Dirk Bokemeyer

Regulation of Mitogen-Activated Protein Kinase Phosphatase-1 in Vascular Smooth Muscle Cells

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.

Novel NCCT Gene Mutations as a Cause of Gitelman’s Syndrome and a Systematic Review of Mutant and Polymorphic NCCT Alleles

Background: Gitelman’s syndrome (GS) is characterized by hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria and these phenotypic features have been shown to be attributable to mutations in the gene encoding the thiazide-sensitive Na/Cl cotransporter (NCCT). Until now, 55 different mutations have been reported and most of the families affected with GS exhibit autosomal recessive inheritance. Methods: All 26 exons of the human NCCT gene were investigated in 2 German NCCT-deficient patients and their families. Mutation detection was performed by either direct automated sequencing of polymerase chain reaction (PCR)-amplified DNA products or by sequence analysis of cloned PCR products. Results: In a 47-year-old German GS female a novel non-conservative missense mutation (S314F) and a complex deletion/insertion in the NCCT gene were found to be associated with the disorder. A further novel non-conservative substitution (S402F) together with a frequently observed R209W exchange were found in a 19-year-old German GS female. Conclusions: The observation of a compound heterozygote state in both females affected and the absence of a GS phenotype in their relatives carrying a single mutant allele is consistent with an autosomal recessive pattern of inheritance.

Y-Box Protein 1 Mediates PDGF-B Effects in Mesangioproliferative Glomerular Disease

The pivotal role of PDGF-B for mesangioproliferative glomerular disease is well established. Here, Y-box protein-1 (YB-1) was identified as a downstream signaling target of PDGF-B. In healthy kidney cells, YB-1 was located predominantly within the nuclear compartment. Subsequent to PDGF-B infusion and in the course of anti-Thy1.1-induced mesangioproliferative glomerulonephritis, relocalization of YB-1 into the cytoplasm was observed. In experimental models that lack profound mesangial cell proliferation (e.g., Puromycin-nephrosis, passive Heyman nephritis, spontaneous normotensive nephrosclerosis, hyperlipidemic diabetic nephropathy), YB-1 remained nuclear. This translocation coincided with upregulation of YB-1 protein levels within the mesangial compartment. Increased YB-1 expression and subcellular shuttling was dependent on PDGF-B signaling via the mitogen-activated protein kinase pathway because these alterations were prevented by specific PDGF aptamers and the mitogen-activated protein kinase pathway inhibitor U0126. Furthermore, PDGF-B strongly induced YB-1 expression in vitro. This induction was important because RNAi-dependent knockdown of YB-1 abolished the mitogenic PDGF-B effect. Taken together, YB-1 seems to represent a specific and necessary PDGF-B target in mesangioproliferative glomerular disease.

Atrial natriuretic peptide blunts the cellular effects of cyclosporine in smooth muscle.

The effect of cyclosporine A to enhance vasoconstrictor-induced calcium (Ca2+) mobilization in vascular smooth muscle cells may contribute to important side effects in cyclosporine therapy such as hypertension and nephrotoxicity. On the other hand, atrial natriuretic peptide (ANP) is known to diminish vasoconstrictor-stimulated Ca2+ mobilization. The present study, therefore, examined the interaction of cyclosporine and ANP on Ca2+ kinetics in cultured rat vascular smooth muscle cells. Intracellular free calcium concentrations ([Ca2+]i) were measured using fura-2. 45Ca2+ was used to estimate Ca2+ efflux and cellular Ca2+ influx. Preincubation of the cells with cyclosporine (10 micrograms/ml) for 12 minutes lowered basal [Ca2+]i from 48 +/- 4 to 28 +/- 3 nM (p < 0.01). However, in the presence of cyclosporine, the angiotensin II (10(-8) M)-stimulated rise of [Ca2+]i was increased from 296 +/- 22 to 460 +/- 47 nM (p < 0.001). ANP (5 x 10(-9) M) blocked the Ca2+ mobilization by angiotensin II (71 +/- 7 versus 69 +/- 7 nM, NS) and also completely inhibited the effect of angiotensin II in the presence of cyclosporine (77 +/- 5 versus 78 +/- 5 nM, NS). Basal efflux as well as angiotensin II-stimulated 45Ca2+ efflux were not altered by preincubation with cyclosporine, indicating that the effect of cyclosporine on [Ca2+]i was not due to an inhibition of 45Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological responses to PDGF-AA versus PDGF-CC in renal fibroblasts

