Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia–reperfusion-induced endothelial cell apoptosis

Activation of cytoskeleton regulator Rho-kinase during ischemia–reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.
Introduction
Experimental ischemia and reperfusion (I/R) induces endothelial cell apoptosis in several organs [1, 2]. We and others have previously shown that activation of RhoA and downstream effector Rho-kinase during experimental I/R plays a major role in I/R injury and associated apoptosis [3–7]. Indeed, Rho-kinase inhibition has beneficial effects on I/R injury in several organs [4, 6–12]. Endothelial cells may be essential for mediating the beneficial effects of Rho-kinase inhibition on I/R injury, because, for instance inhibition of endothelial nitric oxide synthase blocked the beneficial effects of Rho-kinase inhibition on rat myocardial infarct size [12].
Rho-kinase activation during the execution of apoptosis results in increased myosin activity, bundling of F-actin by activated myosins, actin–myosin contractile force generation, cell contraction and ultimately membrane blebbing [13–15]. Recent in vivo and in vitro studies suggest that Rho-kinase and the effects of Rho-kinase on the actin–myosin cytoskeleton may also be involved in the initiation of apoptosis [4, 12, 16–20]. Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway and it upregulates mitochondrial death pathway protein Bax [4, 12, 16, 19]. The effects of Rho-kinase on the cytoskeleton may therefore be important in linking Rho-kinase activation and PI3-kinase/Akt inactivation.
We hypothesized that (1) inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and (2) prevention of F-actin rearrangement facilitates the protective effects of Rho-kinase inhibition (Fig. 1). To test this hypothesis, we assessed whether specific inhibition of Rho-kinase with Y-27632 [21] attenuates apoptosis via phosphorylation of Akt in an in vitro model of simulated I/R. The involvement of the F-actin cytoskeleton was explored by the use of F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide [22].
Fig. 1Proposed mechanism for modulation of I/R-induced apoptosis involving RhoA/Rho-kinase and PI3-kinase/Akt. Simulated I/R activates RhoA/Rho-kinase [3–7], which leads to F-actin rearrangement. F-actin rearrangement facilitates reduction of PI3-kinase/Akt activity and induction of apoptosis. This can be prevented by inhibition of Rho-kinase with Y-27632. The protective effects of Y-27632 can be blocked with PI3-kinase inhibitor wortmannin. Several other pathways for I/R-induced apoptosis are represented by the dashed arrow [2, 19]
Materials and methods
Cell culture
Human Umbilical Vein Endothelial Cells (HUVECs) were isolated, cultured and characterized as previously described [23]. Briefly, HUVECs were cultured on gelatin-coated wells/glass cover slips until confluent at 37°C under 5% CO2/95% air atmosphere. Culture medium was renewed every other day (medium 199 supplemented with 10% heat-inactivated human serum, 10% heat-inactivated new born calf serum, 150 μg/ml crude endothelial cell growth factor, 2 mM l-glutamine, 5 U/ml heparin, 100 IU/ml penicillin, and 100 μg/ml streptomycin).
Simulation of I/R
We used an established in vitro endothelial cell model for simulation of ischemia and reperfusion [24, 25]. Figure 2 shows a schematic representation of the experimental protocol. Cells were washed twice with washing buffer (1.2 mM MgSO4 · 7H2O, 116 mM NaCl, 5.3 mM KCl, 1.13 mM NaH2PO4 · H2O, 1.8 mM CaCl2 · 2H2O, 20 mM HEPES) and treated for 1 h with washing buffer with or without 10 μM Y-27632 (Tocris Cookson Ltd., Bristol, United Kingdom (UK)), 250 nM cytochalasinD (Sigma-Aldrich, Saint Louis, Missouri, United States of America (USA)), 150 nM latrunculinA (Calbiochem, Darmstadt, Germany), 20 nM jasplakinolide (Calbiochem) and 50 nM wortmannin (Sigma-Aldrich). This was followed by 1 h of simulated ischemia by covering cells with 1 ml mineral oil (nitrogen bubbled) and subsequently simulated reperfusion by replacement of culture medium for different time points: no reperfusion for adenosine tri-phosphate (ATP) measurement and F-actin staining, 1 h for ATP measurement, F-actin staining and western blotting to analyze phospho Akt (pAkt) levels and 24 h for quantification of apoptosis. Drugs were also present during simulated reperfusion. As controls, we used cells treated for 1 h with washing buffer followed by treatment with medium (control) or medium supplemented with 10% mineral oil (control oil) to assess any injurious effects of products within the oil.
Fig. 2Schematic representation of the experimental protocol. Cells were pre-treated for 1 h with Y-27632, cytochalasinD, latrunculinA, jasplakinolide or wortmannin. One hour of simulated ischemia was followed by 1–24 h of simulated reperfusion. No reperfusion for ATP measurement and F-actin staining, 1 h for ATP measurement, F-actin staining and analysis of Akt activity by western blotting and 24 h for quantification of apoptosis. The drugs were also present during the reperfusion phase
ATP measurement
We measured ATP after ischemia and I/R (1 h of reperfusion) in order to confirm ischemic substrate deprivation. Cells were grown in a gelatin-coated 12-well chamber and treated according to the simulated I/R protocol. ATP was measured using the ENLITEN ATP Assay System Bioluminiscence Detection Kit for ATP (Promega Corporation, Madison, Wisconsin, USA). Proteins in lysate were measured with BCA Protein Assay Kit (Pierce, Rockford, Illinois, USA) in order to calculate nmol ATP/μg protein.
Quantification of apoptosis
The cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I/R protocol (24 h of reperfusion). Cells were fixed in 4% formaldehyde and permeabilized with 0.2% triton X-100 (Sigma-Aldrich). The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) in Vectashield® Mounting medium (Vector Laboratories, Inc., Burlingame, California, USA). Apoptosis on each glass cover slip was quantified using a DeadEndTM Fluorometric TdT-mediated dUTP Nick End Labeling (TUNEL) System (Promega Corporation) with fluorescence microscopy using a MarianasTM digital imaging microscope and Slidebook 4.2 software (Intelligent Imaging Innovations, Inc., Denver, Colorado, USA). The number of apoptotic nuclei (TUNEL-positive) and the total number of nuclei (DAPI-positive) were counted in three non-overlapping microscope fields/glass cover slip using a 10× air lens (Carl Zeiss B.V., Sliedrecht, The Netherlands) and averaged. The number of apoptotic nuclei was expressed as percentage of the total number of nuclei.
F-actin cytoskeleton staining
The cells were grown in a 24-well chamber on gelatin-coated glass cover slips and treated according to the simulated I/R protocol (1 h of reperfusion). Cells were fixed in 4% formaldehyde and permeabilized with 0.2% triton X-100. F-actin was stained with rhodamin-phalloidin (1:100) (Molecular Probes, Inc., Eugene, Oregon, USA) and the nucleus with DAPI in Vectashield® Mounting medium. Cells were visualized with fluorescence microscopy and Slidebook software.
Analysis of Akt phosphorylation
Cells were grown in a gelatin coated 6-well chamber, treated according to the simulated I/R protocol (1 h of reperfusion) and lysated with lysis buffer (20 mM Tris/HCl pH 8.0, 150 mM NaCl, 90 mM KCl, 2 mM EDTA/NaOH pH 8.0, igepal (1:200), triton X-100 (1:200), 1 mM Na3VO4, 10 mM NaF, protease inhibitors (1:100), phosphatase inhibitors (1:100)). Protein concentration was measured (BCA method) and equal amounts of protein from each sample were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California, USA). Membranes were incubated with polyclonal antibodies against pAkt (serine-473, rabbit, 1:1,000) and total Akt (tAkt, rabbit, 1:1,000) (Cell Signaling Technology, Inc., Danvers, Massachusetts, USA). tAkt was used as loading control. Goat anti-rabbit immunoglobulins horseradish peroxidase (HRP) from DakoCytomation (Glostrup, Denmark) was used for the detection of the primary antibodies at 1:1,000. Detection of the HRP reaction was performed with ECL plus Western Blotting Detection System (Amersham Biosciences, Little Chalfont Buckinghamshire, UK). Imaging and analysis were performed with LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan) and AIDA Image Analyzer (Raytest GmbH, Straubenhardt, Germany). Data were expressed as pAkt/tAkt as percentage of control.
