-fold after 6 weeks. Tubulointerstitial OPN staining was apparent after 21 days of DOCA treatment. Glomerular OPN mRNA and protein was detected after 42 days in parietal and visceral epithelial cells, activated myofibroblasts, and occasionally mesangial cells. Progressive glomerular macrophage infiltration occurred during the development of DOCA hypertension, paralleling the degree of glomerulosclerosis. Glomeruli staining positive for osteopontin contained more macrophages (18.4 +/- 3.4 per cross-section) than osteopontin-negative glomeruli (3.6 +/- 0.5; P < 0.05). Glomerular OPN expression occurs in chronic hypertensive glomerulosclerosis and is associated with macrophage infiltration. The data suggest a role for OPN as a chemoattractant in hypertensive glomerulosclerosis.

Furosemide stimulates macula densa cyclooxygenase-2 expression in rats.

BACKGROUND: During a low salt intake, maintenance of renal blood flow and renin secretion depends on intact formation of prostaglandins. In the juxtaglomerular apparatus, the inducible isoform of cyclooxygenase, cyclooxygenase-2 (COX-2), is restricted to the macula densa and the cortical thick ascending limb of Henle (cTALH) cells, and is inversely regulated by dietary salt intake. This study aimed to elucidate whether the effect of NaCl on macula densa COX-2 expression is mediated by transepithelial transport of NaCl. METHODS: To this end, male Sprague-Dawley rats received subcutaneous infusions of the loop diuretic furosemide (12 mg/day) or were fed with the diuretic hydrochlorothiazide (30 mg/kg day) for seven days each. To compensate for their salt and water loss, the animals had free access to normal water and to salt water (0.9% NaCl, 0.1% KCl). COX-2 expression in kidney cortex was assessed by immunohistochemical staining and by semiquantitative ribonuclease protection assay for COX-2 mRNA. RESULTS: After six days of furosemide infusion to salt-substituted rats, there was no change of extracellular volume. Furosemide led to a fivefold and threefold increase of plasma renin activity and renocortical renin mRNA level, respectively. In parallel, there was a threefold increase of renocortical COX-2 abundance, while the COX-1 mRNA level remained unchanged. Moreover, the percentage of juxtaglomerular apparatuses immunopositive for COX-2 increased threefold in response to furosemide compared with vehicle-infused animals. Hydrochlorothiazide treatment increased plasma renin activity twofold but did not change kidney cortical renin mRNA, COX-2 mRNA, or COX-2 immunoreactivity. CONCLUSION: Our findings suggest that inhibition of salt transport in the loop of Henle, but not in the distal tubule, causes a selective stimulation of COX-2 expression in the macula densa region. This up-regulation may be of relevance for macula densa signaling, which links tubular salt transport rate with glomerular filtration rate and renin secretion.

Acute upregulation of COX-2 by renal artery stenosis.

This study aimed to characterize the influence of acute renal artery stenosis on cyclooxygenase-2 (COX-2) and renin expression in the juxtaglomerular apparatus. For this purpose, male Sprague-Dawley rats received a left renal artery clip, and COX-2 mRNA, COX-2 immunoreactivity, plasma renin activity, and renin mRNA levels were determined. COX-2 mRNA and COX-2 immunoreactivity in the macula densa region in the clipped kidneys increased as early as 6 h after clipping and reached a maximal expression 1-2 days after clipping. Although values for plasma renin activity were elevated markedly at all time points examined, remaining renin mRNA levels were unchanged after 6 h and then increased to reach a maximum value 1-2 days after clipping. In the contralateral intact kidney, renin mRNA and COX-2 immunoreactivity decreased to approximately 50% of their normal values. To investigate a possible causal relationship between the changes of COX-2 and of renin expression, clipped rats were treated with the COX-2 blocker celecoxib (40 mg. kg(-1). day(-1)). This treatment, however, did not change renin mRNA either in the clipped or in the contralateral intact kidney. Our findings indicate that renal artery stenosis causes ipsilaterally an acute upregulation and contralaterally a downregulation of juxtaglomerular COX-2 expression. The lacking effect of celecoxib on renin gene expression does not support the concept of a direct mediator function of COX-2-derived prostaglandins in the control of renin expression during renal hypoperfusion.

Monocyte chemoattractant protein-1 and macrophage infiltration in hypertensive kidney injury.

BACKGROUND: We investigated whether monocyte chemoattractant protein-1 (MCP-1) is expressed in hypertensive nephrosclerosis, and tested the effect of angiotensin II type 1 receptor blockade on MCP-1 expression and macrophage (MPhi) infiltration. METHODS: Rats with two-kidney, one-clip (2K1C) hypertension with and without treatment with the angiotensin II type 1 receptor antagonist valsartan (3 mg/kg/day) were studied. In these animals as well as in spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), hypertensive mRen-2 transgenic rats (TGR), and respective control strains, MCP-1 expression in the kidney was investigated by Northern and Western blots and by immunohistochemistry. Glomerular and interstitial MPhis were counted. RESULTS: In the nonclipped kidney of 2K1C rats, MCP-1 expression was elevated at 14 and 28 days when significant MPhi infiltration was present. MCP-1 was localized to glomerular endothelial and epithelial cells, interstitial and tubular cells, MPhis, and vascular smooth muscle cells. A similar pattern of MCP-1 staining was present in TGR kidneys, whereas MCP-1 expression was not increased in SHR and SHR-SP. Valsartan reduced but did not normalize blood pressure, blocked the induction of MCP-1 protein in 2K1C kidneys, and decreased interstitial MPhi infiltration significantly. CONCLUSION: MCP-1 expression is increased in angiotensin II-dependent models of hypertensive nephrosclerosis and is temporally and spatially related to MPhi infiltration. The angiotensin II type 1 receptor mediates the induction of MCP-1.