BACKGROUND Platelet-derived growth factors (PDGF)-AA and -CC mediate renal fibroblast proliferation and/or renal fibrosis. Whereas PDGF-CC binds to both the PDGF receptors (PDGFRs)-αα- and -αβ, PDGF-AA binds more selectively to the αα-receptor, suggesting potential differences in the biological activities. METHODS We compared signal transduction, gene expression as well as changes in the proteome induced by PDGF-AA and -CC in rat renal fibroblasts, which express both PDGFR subunits. The growth factor concentrations used were chosen based on their equipotency in inducing rat renal fibroblast proliferation. RESULTS Both PDGF-AA and PDGF-CC induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Renal fibroblast proliferation induced by either PDGF-AA or -CC could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase (MAPK)-, Janus-kinase (JAK)/signal transducers and activators of transcription (STAT) and phosphatidyl-inositol-3-kinase (PI3K) pathway, pointing to the involvement of all the three pathways. However, quantitative differences between both the stimulations were minor. Additive or synergistic effects by stimulating simultaneously with PDGF-AA and -CC were not observed. Using a proteomic approach we found eleven differentially expressed proteins, which were quantitatively altered after treatment with either PDGF-AA or PDGF-CC. The regulation of calreticulin and inorganic pyrophosphatase 1 could be verified by western blotting. CONCLUSIONS PDGF-AA and -CC exhibit almost identical biological effects on signal transduction and proteome in cultured renal fibroblasts, suggesting that the ligands exert their activity essentially through the commonly bound PDGFR-αα. Nonetheless, two differentially expressed proteins were identified which might be involved in the development of renal failure.

Hyperosmolality Induced by Betaine or Urea Stimulates Endothelin Synthesis by Differential Activation of ERK and p38 MAP Kinase in MDCK Cells

We have previously shown that cultured porcine inner medullary collecting duct cells produce endothelin (ET) which suppressed arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) generation in an autocrine/paracrine feedback-like fashion. Moreover, hyperosmolality, e.g. induced by sodium chloride and urea, stimulated ET synthesis. Since others showed that hyperosmolality also activates mitogen-activated protein (MAP) kinases and that p38 MAP kinase facilitates cellular influx of betaine to protect the cell from high extracellular solute (urea) concentrations, we were tempted to investigate a potential interaction of MAP kinases with ET production in cultured MDCK cells in response to extracellular hyperosmolality induced by betaine and urea, respectively. Increased extracellular tonicity (602 ± 8 vs. control of 323 ± 3 mosmol/kg H2O) induced by betaine stimulated ERK and, more strongly, p38 kinase activity at 0.5–2 h of incubation with a rise in ET-1 synthesis to 1,713 ± 68 vs. 378 ± 51 fmol/mg protein/24 h under control conditions (p < 0.01). The p38 MAP kinase inhibitor SB203580 suppressed the rise in betaine-induced ET-1 synthesis by 91% to 494 ± 38 fmol/mg protein/24 h, whereas the MEK/ERK inhibitor U0126 suppressed it moderately by 34%. Hypertonicity induced by urea moderately stimulated ERK but not p38 MAP kinase activity at 0.5–2 h and at 24–48 h and resulted in a modest rise in ET-1 synthesis to 681 ± 61 fmol/mg protein/24 h (p < 0.05) which was significantly suppressed by U0126 to 484 ± 16 fmol/mg protein/24 h. These results suggest that a functional interaction between the MAP kinases ERK and p38 MAP kinase and ET-1 synthesis is involved in betaine’s protection of MDCK cells in vitro which may represent an in vivo mechanism of protection from hyperosmotic stress induced by high extracellular solute concentrations.