Statistical analysis
A one-way analysis of variance (ANOVA) was conducted to explore the effects of I/R, Y-27632, cytochalasinD or wortmannin treatment, and their interaction on apoptosis. The t-test was used to further analyse the differences between the groups when ANOVA indicated statistical significance. We adjusted for multiple testing with help of the Bonferroni Holm method. The paired t-test was used for paired ATP data and logarithmic transformed absolute pAkt/tAkt data. Absolute pAkt/tAkt data were logarithmic transformed to obtain a Gaussian distribution. We adjusted for multiple testing with help of the Bonferroni Holm method. The Pearson coefficient of determination (R2) was used to express the relation between the percentage of apoptotic nuclei and the logarithmic transformed absolute pAkt/tAkt data. Differences were considered significant at the P < 0.05 level. Data are presented as means ± standard error of the mean (SEM).
Results
ATP levels and apoptosis following simulated ischemia and I/R
In order to confirm ischemic substrate deprivation in simulated I/R, we measured intracellular ATP levels. Ischemia was simulated by covering the monolayer of cells with nitrogen bubbled mineral oil. Reperfusion was simulated by replacement of culture medium (Fig. 2). Intracellular ATP levels decreased following simulated ischemia compared to the levels in cells treated with medium supplemented with 10% mineral oil (control oil) (0.15 ± 0.04 nmol ATP/μg protein versus 0.22 ± 0.04 nmol ATP/μg protein, P < 0.05, n = 7). Intracellular ATP decreased even further during the reperfusion phase (0.10 ± 0.04 nmol ATP/μg protein, P < 0.05 versus control oil, n = 7). ATP levels of control oil cells were comparable to those of control cells (0.20 ± 0.04 nmol ATP/μg protein, n = 7). Simulated I/R induced a significant percentage of apoptotic nuclei (ANOVA for the effect of I/R on apoptosis, P < 0.0001), while hardly any apoptosis was observed in control and control oil cells (Fig. 3).
Fig. 3Rho-kinase inhibition reduces the degree of apoptosis during I/R via PI3-kinase. Non-treated control (C, n = 11), control oil (C O, n = 2), Y-27632-treated control (C Y, n = 11) and wortmannin-treated control (C W, n = 5) showed hardly any apoptosis. Simulated I/R induced a significant percentage of apoptotic nuclei (minimum P < 0.005 versus the controls, n = 12). Y-27632 treatment reduced the percentage of apoptotic nuclei during simulated I/R (I/R Y, *P < 0.01, n = 12). This protective effect was completely blocked by wortmannin (I/R YW, #P < 0.05, n = 5). The number of apoptotic nuclei is expressed as percentage of total number of nuclei. Data are presented as means ± SEM
The effect of Rho-kinase inhibition on apoptosis
To investigate the role of Rho-kinase during I/R-induced apoptosis, HUVECs were treated with inhibitor Y-27632 during simulated I/R (Fig. 3). Y-27632 had no effect on basal apoptosis, but reduced the degree of apoptosis during I/R (ANOVA for the effects of Y-27632 and the interaction between I/R and Y-27632 on apoptosis, both P < 0.01). To determine whether the protective effects of Rho-kinase inhibition were mediated by PI3-kinase, cells were co-treated with PI3-kinase inhibitor wortmannin. Wortmannin had no effect on basal apoptosis, but blocked the protective effect of Rho-kinase inhibition during I/R (ANOVA for the effect of wortmannin on apoptosis, P < 0.05).
Akt activity in Y-27632-mediated protection against apoptosis
To investigate the effect of Y-27632 treatment during I/R on Akt activity, Akt phosphorylation was analyzed by western blotting (Fig. 4). Simulated I/R reduced Akt activity compared to non-treated or Y-27632-treated control cells. Y-27632 treatment maintained Akt activity in I/R cells. To determine whether this effect was mediated by PI3-kinase, cells were co-treated with wortmannin. Wortmannin blocked the Y-27632-induced maintenance of Akt activity. Akt activity in Y-27632- (Fig. 4) or wortmannin-treated control cells (preliminary data) was comparable to the activity in non-treated control cells.
Fig. 4Rho-kinase inhibition maintains Akt activity during I/R. A representative western blot of pAkt and tAkt is shown in the upper panel and a graph of pAkt/tAkt as percentage of control in the lower panel. Simulated I/R (I/R, n = 15) induced a decrease in pAkt compared to the control and the Y-27632-treated control (C Y, *P < 0.05, n = 15). Y-27632 treatment during simulated I/R maintained pAkt levels (I/R Y, **P < 0.05, n = 14). Wortmannin blocked the Y-27632-induced maintenance of Akt activity (I/R YW, #P < 0.05, n = 12). Data are presented as means ± SEM. Absolute pAkt/tAkt data in arbitrary units (a.u.): control: 0.75 a.u.; C Y: 0.64 a.u.; I/R: 0.32 a.u.; I/R Y: 0.36 a.u.; I/R YW: 0.04 a.u
The F-actin cytoskeleton in Y-27632-mediated protection against apoptosis
To investigate whether Y-27632-mediated protection against apoptosis paralleled the absence of Rho-kinase-mediated F-actin rearrangement, the F-actin cytoskeleton was visualized with rhodamin-phalloidin in non-treated or Y-27632-treated control, simulated ischemia and simulated I/R cells (n = 9, representative pictures are shown in Fig. 5). Y-27632-treated control cells showed a slight decrease in peripheral F-actin bundles compared non-treated control cells, indicating that in vitro inhibition of Rho-kinase was able to reduce F-actin bundle formation. After simulated ischemia in non-treated cells, some F-actin bundles appeared in the cytoplasm. Y-27632-treated ischemic cells showed only F-actin clumps. After simulated reperfusion, even more F-actin bundles appeared in the periphery and the cytoplasm of the non-treated cells. In contrast, Y-27632-treated I/R cells, showed no F-actin bundles.
Fig. 5Y-27632 and cytochalasinD prevent F-actin bundle formation during I/R. Shown is fluorescent imaging of rhodamin-phalloidin stained F-actin structures and DAPI stained nuclei in non-treated, Y-27632-, cytochalasinD-, latrunculinA- and jasplakinolide-treated control, simulated ischemia and simulated I/R cells. The arrow and * indicate F-actin bundles and F-actin clumps, respectively. Control cells: In non-treated cells, most F-actin bundles were seen in the periphery of the cell. Y-27632-treated cells showed a slight decrease in peripheral F-actin bundles. CytochalasinD-treated cells showed only F-actin clumps. Cells treated with latrunculinA or jasplakinolide showed a slight decrease and increase in F-actin bundles, respectively. Ischemic cells: Non-treated cells showed F-actin bundles throughout the whole cell. Cells treated with Y-27632, cytochalasinD or latrunculinA showed only F-actin clumps. Jasplakinolide-treated cells showed F-actin bundles. I/R cells: Non-treated cells showed F-actin bundles throughout the whole cell. In Y-27632-treated cells, no F-actin bundles were seen. CytochalasinD-treated cells showed also no F-actin bundles, only F-actin clumps were visible. LatrunculinA-treated cells showed some F-actin bundles. Jasplakinolide-treated cells showed as many F-actin bundles as non-treated I/R cells
To further investigate the role of the F-actin cytoskeleton in I/R-induced apoptosis, we used F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide (Fig. 5). CytochalasinD-treated control cells showed no F-actin bundles. LatrunculinA-treated control cells showed a slight decrease and jasplakinolide-treated control cells showed a slight increase in F-actin bundles compared to non-treated control cells. CytochalasinD- and latrunculinA-treated ischemic cells showed no F-actin bundles, only F-actin clumps were visible. Jasplakinolide-treated ischemic cells showed F-actin bundles throughout the whole cell. In cytochalasinD-treated I/R cells, no F-actin bundles were visible, only F-actin clumps could be seen. In contrast, latrunculinA-treated I/R cells still showed some F-actin bundles and jasplakinolide-treated I/R cells showed as many F-actin bundles as non-treated I/R cells.
