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Severe hypertension can lead to malignant hypertension (MH) with renal thrombotic microangiopathy and hemolysis. The role of plasma heme release in this setting is unknown. We aimed at evaluating the effect of a mild plasma heme increase by hemin administration in angiotensin II (AngII)-mediated hypertensive rats. Prevalence of MH and blood pressure values were similar in AngII and AngII + hemin groups. MH rats displayed a decreased renal blood flow (RBF), increased renal vascular resistances (RVR), and increased aorta and interlobar arteries remodeling with a severe renal microcirculation assessed by peritubular capillaries (PTC) rarefaction. Hemin-treated rats with or without AngII displayed also a decreased RBF and increased RVR explained only by PCT rarefaction. In AngII rats, RBF was similar to controls (with increased RVR). PTC density appeared strongly correlated to tubular damage score (rho = -0.65, p < 0.0001) and also renal Heme Oygenase-1 (HO-1) mRNA (rho = -0.67, p < 0.0001). HO-1 was expressed in PTC and renal tubules in MH rats, but only in PTC in other groups. In conclusion, though increased plasma heme does not play a role in triggering or aggravating MH, heme release appears as a relevant toxic mediator leading to renal impairment, primarily through PTC endothelial dysfunction rather than direct tubular toxicity.

Calcineurin inhibitors such as cyclosporine A (CsA) are still commonly used after renal transplantation, despite CsA--induced nephrotoxicity (CIN), which is partly related to vasoactive mechanisms. The mineralocorticoid receptor (MR) is now recognized as a key player in the control of vascular tone, and both endothelial cell- and vascular smooth muscle cell (SMC)-MR modulate the vasoactive responses to vasodilators and vasoconstrictors. Here we tested whether vascular MR is involved in renal hemodynamic changes induced by CsA. The relative contribution of vascular MR in acute CsA treatment was evaluated using mouse models with targeted deletion of MR in endothelial cell or SMC. Results indicate that MR expressed in SMC, but not in endothelium, contributes to the increase of plasma urea and creatinine, the appearance of isometric tubular vacuolization, and overexpression of a kidney injury biomarker (neutrophil gelatinase--associated lipocalin) after CsA treatment. Inactivation of MR in SMC blunted CsA--induced phosphorylation of contractile proteins. Finally, the in vivo increase of renal vascular resistance induced by CsA was blunted when MR was deleted from SMC cells, and this was associated with decreased L-type Ca2D channel activity. Thus, our study provides new insights into the role of vascular MR in renal hemodynamics during acute CIN, and provides rationale for clinical studies of MR antagonism to manage the side effects of calcineurin inhibitors., Competing Interests: All the authors declared no competing interests.

Background: Hypertensive nephrosclerosis is one of the most frequent causes of chronic kidney failure. Proteome analysis potentially improves the pathophysiological understanding and diagnostic precision of this disorder. In the present exploratory study, we investigated experimental nephrosclerosis in the two-kidney, one-clip (2K1C) hypertensive rat model., Methods: The renal cortex proteome from juxtamedullary cortex and outer cortex of 2K1C male Wistar-Hannover rats (n = 4) was compared with the sham-operated controls (n = 6), using mass spectrometry-based quantitative proteomics. We combined a high abundant plasma protein depletion strategy with an extended liquid chromatographic gradient to improve peptide and protein identification. Immunohistology was used for independent confirmation of abundance., Results: We identified 1724 proteins, of which 1434 were quantified with at least two unique peptides. Comparative proteomics revealed 608 proteins, including the platelet-derived growth factor receptor-β signalling pathway, with different abundances between the non-clipped kidney of hypertensive 2K1C rats and the corresponding kidney of the normotensive controls (P < 0.05, absolute fold change ≥1.5). Among the most significantly altered proteins in the whole cortex were periostin, transgelin, and creatine kinase B-type. Relative abundance of periostin alone allowed clear classification of 2K1C and controls. Enrichment of periostin in 2K1C rats was verified by immunohistology, showing positivity especially around the fibrotic vessels., Conclusion: The proteome is altered in hypertension-induced kidney damage. We propose periostin, especially in combination with transgelin and creatine kinase B-type, as possible proteomic classifier to distinguish hypertensive nephrosclerosis from the normal tissue. This classifier needs to be further validated with respect to early diagnosis of fibrosis, prognosis, and its potential as a novel molecular target for pharmacological interventions.

Circulating levels of soluble forms of urokinase-type plasminogen activator receptor (suPAR) are generally elevated in sera from children and adults with FSGS compared with levels in healthy persons or those with other types of kidney disease. In mice lacking the gene encoding uPAR, forced increases in suPAR concentration result in FSGS-like glomerular lesions and proteinuria. However, whether overexpression of suPAR, per se, contributes to the pathogenesis of FSGS in humans remains controversial. We conducted an independent set of animal experiments in which two different and well characterized forms of recombinant suPAR produced by eukaryotic cells were administered over the short or long term to wild-type (WT) mice. In accordance with the previous study, the delivered suPARs are deposited in the glomeruli. However, such deposition of either form of suPAR in the kidney did not result in increased glomerular proteinuria or altered podocyte architecture. Our findings suggest that glomerular deposits of suPAR caused by elevated plasma levels are not sufficient to engender albuminuria., (Copyright © 2014 by the American Society of Nephrology.)