Lack of a Role of Neuronal Nitric Oxide Synthase in the Regulation of the Renal Function in Rats Fed a Low-Sodium Diet

Background/Aims: It has been shown that nitric oxide (NO) generated from neuronal NO synthase (nNOS) counteracts angiotensin II mediated vasoconstriction in the pre- and in the postglomerular microcirculation. Previous studies have demonstrated that the nNOS expression in the macula densa of the renal cortex is enhanced by dietary salt restriction. In view of the well-known fact that dietary salt restriction also leads to an activation of the renin-angiotensin system, the present study was performed to assess the role of nNOS-derived NO in the regulation of the renal function in rats maintained on control (C) and low-salt (LS) diets. Methods: Groups of rats were fed either the C or the LS diet. On day 13 after adaptation to the appropriate diet, renal clearance studies were performed to determine the effects of acute nNOS inhibition either by S-methyl-L-thiocitrulline (L-SMTC) or by Nω-propyl-L-arginine (L-NPA) on renal hemodynamics and sodium excretory function. In separate groups of rats maintained on either the C or the LS diet, the mRNA levels of nNOS and of renin in the renal cortex were examined using the semiquantitative reverse-transcriptase polymerase chain reaction. Results: Intrarenal infusion of vehicle (0.9% saline; 4 µl/min) did not change glomerular filtration rate (GFR), renal plasma flow (RPF), or sodium excretion in either C diet or LS diet rats. Acute intrarenal infusion of L-SMTC (0.3 mg/h) and L-NPA (0.01 mg/h) decreased GFR (–14 ± 5 vs. –13 ± 3%), RPF (–19 ± 6 vs. –17 ± 5%), and sodium excretion (–17 ± 5 vs. –16 ± 4%) in C diet rats as compared with control values (p < 0.05). In contrast, in LS rats, intrarenal administration of either L-SMTC or L-NPA did not cause significant changes in GFR, RPF, and sodium excretion. Furthermore, the mRNA expression for nNOS in the renal cortex was moderately increased in LS rats as compared with C rats (densitometric ratios of nNOS mRNA/GAPDH mRNA 0.31 ± 0.01 vs. 0.22 ± 0.04, p < 0.05), in parallel with the renin expression (renin mRNA/GAPDH mRNA ratios 1.4 ± 0.2 vs. 1.0 ± 0.1, p < 0.05). Conclusions: These results indicate that in normotensive rats kept on a normal salt intake nNOS-derived NO modulates both afferent and efferent arteriolar tones. In contrast, rats on an LS diet exhibit an impaired renal vascular responsiveness to nNOS-derived NO or an impaired ability to release NO by nNOS despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the lack of effect of acute nNOS inhibition on renal function suggests that NO derived by nNOS does not participate in counteracting the vasoconstrictor influences of elevated circulating and/or intrarenal angiotensin II levels on pre- and postglomerular microcirculation in rats on an LS diet.

Cardiac hypertrophy in the Prague-hypertensive rat is associated with enhanced JNK2 but not ERK tissue activity.