Since cytochalasinD treatment prevented F-actin rearrangement similarly to Y-27632 treatment, we investigated whether cytochalasinD treatment could also prevent apoptosis during simulated I/R (Fig. 6). CytochalasinD had no effect on basal apoptosis, but reduced the degree of apoptosis during I/R (ANOVA for the effects of cytochalasinD and the interaction between I/R and cytochalasinD on apoptosis, both P < 0.05). Wortmannin blocked the protective effect of cytochalasinD in I/R (ANOVA for the effect of wortmannin on apoptosis, P < 0.0001). In contrast to the protective effect of Y-27632 and cytochalasinD on apoptosis, latrunculinA and jasplakinolide did not affect I/R-induced apoptosis, also not in co-treatment with wortmannin (data not shown). LatrunculinA and jasplakinolide had no effect on basal apoptosis (data not shown).
Fig. 6CytochalasinD reduces the degree of apoptosis during I/R via PI3-kinase. Non-treated control (C, n = 6) and cytochalasinD-treated control (C CytoD, n = 6) showed hardly any apoptosis. Simulated I/R induced a significant percentage of apoptotic nuclei (P < 0.0001 versus the controls, n = 6). Treatment with cytochalasinD reduced the percentage of apoptotic nuclei in simulated I/R (I/R CytoD, *P < 0.05, n = 6). This protective effect was completely blocked by wortmannin (I/R CytoDW, #P < 0.005, n = 6). The number of apoptotic nuclei is expressed as percentage of total number of nuclei. Data are presented as means ± SEM
Furthermore, cytochalasinD maintained Akt activity during simulated I/R (Fig. 7). Co-treatment with wortmannin completely blocked the cytochalasinD-induced maintenance of Akt activity. Akt activity in cytochalasinD-treated control cells was comparable to the activity in non-treated control cells.
Fig. 7CytochalasinD treatment maintains Akt activity during I/R. Simulated I/R (I/R, n = 11) induced a decrease in pAkt compared to the control and the cytochalasinD-treated control (C CytoD, *P < 0.01, n = 11). CytochalasinD treatment during simulated I/R maintained pAkt levels (I/R CytoD, **P < 0.05, n = 11). Wortmannin blocked the cytochalasinD-induced maintenance of Akt activity (I/R CytoDW, #P < 0.01, n = 7). Data are presented as means ± SEM. Absolute pAkt/tAkt data in arbitrary units (a.u.): control: 0.61 a.u.; C CytoD: 0.84 a.u.; I/R: 0.36 a.u.; I/R CytoD: 0.54 a.u.; I/R CytoDW: 0.05 a.u
Interestingly, the percentage of apoptotic nuclei inversely correlated to Akt activity (Fig. 8), indicating that a decrease in Akt activity correlated to an increase in apoptosis.
Fig. 8The percentage of apoptotic nuclei inversely correlates to Akt activity (R2 = 0.68, P < 0.05). Mean percentage of apoptotic nuclei and mean logarithmic transformed absolute pAkt/tAkt levels in non-treated (C), Y-27632 (C Y)- and cytochalasinD (C CytoD)-treated control cells and in non-treated (I/R), Y-27632 (I/R Y)-, cytochalasinD (I/R CytoD)- and wortmannin (I/R YW and I/R CytoDW)- treated I/R cells are shown as *
Discussion
This study shows that (1) inhibition of Rho-kinase attenuates I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and (2) the effects of Rho-kinase inhibition on PI3-kinase/Akt and apoptosis are facilitated by prevention of F-actin rearrangement.
Our data point to a novel mechanistic link between activation of Rho-kinase during I/R and negative regulation of PI3-kinase/Akt, namely the effect of Rho-kinase on F-actin rearrangement (Fig. 1). Prevention of F-actin rearrangement by Rho-kinase inhibition or by treatment with actin depolymerizer cytochalasinD, independent of Rho-kinase, both attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. Indeed, it has been reported previously that cytochalasins inhibited in vivo and in vitro apoptosis induced by a variety of stimuli, including I/R [5, 13, 26]. This study is therefore an extension of and a link between reports on Rho-kinase as negative regulator of the PI3-kinase/Akt pathway and reports on the involvement of Rho-kinase-dependent cytoskeletal rearrangement in the initiation of apoptosis [4, 12, 16–20].
Actin depolymerizer latrunculinA did not affect I/R-induced endothelial cell apoptosis. This may be explained by the nature and degree of F-actin rearrangement during I/R, since F-actin bundles were completely absent in Y-27632- and cytochalasinD-treated I/R cells, while latrunculinA-treated cells still showed some bundles. Indeed, cytochalasinD and latrunculinA depolymerize F-actin by different mechanisms and induce different patterns in vitro [22, 27]. CytochalasinD caps the barbed end of actin filaments, severs actin filaments, sequesters actin monomers or dimers, promotes nucleation and stimulates the ATPase activity of G-actin. In vitro in hamster fibroblasts, it caused a complete collapse of the stress fibers at concentrations comparable to ours [22, 27]. LatrunculinA on the other hand binds to monomeric G-actin to form a non-polymerizable complex and caused concentration-dependent shortening and thickening of stress fibers in the fibroblasts at concentrations comparable to ours [22, 27]. Jasplakinolide, which promotes actin polymerization and stabilizes actin filaments by binding to F-actin [22], induced as many F-actin bundles in I/R as non-treated I/R cells and consequently did not affect I/R-induced endothelial apoptosis. However, in vivo it has been demonstrated that jasplakinolide prevented I/R-induced apoptosis in the rat kidney, while latrunculinB increased I/R-induced apoptosis in rat kidney [28, 29]. Furthermore, we observed hardly any apoptosis in our Y-27632-, cytochalasinD-, latrunculinA- and jasplakinolide-treated control cells, while some reports describe the induction of apoptosis in control cells by latrunculinA or jasplakinolide at much higher concentrations than ours [30, 31], by cytochalasinD in airway epithelial cells, but not in canine kidney epithelial cells [32] or by Y-27632 in airway epithelial cells [32]. Therefore, these discrepancies may be explained by the use of different concentrations, cell types or the use of an in vivo/in vitro model.
Akt activation after 1 h of reperfusion inversely correlated to the percentage of apoptosis after 24 h of reperfusion. Therefore, early reduced Akt activity may have a predictive value for the development of late I/R-induced apoptosis. The preventive effect of Y-27632 and cytochalasinD treatment probably takes place early in the process of apoptosis, because the Y-27632-induced increase in Akt activity is rapid and transient with a maximum after 30 min [12, 16]. Indeed, we could neither detect increased Akt activity in our Y-27632-treated control cells after 3 h nor in our Y-27632-treated I/R cells after 24 h of reperfusion (pilot study, data not shown), possibly because this was to late to detect the peak in Akt activity. I/R-induced apoptosis was maximal as late as after 24–48 h of reperfusion [25]. Therefore, apoptosis was quantified after 24 h of reperfusion. Indeed, we did not see any TUNEL-positive nuclei after 1 h of reperfusion (pilot study, data not shown). Our percentage of apoptotic nuclei was comparable to the percentage reported by the developers of the simulated I/R model [25]. However, less endothelial cell apoptosis was observed in a model of hypoxia/reoxygenation [33], possibly because this induces less severe metabolic deprivation. In addition, PI3-kinase inhibition did not decrease basal Akt activity nor increased basal apoptosis, while it blocked the effects of Rho-kinase inhibition on I/R-induced apoptosis and Akt activity. This suggests that the basal situation is independent of PI3-kinase, while the effects Rho-kinase inhibition during I/R are dependent on PI3-kinase.