This study investigated the role of discoidin domain receptor 1 (DDR1), a collagen receptor that displays tyrosine-kinase activity, in the development of glomerulonephritis. Crescentic glomerulonephritis was induced in DDR1-deficient mice and their wild-type (WT) littermates as controls, by injection of alloimmune sheep nephrotoxic serum (NTS). Histological, functional and transcriptomic studies were performed. Glomerulonephritis produced a 17-fold increase of DDR1 expression, predominantly in glomeruli. DDR1 deletion protected NTS-treated mice against glomerular disease (proteinuria/creatininuria 5.5±1.1 vs. 13.2±0.8 g/mmol in WT, crescents 12±2 vs. 24±2% of glomeruli, urea 16±2 vs. 28±5 mM), hypertension (123±11 vs. 157±8 mmHg), and premature death (70 vs. 10% survival) (all P<0.05). Reciprocal stimulation between DDR1 and interleukin-1b expression in vivo and in cultured podocytes suggested a positive feed-back loop between DDR1 and inflammation. In NTS-treated WT mice, administration of DDR1-specific antisense oligodeoxynucleotides decreased DDR1 expression (-56%) and protected renal function and structure, including nephrin expression (4.2±1.4 vs. 0.9±0.4 arbitrary units, P<0.05), compared to control mice receiving scrambled oligodeoxynucleotides. The therapeutic potential of this approach was reinforced by the observation of increased DDR1 expression in glomeruli of patients with lupus nephritis and Goodpasture's syndrome. These results prompt further interest in DDR1 blockade strategies, especially in the treatment of glomerulonephritis.

The renal urothelium, the monolayered epithelium that covers the papilla, is the direct target of increased pressure during obstruction, yet most studies have mainly focused on tubules, fibroblasts, and inflammatory cells. We studied this epithelium in a unilateral ureteral obstruction mouse mode land found that it was disrupted and had broken tight junctions, enlarged intercellular space, with loss of apicaluroplakins, and marginal lumen desquamation. Shortly after obstruction these urothelial cells proliferated, peaking at day 2. By day 14, the renal urothelium was transformed into a multilayered barrier with newly synthesized uroplakins including the de novo induction of uroplakin II. This proliferation was found to be fibroblast growth factor (FGF)dependent. Renal urothelial cells constitutively express the FGF receptor 2, and obstruction activated the receptor by phosphorylation. Treatment with FGF receptor 2-antisense or vitamin A (an inhibitor of the MAP kinase in the FGFR2 pathway) decreased renal urothelial cell proliferation. Among known FGF receptor 2 ligands, only FGF7 was upregulated.Infusion of FGF7 into control mice caused the formation of a multilayered structure at 7 days, resembling the urothelium 14 days following obstruction. Thus, the pressure/stretching of renal monolayered urothelial cells is a very efficient trigger for proliferation, causing the formation of a bladder-like multistratified barrier with enhanced apical uroplakin plaques. Presumably, this ensures efficient barrier protection and repair.

Progression of chronic kidney disease (CKD) is a major health issue due to persistent accumulation of extracellular matrix in the injured kidney. However, our current understanding of fibrosis is limited, therapeutic options are lacking, and progressive degradation of renal function prevails in CKD patients. Uncovering novel therapeutic targets is therefore necessary.We have previously demonstrated reversal of renal fibrosis with losartan in experimental hypertensive nephropathy. Reversal was achieved provided that the drug was administered before late stages of nephropathy, thereby determining a non-return point of CKD progression. In the present study, to identify factors critically involved in the progression of renal fibrosis, we introduced losartan at the non-return point in L-NAME treated Sprague Dawley rats. Our results showed either reversal or progression of renal disease with losartan, defining 2 groups according to the opposite evolution of renal function. We took advantage of these experimental conditions to perform a transcriptomic screening to identify novel factors potentially implicated in the mechanisms of CKD progression. A secondary analysis of selected markers was thereafter performed. Among the targets identified, periostin, an extracellular matrix protein, presented a significant 3.3-fold higher mRNA expression in progression compared to reversal group. Furthermore, independent of blood pressure, periostin was strongly correlated with plasma creatinine, proteinuria and renal blood flow, hallmarks of hypertensive renal disease severity. Periostin staining was predominant in the injured regions, both in experimental hypertensive and human nephropathy.These results identify periostin as a previously unrecognized marker associated with disease progression and regression in hypertensive nephropathy and suggest measuring periostin may be a sensitive tool to evaluate severity, progression and response to therapy in human kidney disease associated to hypertension.