Mitogen-activated protein (MAP) kinases are important intracellular mediators for proliferation and hypertrophy and therefore may also regulate cardiomyoblast growth in hypertensive heart disease. Thus, the aim of the present study was to examine the activities of MAP kinases, namely extracellular signal-regulated kinase (ERK)1,2, c-Jun NH2-terminal kinases (JNK)1,2 and p38 MAP kinase, in myocardial tissue of 12-week-old Prague normotensive (PNR) and hypertensive rats (PHR), a model of genetic hypertension with marked cardiac hypertrophy. Systolic blood pressure was 121 ± 5 in PNR and 208 ± 15 mm Hg in PHR (p < 0.01). Total heart weight was 247 ± 4 in PNR vs. 316 ± 4 mg/100 g body weight in PHR (p < 0.01). Left and right ventricular weights were 121 ± 5 and 53 ± 3 in PNR vs. 168 ± 4 (p < 0.01) and 57 ± 2 mg/100 g body weight (n.s.) in PHR. Using anti-ERK2 Western blot analysis as well as immunocomplex ERK activity assay, we found no activation of ERK2 in left or right ventricular tissue of PHR and PNR. Similary, p38 MAP kinase phosphorylation and activity were not detectable. In contrast, Western blot analysis using antiphospho-JNK antibodies revealed in myocardial tissue of right and left ventricles significantly greater phosphorylation of JNK2 in PHR than in PNR. This finding was confirmed by immunocomplex JNK activity assay using ATF-2 as substrate, which demonstrated a significant increase in JNK activity in the left ventricle of PHR as compared to PNR (6.4 ± 1.5 vs. 2.5 ± 0.5 OD; each n = 5; p < 0.05). In conclusion, cardiac JNK2 seems to be regulated differently from ERK2 in this rat model. In PHR, as compared to PNR, we found enhanced activity of JNK2 in the left and right ventricles suggesting that JNK2 is involved in hypertensive cardiac disease. The rise in JNK in both ventricles may result indirectly from humoral stimuli, e.g., endothelin-1 and/or angiotensin II, and may contribute to ventricular hypertrophy in this model of spontaneous hypertension.

Cyclosporine A enhances total cell calcium independent of Na-ATPase in vascular smooth muscle cells

The effect of cyclosporine A in enhancing vasconstrictor-induced calcium (Ca2+) mobilization in vascular smooth muscle cells may contribute to important side effects in cyclosporine therapy such as hypertension and nephrotoxicity. As we have previously shown, cyclosporine A stimulates transmembrane Ca2+ influx. Since Ca2+ efflux was not affected by cyclosporine A, we concluded that cyclosporine augments angiotensin II induced Ca2+ mobilization in vascular smooth muscle cells by an increased amount of Ca2+ in angiotensin II sensitive intracellular Ca2+ stores. The present study was therefore designed to examine the effect of cyclosporine A on cellular calcium content and on membrane calcium transport mechanisms. An important mechanism of Ca2+ extrusion from the cell is the Na-Ca exchanger. Its activity is closely related with that of the Na-ATPase. By increasing cellular sodium concentration the blockade of Na-ATPase would in turn activate cellular calcium uptake bx the Na-Ca exchanger. Therefore, we hypothesized that cyclosporine A might exert its effects in the same manner as a circulating Na-ATPase inhibitor. Total cell calcium was measured by atomic absorption and activity of Na-ATPase was estimated by an assay measuring phosphate production. Preincubation of the cells with cyclosporine (10 μg/ml) for 15 min increased total cell calcium from 31.4 ± 5.0 to 46.5 ± 5.3 nmol/mg protein (P < 0.05). Activity of Na-ATPase was not affected by cyclosporine A (3.9 ± 0.2 vs. 4.3 ± 0.2 μol Pi h−1 mg−1 protein). Therefore, cyclosporine A induced Ca2+ influx is not mediated by an inhibition of the Na-ATPase. Cyclosporine-stimulated accumulation of cellular calcium may be mediated, for example, by opening of calcium channels in the plasma membrane. Increased Ca2+ mobilization in the presence of cyclosporine A may be due to an increased amount of Ca2+ avaible from intracellular Ca2+ stores. These results are of substantial significance for understanding the pathophysiological mechanisms of cyclosporine A induced vasoconstriction.

Atrial natriuretic peptide and endothelin : modulators of renal function

In the hormonal regulation of body fluid and cardiovascular homeostasis [3] two new peptide families, namely the atrial natriuretic peptides (ANP) and endothelins (ET), have emerged over the past one and a half decades. They exert their actions at the levels of the brain, adrenals, vasculature, and kidney [1, 2]. This short review on the vasorelaxant, natriuretic, and antiproliferative ANP [1] and the potent vasoconstrictor, diuretic, and mitogenic ET systems [2] is restricted to their potential roles as modulators of renal function.