Although it is unclear by which mechanism F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis, several mechanisms can be proposed. First, RhoA and Rho-kinase may regulate pAkt levels by regulating phosphatase and tensin homologue (PTEN) activity. PTEN is in inactive state in the cytosol and translocates to the membrane upon RhoA or Rho-kinase-induced activation, where it can inhibit the phosphorylation of Akt via PI3-kinase [34]. Disruption of the F-actin bundles by Rho-kinase inhibition or cytochalasinD treatment or the direct effects of Rho-kinase inhibition on PTEN may prevent the translocation and activation of PTEN, thereby preventing its inhibitory effect on Akt phosphorylation. Furthermore, there may be some PI3-kinase independent mechanisms involved in the Y-27632-induced protection against apoptosis. It is reported that F-actin polymerization and actin–myosin contractility are necessary for membrane blebbing and chromatin condensation during execution of apoptosis induced by tumor necrosis factor-α or overexpression of Rho-kinase [13, 26]. Moreover, the actin–myosin cytoskeleton may be involved in facilitating the cellular localization of specific components of the apoptotic machinery and be required for caspase-3 activation [17, 18].
Our study carries some limitations. First, we cannot conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis. Second, Rho-kinase inhibition and F-actin depolymerization could establish only a partial protection against apoptosis, in agreement with the literature [4, 5, 8, 12], possibly because other mechanisms are also involved and the I/R-induced injury is too extensive. Third, we may have underestimated the number of apoptotic cells, because endothelial cells that detached from the basement membrane during simulated I/R and did not reattach later were not counted in the quantification of apoptosis. Indeed, Prahalad et al. [35] describe this reversible cell detachment during ATP depletion of Madin-Darby canine kidney cells.
Conclusion
Prevention of F-actin rearrangement or F-actin bundle formation by Rho-kinase inhibition or by treatment with actin depolymerizator cytochalasinD, independent of Rho-kinase, attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. The present finding suggests that the changes in the F-actin cytoskeleton play a pivotal role in the negative regulation of PI3-kinase and Akt by Rho-kinase. Further studies are required to conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis.

cytoskeleton y-27632 cytochalasind latrunculina jasplakinolide huvec


Therapeutic strategies to slow chronic kidney disease progression

Childhood chronic kidney disease commonly progresses toward end-stage renal failure, largely independent of the underlying disorder, once a critical impairment of renal function has occurred. Hypertension and proteinuria are the most important independent risk factors for renal disease progression. Therefore, current therapeutic strategies to prevent progression aim at controlling blood pressure and reducing urinary protein excretion. Renin-angiotensin-system (RAS) antagonists preserve kidney function not only by lowering blood pressure but also by their antiproteinuric, antifibrotic, and anti-inflammatory properties. Intensified blood pressure control, probably aiming for a target blood pressure below the 75th percentile, may exert additional renoprotective effects. Other factors contributing in a multifactorial manner to renal disease progression include dyslipidemia, anemia, and disorders of mineral metabolism. Measures to preserve renal function should therefore also comprise the maintenance of hemoglobin, serum lipid, and calcium-phosphorus ion product levels in the normal range.
Natural course of chronic kidney disease
Progression of chronic kidney disease (CKD) toward end-stage renal failure is common in CKD patients, and once significant impairment of renal function has occurred, it tends to progress irrespectively of the underlying kidney disorder. However, information on the natural course of CKD progression in children is still limited. The prospective, population-based ItalKid registry, including almost 1,200 CKD children with various renal diseases over a 10-year period, reported a prevalence of 23% of patients suffering from severe kidney disease with chronic renal insufficiency (CRI). The incidence of renal replacement therapy was 7.3 per year per 100 patients with CRI, and the risk of developing end-stage renal disease (ESRD) by age 20 was 68% [1]. The decline of renal function was not linear but rather characterized by a sharp decline during puberty and at early postpubertal age. This finding supports the general clinical impression that in many children with renal hypodysplasia, kidney function deteriorates more rapidly around the time of puberty. This notion received further support by a recently published retrospective analysis of 176 children with renal hypodysplasia [2]. The authors postulated that the natural course of chronic renal failure in these patients can be divided into three time periods: an initial period, usually lasting the first 3 years of life characterized by an improving renal function, a subsequent period of stable renal function attained by 50% of patients for a mean of 8 years, and a phase with renal function gradually deteriorating toward ESRD. The latter period started just after infancy in 48% and around puberty in 23%. In 30% of patients, renal function remained stable even beyond puberty.
Factors affecting renal disease progression
There is clear evidence from clinical studies that hypertension and proteinuria are key players in the pathophysiology of CKD progression in humans [3–5]. The renin-angiotensin system (RAS) is intrinsically involved in the process, and other potential contributors include genetic background, renal anemia, altered mineral homeostasis, dyslipidemia, chronic inflammation, and oxidative stress as well as general cardiovascular risk factors such as diabetes, smoking, and obesity. As a consequence of the mechanistic insights in renal disease progression obtained by experimental work, several principal renoprotective strategies have emerged in recent years (Fig. 1). These are based mainly on clinical evidence established in adult patients, but growing evidence supports their efficacy also in children. Efficient control of blood pressure and minimization of proteinuria appear as the two most important measures to preserve residual kidney function. Other issues, such as prevention and treatment of renal anemia, uremic dyslipidemia, and disorders of mineral metabolism, have an experimental basis, although their clinical importance is less clear to date. In the following, we review current treatment strategies to slow renal disease progression in childhood CKD.
Fig. 1Pathophysiology, consequences, and treatment options of hypertension and proteinuria in chronic kidney disease. EPO erythropoietin, P reduction  serum phosphate reduction, RAS renin angiotensin system, Non-DHP CCP non dihydropyridine calcium channel blockers, TGFβ transforming growth factor β, TIMP tissue inhibitors of metalloproteinases, ET1 endothelin 1, PAI plasminogen activator inhibitor, ⊥ inhibitory effect
Hypertension
Hypertension is an independent risk factor of renal failure progression in adults [3–5]. Whereas the degree of hypertension correlates with the severity of the underlying renal disease, interventional studies have provided evidence that high blood pressure actively contributes to renal failure progression in human CKD. In pediatric nephropathies, renal hypertension is common, although typically less severe than in adult kidney disorders. Hypertension prevalence estimates in children with CKD range from 20% to 80% depending on the degree of renal dysfunction and underlying renal disease [6, 7]. However, even children with CKD stage 2 or renal hypodysplasia may present with significantly elevated blood pressure [8]. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood demonstrated that in CKD children, a systolic blood pressure greater than 120 mmHg was associated with a significantly faster glomerular filtration rate (GFR) decline [9].
Numerous studies in adults have provided proof to the concept that consequent antihypertensive therapy slows down the rate of renal-failure progression [10]. A close linear relationship between the blood pressure level achieved by antihypertensive treatment and the rate of renal failure progression in adult CKD patients has been noted, which appears to persist well into the normal range of blood pressure [11, 12].