The interactions between tubulointerstitial infiltrating cells and the extracellular matrix play an important role in regulating renal fibrosis. Discoidin domain receptor 1 (DDR1) is a nonintegrin tyrosine kinase receptor for collagen implicated in cell adhesion, proliferation, and extracellular matrix remodeling. We have previously demonstrated that transgenic mice lacking DDR1 are protected from hypertension-associated renal fibrosis. The purpose of this study was to determine the role of DDR1 in renal inflammation and fibrosis related to primitive tubulointerstitial injury. After 12 days of unilateral ureteral obstruction (UUO), kidney histopathologic and real-time quantitative PCR analyses were performed in DDR1(-/-) and wild-type mice. DDR1 expression was strongly increased in the obstructed kidney. Wild-type mice developed important perivascular and interstitial inflammation and fibrosis. In comparison, DDR1(-/-) mice displayed reduced accumulation of fibrillar collagen and transforming growth factor β expression. F4/80(+) cell count and proinflammatory cytokines were remarkably blunted in DDR1(-/-) obstructed kidneys. Leukocyte rolling and adhesion evaluated by intravital microscopy were not different between DDR1(-/-) and wild-type mice. Importantly, macrophages isolated from DDR1(-/-) mice presented similar M1/M2 polarization but displayed impaired migration in response to monocyte chemoattractant protein-1. Together, these data suggest that DDR1 plays an important role in the pathogenesis of renal disease via enhanced inflammation. Inhibition of DDR1 expression or activity may represent a novel therapeutic target against the progression of renal diseases., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The Notch3 receptor participates in the development and maturation of vessels. Mutations of Notch3 in humans are associated with defective regulation of cerebral blood flow. To investigate the role of Notch3 in the regulation of renal hemodynamics, we used mice lacking expression of the Notch3 gene (Notch3-/- mice). Bolus injections of norepinephrine and angiotensin II increased renal vascular resistance and decreased renal blood flow in a dose-dependent manner in wild-type mice. In sharp contrast, renal vascular resistance of Notch3-/- mice varied little after boluses of norepinephrine and angiotensin II. Inversely, bradykinin and prostacyclin relaxed renal vasculature in wild-type mice. Both vasodilators had a negligible effect on renal vascular resistance of Notch3-/- mice. Afferent arterioles freshly isolated from Notch3-/- mice displayed decreased thickness of vascular wall compared with wild -type mice and showed a deficient contractile response to angiotensin II. To examine the physiopathological consequences of the above-described deficiency, hypertension was induced by continuous infusion of angiotensin II. Angiotensin II gradually increased blood pressure in both strains, but this increase was lesser in the Notch3-/- mice. Despite this blunted systemic effect, Notch3-/- mice displayed high mortality rates (65%) attributed to heart failure. In the kidney, the surviving Notch3-/- mice showed focal structural alterations characteristic of nephroangiosclerosis. These data show that Notch3 is necessary for the adaptive response of the renal vasculature to vasoactive systems. A deficiency in the expression of Notch3 could have important physiopathological consequences in the adaptation of the cardiac and renal function to chronic increase of blood pressure.

The present study investigated mechanisms of regression of renal disease after severe proteinuria by focusing on the interaction among EGF receptors, renal hemodynamics, and structural lesions. The nitric oxide (NO) inhibitor N(G)-nitro-l-arginine-methyl ester (l-NAME) was administered chronically in Sprague-Dawley rats. When proteinuria exceeded 2 g/mmol creatinine, animals were divided into three groups for an experimental period of therapy of 2 wk; in one group, l-NAME was removed to allow reactivation of endogenous NO synthesis; in the two other groups, l-NAME removal was combined with EGF or angiotensin receptor type 1 (AT(1)) antagonism. l-NAME removal partially reduced mean arterial pressure and proteinuria and increased renal blood flow (RBF), but not microvascular hypertrophy. Progression of structural damage was stopped, but not reversed. The administration of an EGF receptor antagonist did not have an additional effect on lowering blood pressure or on renal inflammation but did normalize RBF and afferent arteriole hypertrophy; the administration of an AT(1) antagonist normalized all measured functional and structural parameters. Staining with a specific marker of endothelial integrity indicated loss of functional endothelial cells in the l-NAME removal group; in contrast, in the animals treated with an EGF or AT(1) receptor antagonist, functional endothelial cells reappeared at levels equal to control animals. In addition, afferent arterioles freshly isolated from the l-NAME removal group showed an exaggerated constrictor response to endothelin; this response was blunted in the vessels isolated from the EGF or AT(1) receptor antagonist groups. The EGF receptor is an important mediator of endothelial dysfunction and contributes to the decline of RBF in the chronic kidney disease induced by NO deficiency. The EGF receptor antagonist-induced improvement of RBF is important but not sufficient for a complete reversal of renal disease, because it has little effect on renal inflammation. To achieve full recovery, it is necessary to apply AT(1) receptor antagonism.