Elastic fiber proteins in the glomerular mesangium in vivo and in cell culture.

BACKGROUND: Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity. Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability. Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli. The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins. METHODS: We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys. In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry. RESULTS: Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane. Several of these proteins, as well as elastin, were also expressed in the renal vasculature. While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft. Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry. mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells. This process is regulated by growth factors. Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft.

Distinct structural forms of type I collagen modulate cell cycle regulatory proteins in mesangial cells.

BACKGROUND: Extracellular matrix molecules profoundly regulate cell behavior, including proliferation. In glomerulonephritis, type I collagen accumulates in the mesangium and is constantly structurally modified and degraded during the course of the disease. METHODS: We studied how two structurally distinct forms of type I collagen, monomer versus polymerized fibrils, affect cell proliferation, mitogen-activated protein kinase (MAPK) activation, and expression of G1-phase regulatory proteins in cultured rat mesangial cells (MCs). To analyze the possible involvement of collagen-binding integrins in type I collagen-derived growth signals further, distribution patterns of integrin chains were examined by immunocytochemistry. RESULTS: Polymerized type I collagen completely prevented the increase of DNA synthesis and cell replication induced by 5% fetal calf serum (FCS) or 25 ng/mL platelet-derived growth factor (PDGF) in MCs on monomer type I collagen. Protein expression of cyclins D1 and E was markedly down-regulated in MCs plated on polymerized type I collagen for eight hours in 5% FCS, as compared with MCs on monomer type I collagen. Incubation with 5% FCS reduced expression of the cdk-inhibitor protein p27Kip1 on monomer but not on polymerized type I collagen. Moreover, polymerized type I collagen markedly reduced cyclin E-associated kinase activity in the presence of 5% FCS. Polymerized type I collagen diminished the PDGF-induced phosphorylation and nuclear translocation of p42/p44 MAPK, but did not affect phosphorylation of PDGF beta-receptors. In MCs plated on monomer type I collagen, alpha1, alpha2, and beta1 integrin chains were recruited into focal contacts. However, on polymerized type I collagen, alpha2 and beta1, but not alpha1, integrin chains were condensed into focal contacts. CONCLUSIONS: The growth-inhibitory effect of polymerized type I collagen is characterized by rapid changes of expression and/or activation of MAPK and G1-phase regulators and could result from the lack of alpha1beta1 integrin signaling in MCs on polymerized type I collagen. Conceivably, deposition of polymerized type I collagen might reflect a reparative response to control MC replication in glomerular inflammation.

Upregulation of cyclooxygenase-1 and the PGE2 receptor EP2 in rat and human mesangioproliferative glomerulonephritis.

OBJECTIVE AND DESIGN: Glomerular expression and localization of the two cyclooxygenase isoforms, Cox-1 and Cox-2, and the prostaglandin E2 receptor EP2 were investigated in a rat model of transient mesangioproliferative glomerulonephritis. Cox expression was also studied in biopsies from patients with IgA nephropathy. MATERIALS AND TREATMENT: After induction of glomerulonephritis by i.v. injection of a monoclonal anti-Thy1.1 antibody, rats were sacrificed at day 2, 6, 12 and 56. Changes in protein expression were detected by immunohistochemistry. Glomerular mRNA levels were analyzed by real time polymerase chain reaction (PCR). RESULTS: In normal rat kidney, immunoreactivity of Cox-1 was detected predominantly in collecting duct cells and that of Cox-2 in the macula densa. Cox-1 staining showed a massive transient increase in diseased glomeruli at day 6, localized mainly to mesangial cells coinciding with cell proliferation, which also peaked at day 6. Upregulation of Cox-1 was also evident at the mRNA level (4 fold). Cox-2 expression in the macula densa region transiently increased at day 6, but no significant upregulation of Cox-2 was observed in glomerular cells at any time point. Prostaglandin E2 receptor EP2 mRNA and protein were detected in rat glomeruli. EP2 immunoreactivity was prominent on podocytes in normal rats while at day 6 of the disease also mesangial cells stained positive. In biopsies of patients with IgA nephritis, predominant expression of Cox-1, but not Cox-2, was found in glomeruli, whereas Cox-2 was strongly expressed in infiltrating interstitial cells. CONCLUSIONS: The upregulation of glomerular Cox-1 but not Cox-2 and the parallel induction of the EP-2 receptor, which was shown to mediate cAMP accumulation in mesangial cells, suggest that induction of prostaglandin formation may contribute to the resolution rather than to the progression of anti-Thy1.1 nephritis. The expression pattern of Cox-1 and Cox-2 in human IgA nephritis points to a role for both Cox isoforms in human glomerular inflammation.