The firm evidence of a favorable effect of intensified blood pressure control in patients with CKD has resulted in generally lower target blood pressure recommendations in this patient group. In the most recent guidelines by the Joint National Committee in the US (JNC7) [13] and the Guidelines of the European Hypertension Society [14], 120/80 mmHg has been defined as the upper limit of the ‘optimal’ blood pressure range, particularly when proteinuria is present, and any blood pressure > 130/80 in CKD patients should be actively lowered by therapeutic intervention [15]. These blood pressure targets are equivalent to the 50th to 75th distribution percentile in the general young adult population. It is as yet unknown whether these blood pressure targets hold true for the pediatric population and whether glomerular damage in children correlates with absolute or age-specific relative blood pressure. The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines on blood pressure control in CKD children [15] adopted the recommendations of the task force that target blood pressure should be <90th percentile for normal values [16] adjusted for age, gender, and height percentile. Assuming that equivalent blood pressure percentiles should be targeted in children as in adults, the adult recommendations would, for instance, correspond to an acceptable upper blood pressure level of 106/66 mmHg (75th percentile) in an 8-year old child with CKD. The final results of the Effect of Strict Blood Pressure Control and ACE Inhibition on Progression of Chronic Renal Failure in Pediatric Patients (ESCAPE) trial, to become available in mid-2008, will provide pediatric evidence as to whether intensified blood pressure control (targeting to below the 50th percentile of 24-h mean arterial pressure) will confer a renoprotective advantage over a more conventional target (50th to 95th percentile) [17]. The integrity of the normal diurnal blood pressure pattern may play a significant role in renal failure progression in addition to and independent of the absolute blood pressure level. Nondipping, a well known independent cardiovascular risk factor and common characteristic of renoparenchymal hypertension, is associated with more rapid progression of renal failure in adult CKD patients [18, 19], and nondipping is believed to reflect sympathetic hyperactivation in CKD.
Proteinuria
Population-based studies in healthy individuals have demonstrated that proteinuria is a powerful independent risk factor for ESRD and overall mortality [20–22]. Proteinuria is certainly predictive of the renal prognosis in adults with diabetic and nondiabetic kidney disorders [23–25]. Urinary protein excretion was the only baseline variable correlated with GFR decline and progression to ESRD in the Ramipril Efficacy in Nephropathy (REIN) trial [26]. However, the spectrum of underlying renal disorders in children differs markedly from adults. In the pediatric CKD population, congenital renal hypodysplasia with or without urinary tract abnormalities is the leading underlying renal disorder, affecting more than 60% of children. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood first demonstrated in 200 children with CKD stage 3–4 that proteinuria and hypertension are major independent determinants of GFR decline in pediatric nephropathies [9]. The ItalKid Project confirmed that proteinuria predicts renal disease progression in children with renal hypodysplasia [27]. In addition, there is evidence from the ESCAPE trial that residual urinary protein excretion during angiotensin-converting enzyme (ACE) inhibition is quantitatively associated with renal failure progression [28]. Even in children with normal kidney function, persistent proteinuria in the nephrotic range is a risk factor for progressive renal injury, and early detection and treatment of proteinuria is essential [29]. In nonproteinuric children with CRI of nonglomerular origin, the level of protein excretion does not appear to play a major role in CKD progression, which seems to be best predicted by rapid somatic growth, age, and blood pressure [30].
In line with evidence from animal models, multiple clinical studies have confirmed that proteinuria is not only a marker but also an important mechanism of CKD progression. Reduction of proteinuria is associated with a slowing of GFR loss in the long term [25, 31–33]. In the Modification of Diet in Renal Disease (MDRD) trial, for each 1 g/d reduction in proteinuria observed within 4 months of antiproteinuric treatment (i.e. blood pressure reduction and dietary interventions), the subsequent GFR decline was slowed by about 1 ml/min per 1.73 m2 per year [25]. In the REIN study, reduction of proteinuria at 3 months of ACE inhibitor therapy by 1 g/d resulted in slowing down GFR decline by 2 ml/min per year [34]. This degree of proteinuria reduction appears to be associated with the maximal renoprotective effect [35, 36]. Hence, the goal of any antiproteinuric treatment is to reduce proteinuria as much as possible, ideally to <300 mg/m2/day.
Dyslipidemia
Epidemiological studies suggest that dyslipidemia is an independent risk factor not only for cardiovascular disease but also for progressive chronic renal failure [37]. The dyslipidemic pattern differs between the major renal disease entities [38], and the degree of dyslipidemia parallels the degree of renal function impairment. In animal models, hypercholesterolemia clearly accelerates the rate of progression of kidney disease [39]. A high-fat diet causes macrophage infiltration and foam-cell formation in rats, leading to glomerulosclerosis [40]. Dyslipidemia may damage glomerular capillary endothelial and mesangial cells as well as podocytes. Macrophages are the major cell types expressing scavenger receptors; however, mesangial cells, as well, express receptors for low-density lipoprotein (LDL) and oxidized LDL, which upon activation induce mesangial cell proliferation, increase mesangial matrix deposition, and enhance production of chemokines, cytokines, or growth factors and increase oxidative stress.
A relationship between serum cholesterol levels and GFR decline was shown in adult patients with type 1 diabetes and overt nephropathy [41]; patients with a total cholesterol level >7 mmol/L showed an at least three times faster decline in GFR than subjects with lower cholesterol levels. For the general adult population, the Arteriosclerosis Risk in Communities (ARIC) study demonstrated that elevated triglycerides and low high-density lipoprotein (HDL) cholesterol but not LDL cholesterol were associated with an increased risk of renal dysfunction [37]. In a cohort of more than 11,000 middle-aged adults with normal kidney function, hypertriglyceridemia was associated with a 1.68 times increased risk of a 0.4 mg/dl increase in serum creatinine within the 3-year observation period [37].
There are also observations that insulin resistance syndrome may underlie or mediate the association between lipids and loss of renal function. In humans, a strong relationship between metabolic syndrome and the risk for chronic renal disease and microalbuminuria was found in a large nondiabetic general population [42].
Anemia
There is increasing evidence that anemia is an independent risk factor for progression of chronic renal failure. Anemia is a surrogate marker for tissue hypoxia that might perpetuate preexisting renal tissue damage. In patients with reduced nephron number, hypoxia of tubular cells is favored by an increase of oxygen consumption by tubular cells of the remaining nephrons, a decrease in the number of interstitial capillaries [43], and an accumulation of extracellular matrix between interstitial capillaries and tubular cells, which hampers oxygen diffusion. Hypoxia appears to have at least three consequences: It stimulates production of profibrotic molecules such as transforming growth factor (TGF)-β or endothelin-1 by tubular cells, synthesis of extracellular matrix [44], and increased oxygen consumption, which also enhances production of reactive oxygen species (ROS) that may play an additional role in CKD progression.
The renoprotective effect of erythropoietin (EPO) in CKD might be partially related to an attenuation of interstitial fibrosis and tubuloepithelial cell loss by improved oxygen supply and reduced oxidative stress via correction of anemia. In addition, EPO might exert direct protective effects on tubular cells and might help maintain integrity of the interstitial capillary network and stimulate regenerative progenitor cells [45]. The combination of antiapoptotic effects of rhuEPO in renal tissue and stimulation of regenerative progenitor cells may play a role in organ protection.
Oxidative stress
Oxidative stress is defined as an imbalance between ROS and endogenous levels of antioxidant substances. High oxidative stress and low availability of the substrate of nitric oxide (NO) synthase, L-arginine, and an accumulation of endogenous NO inhibitors such as asymmetric dimethylarginine (ADMA) may induce endothelial dysfunction. Several studies reported on increased oxidative stress in CKD patients. This increase appears to correlate with the extent of deterioration of renal function [46]. Increased oxidative stress contributes to the release of proinflammatory and profibrotic molecules and thereby directly enhances the production of extracellular matrix by fibroblastic cells. This may lead to accelerated progression of CKD, hypertension, and cardiovascular complications, and it was suggested that increased oxidative stress in CKD patients may be both a cause and an effect of renal injury. Anemia, hypercholesterolemia, and chronic inflammation are conditions known to further promote oxidative stress.