Hypertension is associated with vascular remodeling characterized by rearrangement of extracellular matrix proteins. To evaluate how matrix metalloproteinase (MMP)-9 contributes to the progression of hypertensive vascular disease in vivo, wild-type (wt) or MMP-9(-/-) mice were treated with angiotensin II (Ang II; 1 microg/kg per minute, by minipump) plus a 5% NaCl diet during 10 days. Baseline blood pressure was equivalent in wt and knockout mice, but Ang II treatment increased systolic blood pressure to a greater extent (P<0.05) in MMP-9(-/-) mice (94+/-6 to 134+/-6 mm Hg; P<0.001) than in wt animals (93+/-4 to 114+/-6 mm Hg; P<0.01). In wt mice, Ang II treatment increased the carotid artery pressure-diameter relationship significantly, and maximal diameter reached 981+/-19 microm (P<0.01 versus sham; 891+/-10 microm). In contrast, in MMP-9(-/-) mice, carotid artery compliance was actually reduced after Ang II (P<0.05), and maximal diameter only reached 878+/-13 microm. Ang II treatment induced MMP-2 and increased carotid media thickness equally in both phenotypes. However, MMP-9 induction and in situ gelatinase activity were only enhanced in Ang II-treated wt mice, and vessels from these mice also produced more collagen I breakdown products than their MMP-9(-/-) counterparts (P<0.05). Inversely, staining for collagen IV was particularly enhanced in vessels from MMP-9(-/-) mice treated with Ang II. These results demonstrate the following: (1) the onset of Ang II-induced hypertension is accompanied by increased MMP-9 activity in conductance vessels; (2) absence of MMP-9 activity results in vessel stiffness and increased pulse pressure; and (3) MMP-9 activation is associated with a beneficial role early on in hypertension by preserving vessel compliance and alleviating blood pressure increase.

A frequent complication of hypertension is the development of chronic renal failure. This pathology usually is initiated by inflammatory events and is characterized by the abnormal accumulation of collagens within the renal tissue. The purpose of this study was to investigate the role of discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor that displays tyrosine-kinase activity, in the development of renal fibrosis. To this end, hypertension was induced with angiotensin in mice that were genetically deficient of DDR1 and in wild-type controls. After 4 or 6 wk of angiotensin II administration, wild-type mice developed hypertension that was associated with perivascular inflammation, glomerular sclerosis, and proteinuria. Systolic pressure increase was similar in the DDR1-deficient mice, but the histologic lesions of glomerular fibrosis and inflammation were significantly blunted and proteinuria was markedly prevented. Immunostaining for lymphocytes, macrophages, and collagens I and IV was prominent in the renal cortex of wild-type mice but substantially reduced in DDR1 null mice. In separate experiments, renal cortical slices of DDR1 null mice showed a blunted response of chemokines to LPS that was accompanied by a considerable protection against the LPS-induced mortality. These results indicate the importance of DDR1 in mediating inflammation and fibrosis. Use of DDR1 inhibitors could provide a completely novel therapeutic approach against diseases that have these combined pathologies.

Hypertension is frequently associated with the development of renal fibrosis leading to chronic renal failure. The objective of the present study was to evaluate the role of blood pressure and renal hemodynamics on the development of renal lesions during hypertension. To this end, rats were treated with a NO synthase inhibitor, L-NAME, for 4 weeks. At this time point, systolic blood pressure reached 170 mmHg, renal blood flow dropped to 3.3 +/- 0.7 ml/min and kidneys displayed glomerular and tubulo-interstitial lesions as evidenced by histological analysis. Thereafter, L-NAME treatment was combined with an AT1 receptor antagonist, losartan (30 mg/kg/d), for an additional period of 4 weeks. Treatment with losartan for 4 additional weeks did not significantly modify hypertension (168 mmHg) either the degree of tubulo-interstitial lesions; in contrast, a significant regression of ischemic and sclerotic glomerular lesions was observed. In parallel, renal blood flow was significantly improved by losartan (5.2 +/- 0.8 ml/min). In addition a negative correlation was observed between renal blood flow and index of glomerulosclerosis (r = -0.82), whereas tubulo-intarstitial damage was positively correlated to systemic pressure (r = 0.93). In conclusion, inhibition of the local effects of angiotensin II alleviates the fall of renal blood flow consecutive to NO deficiency and reduces the morphological and functional lesions of glomeruli, independently of the changes in blood pressure. In contrast, tubulo-interstitial lesions are not correlated with the levels of renal blood flow and do not regress with the blockade of AT1 receptors when rats remain hypertensive.

Background: Renal fibrosis, a common complication of hypertension and diabetes is considered as a non-curable disease and is characterized by the abnormal accumulation of collagen I within the kidney. Chronic inhibition of nitric oxide (NO) synthesis is a model of hypertension associated with the development of nephroangiosclerosis. The present study investigated whether halt of NO inhibition leads to the regression of renal sclerotic lesions., Methods: The NO deficiency (N(G)-nitro-L-arginine methylester; L-NAME) model of hypertension was applied in transgenic mice harbouring the luciferase reporter gene under the control of the collagen I-alpha2 chain promoter., Results: Systolic pressure gradually increased following the administration of L-NAME, and reached 160 mmHg after 8-10 weeks. Activation of collagen I gene within the renal vasculature preceded the blood pressure increase and was accompanied by the appearance of sclerotic glomeruli and tubulointerstitial infiltration. After renal lesions had been established (20 weeks), animals were divided in three subgroups for an additional experimental period of 10 weeks: first group continued to receive L-NAME, in the second, L-NAME administration was stopped to allow endogenous NO synthesis and in the third the removal of L-NAME was combined with endothelin receptor antagonism. Removal of L-NAME decreased, without normalizing, systolic pressure and collagen I gene activity; renal morphology was substantially improved, and tubulointerstitial infiltration disappeared. Combination of L-NAME removal with endothelin antagonism normalized collagen I gene expression and further improved renal morphology without further decreasing blood pressure., Conclusion: Manipulating the balance between NO/vasoconstrictors in favour of NO could provide a curative approach against renal inflammatory and fibrotic complications associated to hypertension.

Background: Sickle cell disease (SCD) affects the kidney by acute mechanisms as well as by insidious renal medullary/papillary necrosis, resulting in tubular defects, which increase the risk of dehydration and subsequent sickle crisis. Hypoxia has been reported to stimulate endothelin-1 (ET-1) synthesis by endothelial cells and also in the renal tubule., Methods: This case-control study measured ET-1 in urine as a marker of its renal synthesis in asymptomatic SCD patients. Baseline plasma and urinary ET-1 levels were measured and followed during a water deprivation study and a subsequent administration of desmopressin., Results: Urine and plasma levels of ET-1 were elevated in patients with SCD, compared with carefully matched African-French and African controls, and urine ET-1 excretion was associated with a marked urine-concentrating defect. Moreover, urinary ET-1 output was correlated with microalbuminuria in SCD patients., Conclusions: ET-1 is known to antagonize the tubular effects of vasopressin and to promote renal scarring; increased renal production of ET-1 could produce nephrogenic diabetes insipidus and dehydration in SCD patients through a combination of fibrosis and functional resistance to vasopressin. This study provides a rationale for trials with endothelin receptor antagonists in sickle cell disease nephropathy.

Hypertension is frequently associated with the development of renal vascular and glomerular fibrosis. The purpose of the present study was to investigate whether epidermal growth factor receptor (EGFR) activation participates in the development of renal fibrosis and to test if blockade of EGFR activation would have therapeutic effects. Experiments were performed during nitric oxide (NO) deficiency-induced hypertension in rats (L-NAME model). After 4 weeks of L-NAME treatment, animals developed hypertension associated to deterioration of renal structure and function. Over the same period, EGFR was activated twofold within glomeruli. This activation was accompanied by increased activity of the mitogen-activated protein kinase (MAPK) p42/p44 pathway and exaggerated collagen I expression. Gefitinib, an EGFR-tyrosine kinase inhibitor, given concomitantly with L-NAME, normalized MAPK activation and collagen I expression and prevented the decline of renal function and the development of fibrosis. Since endothelin mediates the L-NAME-induced fibrogenesis, the endothelin-EGFR interaction was tested in transgenic mice expressing luciferase under the control of collagen I-alpha2 promoter: In renal cortex of these animals, the endothelin-induced collagen I gene activity was inhibited by an EGFR-phosphorylation inhibitor. These results provide the first evidence that EGFR activation plays an important role in the progression of renal vascular and glomerular fibrosis.

Renal fibrosis is one of the major complications associated with the development of hypertension. The objective of the present study was to determine whether and by which mechanisms treatment with AT1 receptor antagonists makes possible the regression of renal vascular and glomerular fibrosis. Experiments were performed in the hypertensive model of nitric oxide (NO) deficiency in rats. After 4 wk of hypertension, mortality rates averaged 20%; the surviving animals displayed a decline of renal function (urine protein/creatinine, 1.89 +/- 0.63 versus 0.24 +/- 0.03 mg/mmol; creatininemia, 110 +/- 14 versus 38 +/- 2 mmol/L in hypertensive animals and control, respectively; P < 0.01) and an exaggerated gene and protein expression of TGF-beta, collagen I, and collagen IV (P < 0.001) within the renal vasculature associated with the development of glomerulosclerosis (sclerotic index, 2.26 +/- 0.29 versus 0.12 +/- 0.04; P < 0.001). In addition, activities of matrix metalloproteinases 2 and 9 were increased twofold in renal vessels and glomeruli (P < 0.01). Afterwards, losartan, an antagonist of angiotensin receptor type I, or hydralazine were administered in subgroups of hypertensive animals. After 1 wk of angiotensin II antagonism, collagen I, collagen IV, and TGF-beta gene and protein expressions were decreased and glomerulosclerosis was less marked (sclerotic index 1.04 +/- 0.45), whereas activities of metalloproteinases remained twofold higher than controls (P < 0.01). Hydralazine failed to improve renal function despite a similar degree of systolic pressure decrease. After 4 wk of losartan, the renal functional and histologic parameters were completely normalized, whereas they remained damaged in the hypertensive animals in which the mortality rate reached 85%. These data suggest that the progression of renal vascular fibrosis is a reversible process, at least in the NO deficiency model. The mechanism of the regression appears to be dual: inhibition of collagen synthesis due to AT1 receptor antagonism and activation of metalloproteinases that is probably associated with the degree of fibrosis independently of AT1 blockade.