Nutrition
Nutrition has been considered an important instrument for slowing down renal disease progression in individuals with impaired renal function. In animals, high protein diet results in renal scarring, whereas restriction of dietary protein diminishes or even prevents progressive renal damage [47]. In end-stage renal failure, uremic symptoms can be often diminished and renal replacement therapy postponed by the restriction of dietary protein intake. This led to the hypothesis that restriction of protein intake might also slow down the progression of chronic renal failure in patients with CKD stages 2–4.
Disorders of calcium-phosphate metabolism
Disorders of the calcium-phosphate metabolism are additional risk factors for renal disease progression. On one hand, renal insufficiency causes disturbances of the calcium-phosphate homoeostasis and alters serum lipid profiles. On the other hand, the resulting vasculopathy and hypertension promote progression of chronic renal failure toward end-stage renal disease. Several factors related to disturbed calcium-phosphorus metabolism, such as hyperphosphatemia, hyperparathyroidism, lack of active vitamin D, and possibly the phosphaturic hormone FGF23, may be considered to be—at least to a minor extent—involved in the progression of renal dysfunction [48].
Treatment strategies and their impact on renal disease progression
Several antihypertensive and antiproteinuric therapies have proven effective. Blood pressure control per se has a proteinuria-lowering effect, as demonstrated by three large trials: the MDRD study [25], the Appropriate Blood Pressure Control in Diabetes (ABCD) study [49], and the African American Study of Kidney Disease and Hypertension (AASK) [32]. A low blood pressure goal, i.e. <125/75 mmHg in adults, either reduced proteinuria absolutely by 50% [25] or prevented the two- to threefold increase in proteinuria observed in patients with the more conventional blood pressure goal of 140/90 mmHg [49]. A low blood pressure goal appears to be very well tolerated by the vast majority of patients and in terms of cardiovascular outcomes; the “J curve” phenomenon (a slight increase of cardiovascular events in patients achieving a very low blood pressure level) seems to be confined to aged patients with advanced atherosclerosis.
The goal of any antiproteinuric treatment is to reduce proteinuria as much as possible, ideally to <300 mg/m2/day. This degree of proteinuria reduction appears to be associated with the maximal renoprotective effect [35, 36]. Whereas the different classes of antihypertensive agents are comparable with respect to their blood pressure-lowering efficacy, they differ markedly regarding their effects on proteinuria and CKD progression [32, 35, 50, 51].
Blockade of the renin-angiotensin system
Antagonists of the RAS, such as ACE inhibitors and, more recently, angiotensin II type I receptor blockers (ARB) have become pharmacotherapeutics of first choice in adults [15] as well as children with CKD by virtue of their pharmacological properties. They significantly reduce blood pressure as well as urinary protein excretion and have an excellent safety profile, which is almost indistinguishable from placebo. In adults with essential hypertension, treatment with RAS antagonists has been associated with the best quality of life among all antihypertensive agents.
RAS antagonists suppress the local angiotensin II tone (ACE inhibitor) or action (ARB). This results in a reduction of intraglomerular pressure and proteinuria, diminished local release of cytokines and chemokines, and alleviated activation of inflammatory pathways, with consequently attenuated glomerular hypertrophy and sclerosis, tubulointerstitial inflammation, and fibrosis [8], as well as in a normalized central nervous sympathetic tone by reduced renal afferent nerve stimulation. In addition, oxidative stress is reduced independently of the blood-pressure-lowering effect [52].
In adults with diabetic or nondiabetic kidney disease, several randomized trials demonstrate a more effective reduction of proteinuria, usually by 30–40%, by ACE inhibitors compared with placebo and/or other antihypertensive agents [35]. This is associated with a significantly reduced rate of renal failure progression in the long term [31, 35, 53–61].
Very similar results were obtained in randomized studies comparing ARBs with placebo or conventional antihypertensive agents in diabetic nephropathy [51, 62, 63]. It has been reasoned that ACE inhibitors might have a specific renoprotective advantage by inducing accumulation of vasodilatory and antifibrotic bradykinins; however, the course of GFR was similar in two clinical trials comparing ACE inhibitors and ARB therapy [64, 65]. The size of the advantage of RAS antagonists over other antihypertensive agents is still under debate [66]. The risk of doubling serum creatinine or attaining ESRD is typically reduced by 30–40%, but the superiority of RAS antagonists is related to the prevailing degree of proteinuria [35, 36]. In adults, ACE inhibitors are believed to provide better renoprotection than other antihypertensive agents in patients with proteinuria exceeding 500 mg/day.
However, there is some evidence that previous studies may not have used sufficiently high ACE inhibitor doses to achieve effective RAS suppression at the kidney tissue level and obtain a maximal renoprotective effect. Furthermore, at least a subset of patients appears to develop partial secondary resistance to ACE inhibition (aldosterone escape by compensatory upregulation of ACE-independent angiotensin II production) [67–69]. It is currently an open issue whether such patients would benefit from the primary use of ARBs alone or in combination with ACE inhibitors.
Whereas the maximal antiproteinuric and renoprotective effects of ACE inhibitors and ARBs seem to occur at doses that are supramaximal with respect to maximal antihypertensive action, regulatory authority approval is usually available only for the indication of hypertension in the respective dose range. Therefore, it is generally recommended to administer these drugs, after confirming tolerability in a short run-in period, at their highest approved doses [32, 70].
Limited information is available regarding the efficacy of RAS antagonists for renoprotection in children with CKD. Small uncontrolled studies showed stable renal function in children with sequelae of hemolytic uremic syndrome during long-term ACE inhibitor treatment [71], stable GFR during 2.5 years of losartan treatment in children with proteinuric CKD [72], and attenuated histopathological progression in children with IgA nephropathy receiving combined RAS blockade [73]. Data from the ItalKid Study did not show a significant modification of CKD progression by ACE inhibitor treatment in children with hypodysplastic kidney disease [74] compared with matched untreated subjects. However, the overall CKD progression rate in the total cohort was very slow (< –2 ml/min per 1.73 m2 per year), thereby making the detection of significant differences (ACE inhibitors -1.08 vs. non-ACE inhibitors 1.80; not significant) difficult. In addition, no information was available with respect to the types and dosages of ACE inhibitors used and the prevailing degree of proteinuria.
The ESCAPE trial demonstrated efficient blood pressure and proteinuria reduction by ramipril in almost 400 children with CKD [17]. However, an interim analysis of the 3-year results revealed a gradual rebound of proteinuria after the second treatment year. This effect was dissociated from a persistently good blood pressure control and may limit the long-term renoprotective efficacy of ACE inhibitor monotherapy in pediatric chronic kidney disorders [28].
Aldosterone antagonists also lower blood pressure by RAS suppression. Whereas the use of spironolactone is limited by endocrine side effects, the new aldosterone antagonist, eplerenone, has minimal affinity for progesterone and androgen receptors. Apart from the risk of hyperkalemia, reported side effects are similar to placebo [75]. Combined therapy of eplerenone and an ACE inhibitor increases patient survival in adults with congestive heart failure [76]. However, combination therapy appears limited in CKD patients due to the potentiated risk of hyperkalemia [77, 78].
Aliskiren, a renin-antagonist, blocking the conversion from angiotensinogen to angiotensin I, has been shown to effectively lower blood pressure in animals and humans. The effect on blood pressure is comparable with that of ARBs, and combination therapy of aliskiren and valsartan at maximum recommended doses provided significantly greater reductions in blood pressure than did monotherapy, with a tolerability profile similar to that of aliskiren or valsartan alone [79]. However, there are no data on the effect of aliskiren on renal disease progression in adults nor on its applicability in children available to date.