Vascular remodeling and rearrangement of the extracellular matrix formation are among the major adaptive mechanisms in response to a chronic blood pressure increase. Vasoactive peptides, such as endothelin, participate in hypertension-associated vascular fibrosis by stimulating collagen I formation and increasing contractility of arterial wall. In the present study, we tested the hypothesis that activation of the epidermal growth factor (EGF) receptor pathway mediates these events. Experiments were performed in transgenic mice harboring the luciferase gene under the control of the collagen I-alpha2 chain promoter. Endothelin induced a rapid phosphorylation of the mitogen-activated protein kinase (MAPK)/ERK and increased collagen I gene activity in freshly isolated aortas. This effect of endothelin was totally inhibited by an endothelin receptor antagonist, an EGF receptor phosphorylation inhibitor, and a blocker of the MAPK/ERK cascade. In parallel experiments, inhibition of EGF receptor phosphorylation decreased the endothelin-induced pressor effect in isolated aortic rings and in anesthetized animals in vivo. In addition, the endothelin-induced increase of blood pressure was blunted in the waved-2 mice, a strain expressing functionally impaired EGF receptors. Our results provide the first evidence that the EGF receptor mediates at least two of the major actions of endothelin in the vascular tissue: contractility and fibrogenesis.

Hypertension is frequently associated with the development of renal vascular fibrosis. This pathophysiologic process is due to the abnormal formation of extracellular matrix proteins, mainly collagen type I. In previous studies, it has been observed that the pharmacologic blockade of angiotensin II (Ang II) or endothelin (ET) blunted the development of glomerulo- and nephroangiosclerosis in nitric oxide-deficient hypertensive animals by inhibiting collagen I gene activation. The purpose of this study was to investigate whether and how AngII interacts with ET to activate the collagen I gene and whether transforming growth factor-beta (TGF-beta) could be a player in this interaction. Experiments were performed in vivo on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha 2 chain promoter (procol alpha 2[I]). Bolus intravenous administration of AngII or ET produced a rapid, dose-dependent activation of collagen I gene in aorta and renal cortical slices (threefold increase over control at 2 h, P < 0.01). The AngII-induced effect on procol alpha 2(I) was completely inhibited by candesartan (AngII type 1 receptor antagonist) and substantially blunted by bosentan (dual ET receptor antagonist) (P < 0.01), whereas the ET-induced activation of collagen I gene was blocked only by bosentan. In subsequent experiments, TGF-beta (also administered intravenously) produced a rapid increase of procol alpha 2(I) in aorta and renal cortical slices (twofold increase over control at 1 h, P < 0.01) that was completely blocked by decorin (scavenger of the active form of TGF-beta). In addition, decorin attenuated the activation of collagen I gene produced by AngII (P < 0.01). These data indicate that AngII can activate collagen I gene in aorta and renal cortex in vivo by a mechanism(s) requiring participation and/or cooperation of ET and TGF-beta.

Mesangial cells from nonobese diabetic (NOD) mice (D-NOD) that develop diabetes at 2-4 mo express an increased density of atrial natriuretic peptide (ANP) clearance receptors [natriuretic peptide C receptor (NPR-C)] and produce less GMP in response to ANP than their nondiabetic counterparts (ND-NOD). Our purpose was to investigate how both phenotypic characteristics were regulated. Epidermal growth factor (EGF) and heparin-binding (HB)-EGF, but not platelet-derived growth factor or insulin-like growth factor I, inhibited (125)I-ANP binding to ND-NOD and D-NOD mesangial cells, particularly in the latter. NPR-C density decreased with no change in the apparent dissociation constant, and there was also a decrease in NPR-C mRNA expression. The EGF effect depended on activation of its receptor tyrosine kinase but not on that of protein kinase C, mitogen-activated protein kinases, or phosphoinositide-3 kinase. Activation of activator protein-1 (AP-1) was necessary, as shown by the inhibitory effect of curcumin and the results of the gel-shift assay. The cGMP response to physiological concentrations of ANP was greater in EGF-treated D-NOD cells. These studies suggest that EGF potentiates the ANP glomerular effects in diabetes by inhibition of its degradation by mesangial NPR-C via a mechanism involving AP-1.

Background: Hypertension is frequently associated with renal vascular fibrosis. The purpose of this study was to investigate whether angiotensin II (Ang II) is involved in this fibrogenic process., Methods and Results: Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha(2) chain promoter [procolalpha(2)(I)]. Hypertension was induced by chronic inhibition of NO synthesis (N(G)-nitro-L-arginine methyl ester, L-NAME). Procolalpha(2)(I) activity started to increase in the renal vasculature after 4 weeks of L-NAME treatment (P<0.01) and at 14 weeks reached 3- and 8-fold increases over control in afferent arterioles and glomeruli, respectively (P<0.001). Losartan, an AT(1) receptor antagonist, given simultaneously with L-NAME prevented the increase of procolalpha(2)(I) levels and attenuated the development of renal vascular fibrosis without normalizing systolic pressure increase. Because we found previously that endothelin mediated renal vascular fibrosis in the L-NAME model, the interaction between Ang II, endothelin, and procolalpha(2)(I) was investigated in ex vivo and short-term in vivo experiments. In both conditions, the Ang II-induced activation of procolalpha(2)(I) in renal cortex was blocked by an endothelin receptor antagonist., Conclusions: During chronic inhibition of NO, the collagen I gene becomes activated, leading to the development of renal vascular fibrosis. Ang II is a major player in this fibrogenic process, and its effect on collagen I gene is independent of systemic hemodynamics and is at least partly mediated by the profibrogenic action of endothelin.