Calcium-channel blockers
Calcium-channel blockers (CCBs) are safe and can achieve blood pressure goals in patients with CKD. However, CCBs of the dihydropyridine type (amlodipine, nifedipine) fail to reduce progression of chronic renal failure and may even increase proteinuria and promote more rapid CKD progression [33]. Therefore, dihydropyridine CCBs may be acceptable as first-line antihypertensive monotherapy only in nonproteinuric patients and should be avoided unless in combination with RAS antagonists to improve blood pressure control in proteinuric patients [70]. In contrast, nondihydropyridine CCB (diltiazem, verapamil) may have some antiproteinuric effect and may be therefore renoprotective [33]. However, data are not conclusive. An antiproteinuric effect was not observed in type 2 diabetes [80], and amlodipine exerted a renoprotective effect comparable with ACE inhibitors in one study [81].
Beta-blockers
CKD is often a state of overactivation of the sympathetic nervous system, and antiadrenergic drugs play an important role in its management. Beta-blockers are effective in lowering blood pressure in CKD patients by blockade of postsynaptic beta receptors, resulting—among others effects—in a reduction of pulse rate, cardiac output, afterload, and renal renin release. Metoprolol and atenolol were the first antihypertensive agents for which beneficial effects on the decline of renal function in CKD patients were demonstrated [54]. In the AASK trial, the beta-blocker metoprolol had an antiproteinuric effect almost comparable with ramipril, in marked contrast to amlodipine [32]. The antiproteinuric action may be due to sympathicoplegic effects. Newer beta-blockers, such as carvedilol, have even improved antiproteinuric effects compared with atenolol [82, 83].
Combination therapy
Because hypertension is a multifactorial disorder, monotherapy is often not effective in lowering blood pressure or reducing proteinuria to the target range. Treatment with a single antihypertensive agent usually controls blood pressure in less than half of the patients. According to the JNC7 guidelines, subjects with blood pressure >20/10 mmHg above the normal range (i.e. >160/100 mmHg in adults) should be started on combination drug therapy [13]. In CKD patients, RAS antagonists are most commonly combined with a diuretic or with a CCB, whereas their combination with a beta-blocker usually does not exert an additive effect on blood pressure control. Fixed-dose combination preparations are becoming increasingly popular in antihypertensive therapy and may help maximize treatment adherence and efficacy.
Combined RAS blockade using ACE inhibitors and ARB concomitantly has only a minor effect on blood pressure (3–4 mmHg vs. monotherapy) but increases the antiproteinuric effect of ACE inhibitors or ARB monotherapy by 30–40% [64, 84–86]. The prospective randomized Combination Treatment of Angiotensin II Receptor Blocker and Angiotensin-Converting-Enzyme Inhibitor in Non-diabetic Renal Disease (COOPERATE) trial, performed in adults with nondiabetic nephropathies, suggested that combination therapy may also provide better long-term renoprotection [64]. However, in most ACE inhibitor/ARB combination studies, it remained unclear whether maximally efficient single-drug doses were used, a formal prerequisite to demonstrate true synergism of the two drug classes. The few published studies assessed the effects of single-drug dose escalation followed by combined administration of an ACE inhibitor and an ARB at maximally effective single doses found synergistic antiproteinuric effects of combined treatment [87]. Otherwise, a recent study demonstrated additional proteinuria reduction by escalating candesartan exposure to an ultrahigh dose [88]. Notably, raising the dose from 16 to 32 mg daily had no effect on proteinuria, whereas a further increase from 32 to 64 mg was highly effective, suggesting that the dose–response relationship may be nonlinear. Hence, the issue of whether ACE inhibitor and ARB combination therapies have a synergistic renoprotective potential remains an exciting field of clinical research.
Restoration of blood pressure day–night rhythm
In view of the fact that nondipping of nocturnal blood pressure is an independent risk factor for CKD progression, effects of the timing of application of antihypertensive drugs may be an issue of interest. Even using agents with a long half-time and recommended administration on a once-daily basis, evening administration lowers nighttime blood pressure more effectively, increasing the day–night ratio and partially restoring the physiological nocturnal dipping pattern. However, these effects seem to differ for individual antihypertensive drug classes. Whereas bedtime administration of CCBs and ACE inhibitors tends to restore the dipping pattern, evening dosing of beta-blockers has no effect on the circadian blood pressure rhythm [89]. In a substudy of the Heart Outcomes Prevention Evaluation (HOPE) trial, adult patients were evaluated by ambulatory blood pressure monitoring (ABPM) after evening administration of the ACE inhibitor ramipril. A more marked blood pressure reduction during nighttime was observed, compatible with the notion that the beneficial effects of ramipril on cardiovascular morbidity and mortality in the HOPE study were related to the 8% increase in the day–night ratio of blood pressure obtained with evening dosing [90]. Although this association appears firm, it is as yet unclear whether pharmacological restoration of the dipping pattern will result in any long-term clinical benefit for cardiovascular health in general and for renal function preservation in CKD. However, it is of note that the antiproteinuric efficacy of the ARB valsartan was found correlated with the increase in blood pressure day–night ratio induced by evening dosing [91].
Treatment of dyslipidemia
General measures to prevent dyslipidemia in CKD patients include prevention or treatment of malnutrition and correction of metabolic acidosis, hyperparathyroidism, and anemia, all of which may contribute to dyslipidemia [92–94]. In addition, referring to evidence from the general population, therapeutic life-style modification is recommended for adults and children with CKD-related dyslipidemia [95]. However, the lipid-lowering effect of life-style modifications in CKD patients is usually not impressive. Lipid-lowering medical treatment is commonly prescribed in adults with CKD based on the evident benefit of this approach for primary and secondary prevention of cardiovascular disease in the general adult population. Statin therapy is effective in reducing cardiovascular morbidity and mortality in adults with moderate to severe CKD although not in patients with ESRD [96, 97].
With respect to renoprotection, experimental evidence suggests that statins may retard renal disease progression not only by their lipid-lowering but also by lipid-independent pleiotropic effects. Statins inhibit signaling molecules at several points in inflammatory pathways. Anti-inflammatory effects, reduction of oxidative stress, and improved endothelial function are thought to be partially responsible both for CVD risk reduction and improved renal function [98]. Furthermore, there is also evidence for synergistic effects of statins and RAS inhibitors on the prevention of renal disease progression [99]. However, a recent meta-analysis of published clinical trials concluded that the intrinsic antiproteinuric and renoprotective effects of statins, albeit significant, are quantitatively small [100]. To date, no studies have evaluated the usefulness of statins in children with progressive nephropathies.
Erythropoietin treatment
In rats undergoing acute ischemic renal injury, pretreatment with recombinant human erythropoietin (rhuEPO) reduces renal dysfunction and morphological damage. This effect appears to be mainly mediated by a reduction of apoptotic cell death [101]. Darbepoetin, a long-acting EPO analog, ameliorated podocyte injury and decreased proteinuria by maintenance of the podocyte actin cytoskeleton and nephrin expression in puromycin-induced nephrotic rats [102]. Even more interesting than treatment of acute renal failure may be tissue protection in chronic renal failure. In a recently published clinical trial, early initiation of rhuEPO therapy in patients with CKD and mild to moderate anemia significantly slowed down the progression of renal disease and delayed the need for renal replacement therapy [103]. However, other data in patients with more advanced CKD and high-dose rhuEPO treatment revealed no beneficial effect on renal survival [104]. The role of EPO in pediatric CKD progression has not been defined yet.