Background: Experimental evidence shows that natriuretic peptides (NPs) play a pathophysiological role in the glomerular hemodynamic abnormalities that occur in diabetes mellitus., Methods: In this study, the cGMP response to NPs and the different subtypes of NP receptors were examined in mesangial cells derived from a genetic model of diabetes, the nonobese diabetic (NOD) mouse. Multiple mesangial cell lines were derived from diabetic (D-NOD) and nondiabetic (ND-NOD) adult mice and were studied at different passages., Results: cGMP accumulation after stimulation by atrial NP (ANP) or C-type NP (CNP) was markedly inhibited in D-NOD cells irrespective of the glucose concentration (6 or 20 mM) in the culture medium. In contrast, NP receptor density measured from [125I]-ANP saturation binding curves was 7.5 times greater in D-NOD than in ND-NOD cells. No change in KD (200 pM in both cell lines) was observed. Competitive inhibition studies showed that 4-23 C-ANP, which is specific of clearance receptors (NPR-C), displaced 90% of the maximum fraction bound, suggesting the predominance of NPR-C in both cell lines. Further identification was obtained from RNase protection assay and reverse transcription-polymerase chain reaction, which also demonstrated the higher expression of NPR-C mRNA in D-NOD cells. In contrast, NPR-A mRNA was not modified. Increased expression of NPR-C in D-NOD cells was associated with an increase of ANP internalization rate at 37 degrees C, indicating that these receptors were functional., Conclusions: These studies demonstrate that the constitutive overexpression of NPR-C in D-NOD mesangial cells is associated with a decreased response of cGMP to ANP or CNP treatment. This could be due to the lesser availability of the peptides for binding to NPR-A or NPR-B or to an inhibitory effect on NP-dependent guanylate cyclase activity via the activation of NPR-C.

Bradykinin (BK) plays a key role in collecting duct functions. Using an established line of principal cells of the rabbit collecting duct (R.C. SV3), we examined the characteristics of the BK receptors in these cells. [3H]-BK bound specifically to R.C. SV3. Saturation binding analyses allowed KD (968 +/- 232 pM) and Bmax values (356 +/- 43 fmol/mg protein) to be calculated. Competitive displacement of [3H]-BK was observed with Hoe-140, a specific type 2 BK receptor (BKR-2) antagonist, but not with des arg9-BK, a BKR-1 agonist. The presence of BKR-2 was confirmed by the reverse-transcription polymerase chain reaction technique. BK stimulated cytosolic calcium and inositol phosphate formation in a dose-dependent manner (from 1 nM to 1 microM). BK also inhibited the arginine vasopressin dependent increase of cyclic adenosine monophosphate. This effect could not be related to the production of prostaglandin E2. These results demonstrate the presence of high-affinity BKR-2 in the principal cells of the rabbit collecting duct that are linked to phospholipase C activity and are involved in arginine vasopressin related regulatory loops.

We examined the effect of prostaglandin E2 (PGE2) on bradykinin (BK) binding, BK-dependent intracellular calcium and inositol phosphate (i.p.) concentrations and BK mRNA in human glomerular visceral epithelial cells (hGVEC). PGE2 (10 nM) produced a concentration-dependent increase in [3H]-BK specific binding after a lag time of 24 h with a threshold at 0.1 nM. This increase appeared to be mediated exclusively by an increase in BK receptor (BKR)-2 expression. Scatchard analysis of [3H]-BK saturation binding showed that PGE2 produced an increase in the receptor site density without a change in the apparent dissociation constant. PGE2 also markedly stimulated cAMP production. This effect was thought to mediate the increase in expression of BKR-2 as 8-bromo cAMP and various cAMP-stimulating agents acted similarly. PGE2 did not change the BK-dependent intracellular IP3 and cytosolic calcium increases. The overexpression of BKR-2 in the presence of PGE2 was associated with an increase in mRNA as shown by the nuclease protection assay without any change in mRNA half-life. Cycloheximide, an inhibitor of protein synthesis, enhanced BKR-2 mRNA expression. In conclusion, treatment with PGE2 stimulates the synthesis of BKR-2 in hGVEC, possibly by interfering with an inhibitory protein involved in BKR-2 transcription.

The aim of the present study was to analyze the expression of natriuretic peptide receptors in human collecting duct, by using a newly established SV40 cell line (HCD). ANP and C-type natriuretic peptide (CNP) induced a concentration-dependent increase in cGMP suggesting the presence of type-A (NPR-A) and type-B (NPR-B) receptors, respectively. Threshold concentrations were 1 pM and 1 nM, respectively, and stimulated over basal cGMP ratios were 500 and 160 at 0.1 microM ANP and CNP. The urodilatin concentration-response curve was similar to that of ANP. [125I]-ANP bound specifically to HCD cells in a time-dependent fashion, reaching a plateau-phase between one and two hours at 4 degrees C. Equilibrium saturation binding curves suggested a single group of receptor sites (Kd = 421 +/- 55 pM, Bmax = 49.2 +/- 8.8 fmol/mg protein, Hill coefficient = 1.44 +/- 0.1, N = 6). Binding of [125I]-ANP was not displaced by CNP or by C-ANP (4-23), a specific ligand of clearance receptors (NPR-C), and thus occurred mainly via NPR-A. Neither Northern blot analysis nor RT-PCR could detect NPR-C mRNA, although the latter was clearly identified in control human glomerular visceral epithelial cells. In contrast, PCR products with the expected lengths were obtained for NPR-A and NPR-B. In conclusion, HCD cells express both NPR-A and NPR-B, as demonstrated by mRNA and cGMP production studies, but fail to produce NPR-C. This suggests that the human cortical collecting duct is a target for ANP, CNP and urodilatin.

Our aim was to examine whether the human glomerulus was a target for C-type natriuretic peptide (CNP) and how A, B and C receptors of natriuretic peptides (ANPR-A, ANPR-B, ANPR-C) were distributed in glomerular mesangial and epithelial cells. CNP stimulated cyclic GMP production in cultured human mesangial and epithelial cells with similar threshold concentrations (1 nM) and maximum effects (basal value x 30 at 1 microM). In contrast, atrial natriuretic peptide (ANP) was only stimulatory in epithelial cells. [125I] CNP bound specifically to mesangial cells with a Kd of 0.47 nM and Bmax of 42 fmol/mg. Equilibrium of binding was obtained after four to five hours at +4 degrees C and nonspecific binding represented 10 to 20% of total binding. HS142-1 (100 micrograms/ml), a specific inhibitor of ANPR-A and ANPR-B, suppressed 90% of CNP-dependent cyclic GMP production whereas it had little effect on [125I]-CNP binding, suggesting that C receptors were largely predominant in mesangial cells. No biological effect of CNP on mesangial cells, including change in basal or angiotensin II-induced contractility and inhibition of basal or serum-dependent proliferation, could be demonstrated. Similar results were obtained with 8-bromo-cyclic GMP and sodium nitroprusside. Intraglomerular localization of ANPR-A, ANPR-B and ANPR-C mRNA was studied using reverse transcriptase-polymerase chain reaction with amplification of their corresponding cDNA by different primers. Amplification products were identified on gel electrophoresis by their predicted sizes and sequencing. ANPR-A, ANPR-B and ANPR-C mRNA were present in epithelial cells whereas only ANPR-B and ANPR-C mRNA were detected in mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Because dipeptidylpeptidase IV (DPP IV) is present in vivo on glomerular visceral epithelial cells and possesses immunogenic properties, as shown by the capability of anti-DPP IV antibody to induce the Heymann model of glomerulonephritis, we studied the expression and regulation of DPP IV in cultured human glomerular visceral epithelial cells. DPP IV is an ectoenzyme, as indicated by the rapid detection of the product of the reaction in the incubation medium of intact cells and the staining of paraformaldehyde-fixed cells in the presence of a specific anti-DPP IV antibody. DPP IV activity was inhibited by diisopropylfluorophosphate and phenylmethyl sulphonylfluoride. Its optimum pH was alkaline (7.7-8) and it exhibited a Km value of 0.94 mM. DPP IV expression was induced in cells treated by interferon-gamma (IFN-gamma). The effect was significant after a 3-day treatment with 100 U/ml. It increased with time, reaching a plateau after 11 days, and was dose-dependent with a maximum at a concentration of 1000 U/ml. Staining of the cells with anti-DPP IV antibody was also increased after a 6-day treatment with 100 U/ml IFN-gamma. It was shown by Northern analysis that, after 24 hr of exposure to 500 U/ml of IFN-gamma, DPP IV mRNA transcript was stimulated. Transcriptional activation by IFN-gamma did not require new protein synthesis. Interleukin-1 (IL-1) and cyclic AMP had a small stimulatory effect, whereas dexamethasone and phorbol esters were inefficient. These results suggest that DPP IV of glomerular epithelial cells may be up-regulated by IFN-gamma from activated T lymphocytes in glomerular diseases and during lymphocyte-mediated graft rejection.

We have examined the intracellular accumulation and the release of cGMP and cAMP from the apical and basolateral poles of SV40-transformed human glomerular visceral epithelial cells (HGVEC) cultured on filters. After treatment of the cells by atrial natriuretic peptide (ANP), cGMP did not accumulate in the cells and was rapidly released mainly into the apical medium (86% of its total secretion). Its apical secretion was inhibited by 83% in the presence of probenecid, an organic acid transport inhibitor, and by 90% in the presence of nocodazole, a microtubule disrupter. cAMP was released more slowly than cGMP after it had accumulated in the cells. Apical polarization of the secretion still existed but was less marked than for cGMP (70% of its total secretion). Similarly, probenecid and nocodazole inhibited to a lesser degree cAMP egression (66 and 33%, respectively, for the apical secretion). Polarization of HGVEC was confirmed by the vectorialized secretion of lactate dehydrogenase. These results indicate that cGMP release from HGVEC is essentially apical and depends on a probenecid-sensitive organic acid transport and also on an intact microtubular network. cAMP release appears to be less polarized and less sensitive to the two drugs studied. Vectorially secreted cGMP in the urinary space in response to ANP could act downstream in the nephron and thus permit ANP to stimulate cells which do not necessarily possess ANP receptors.