Nutrition and vitamin D supplementation
For decades, low-protein diets have been prescribed for preventing CKD progression. However, the effects of these diets on CKD progression and delay of ESRD are still inconclusive. One of the largest trials, the MDRD trial, could not prove efficacy of a low-protein diet on progression in nondiabetic kidney disease [105], whereas a recent Cochrane Review [106] found a risk reduction of renal death in patients with protein restriction. Thus, the progression rate was not significantly influenced by protein restriction, whereas renal replacement therapy could be postponed. In children, reducing protein intake to the maximal acceptable lower limit was ineffective to slow down renal disease progression [9, 107]. Further reductions may be effective but not acceptable. Furthermore, therapeutic strategies of protein reduction in children may be conflicting, since a low-protein diet bears the risk of low calorie intake, whereas a high calorie intake is needed for optimal growth. Therefore, at present, it seems not to be justified to prescribe low-protein diets to children early in the course of chronic renal failure.
Some studies in adult CKD patients suggest that dietary phosphorus restriction may stabilize kidney function [108]. However, conclusions in this regard could not be drawn from studies in children [109]. A high calcium–phosphorus product may be detrimental to renal survival by aggravating intrarenal vasculopathy as well as by causing tubulointerstitial calcifications, which may stimulate tubulointerstitial inflammation and fibrosis. In view of these pathophysiological associations, it is currently discussed whether calcium-free phosphate binders may have some renoprotective potential in patients with CKD. Sevelamer may prove beneficial beyond phosphate lowering due to its pleiotropic effects, which include lipid-lowering and anti-inflammatory properties. Treatment with nonhypercalcemic doses of active vitamin D attenuates renal failure progression in chronically uremic rats. This effect may be brought about by the immune modulatory and antifibrotic properties of vitamin D. In addition, a negative endocrine regulation of the RAS through 1,25-Dihydroxyvitamin D3 has been reported [110]. In humans, an antiproteinuric effect of oral paricalcitol was demonstrated in adult CKD patients [111]. These exciting experimental and early clinical findings provide an additional rationale beyond mineral metabolism for close monitoring and early intervention to maintain mineral, vitamin D, and PTH homeostasis in CKD [109].
Conclusion
In conclusion, hypertension and proteinuria are key players in renal disease progression. Therapeutic strategies to prevent progression should comprise blood pressure control and lowering of proteinuria. RAS antagonists preserve kidney function, not only by lowering blood pressure but also through antiproteinuric and antifibrotic properties. Other factors contributing to renal disease progression in a multifactorial manner include anemia, dyslipidemia, and disorders of mineral metabolism, and measures to preserve renal function should therefore also comprise the maintenance of hemoglobin and serum lipid and calcium-phosphorus ion product levels in the normal range.
Questions
(Answers appear following the reference list.)
Progression of chronic renal failure toward ESRD is common in children with CKD and:
Declines linearlyNonlinearly and often characterized by a sharp decline in renal function during pubertyInevitablyStrongly, depending upon the underlying renal diseaseResults in ESRD in less than 50% of patients at age 20 years
The most important factors influencing renal disease progression are:
Age at onset of chronic renal failure, gender, and underlying renal diseaseResidual renal function and blood pressureBlood pressure and proteinuriaRapid somatic growth during puberty, and age
The different antihypertensive drug classes are comparable regarding:
Antihypertensive efficacyAntiproteinuric efficacySide effects and safety profileEffect on CKD progression
The antihypertensive and antiproteinuric effects of RAS antagonists are:
Strictly dose dependentBasically mediated by reduction of systemic hypertensionMediated by bradykinin releaseAlso mediated by antifibrotic and vasodilatory effectsDue to restoration of the often disturbed day–night blood pressure pattern (dipping) in CKD patients
Angiotensin receptor antagonists in children:
Are approved for the indication proteinuria and hypertensionDo not exert additional antiproteinuric effect when combined with ACE inhibitorsShould be given at the highest approved dose for maximal antiproteinuric effectShould not be combined with ACE inhibitorsHave the same side effect profile as ACE inhibitors
Decline of GFR:
Is independent of serum cholesterol levelCan be reversed by dietary protein restrictionCan be reversed by the use of statins in younger childrenMay be accelerated by prescription of erythropoietinMay be retarded by the lipid-independent pleiotropic anti-inflammatory effects of statins

chronic kidney disease
progression
hypertension
proteinuria
prevention
children


Preconception Care and Women with or at Risk for Diabetes: Implications for Community Intervention

Introduction
Diabetes is a chronic and, often, disabling disease, which has reached epidemic proportions in America and worldwide. When a person has diabetes their body cannot produce or properly use insulin – a hormone needed to convert sugar, starches, and other foods into energy. This leads to high levels of sugar in the bloodstream, which can result in serious complications and premature death, if diabetes is not controlled. There are three main types of diabetes [1]. In type 1 diabetes, the body does not produce any insulin; daily injections of insulin are required for survival. Typically beginning in childhood or young adulthood, type 1 accounts for approximately 5–10% of all cases of diabetes. Autoimmune, genetic, and environmental factors influence type 1 diabetes risk [1].
In type 2 diabetes, which accounts for 90–95% of all diagnosed cases, the body’s cells do not secrete or use insulin adequately. Risk factors for type 2 diabetes include obesity, physical inactivity, family history of diabetes, and history of gestational diabetes (GDM). GDM, defined as diabetes that develops or is first recognized during pregnancy, is the third type of diabetes. Risk factors for GDM include obesity, pregnancy weight gain, age, and family history of diabetes [1–5].
In the weeks after pregnancy, 5–10% of women who had GDM are diagnosed with type 2 diabetes [1]. Subsequently, women with a history of GDM have a 20–50% chance of developing type 2 diabetes five to 10 years after the index pregnancy, with a lifetime risk near 80% [1, 4–7]. Both type 2 diabetes and GDM are diagnosed more frequently in African Americans, Hispanic/Latino Americans, and American Indians compared to non-Hispanic whites [1].
National Health and Nutrition Examination Survey III data for nonpregnant women aged 20–49 years indicate that during the period 1988–1994, 27.6% of Mexican American women and 22.4% of African American women of childbearing age had diabetes or impaired glucose tolerance, in comparison to 10.1% of non-Hispanic white women [8]. Approximately one third of women of childbearing age have undiagnosed diabetes [9]. Additionally, between 3 and 8% of pregnant women have gestational diabetes (GDM) [2, 3, 9, 10].
A study of women with pregestational diabetes (type 1 and type 2) found that 60% of the women had suboptimal glucose control before conception [11]. Women who had a poor outcome in a previous pregnancy were more likely to enter a subsequent pregnancy with poor glucose control then were women with good outcomes [11].
Diabetes during pregnancy is associated with increased risk for miscarriages, stillbirth, macrosomia and obstetric complications [12–16], intrauterine developmental and growth abnormalities, birth and neonatal complications, and later development of obesity and type 2 diabetes [3, 10–15, 17, 18]. Treatment to normalize maternal blood glucose prior to conception and throughout pregnancy is necessary to reduce the likelihood of maternal, obstetric, and infant complications [12–16]. While treatment and monitoring are common practice during prenatal care, many women and their families may not know about the importance or even the existence of preconception care interventions for women of childbearing age who have or are at risk for diabetes.
Greater awareness of the potential contribution of preconception care to diabetes prevention and control may help reduce the devastating impact of diabetes and its complications on the lives of women and their families. The objectives of this paper are to: 1) review barriers that can impede a woman’s ability to receive preconception care, and 2) recommend novel interventions to reach reproductive-aged women with or at risk for diabetes.
Prevention trials have demonstrated that type 2 diabetes and its complications can be prevented or at least delayed through healthful dietary practices, regular moderate physical activity, weight loss, and medication use [19, 20]. The clinical practice guidelines of the American College of Obstetrics and Gynecologists [21] and the American Diabetes Association [15] suggest that preconception care is an ideal primary prevention opportunity during which modifiable risk factors can be identified and reduced.
Preconception care may be defined as a window of opportunity for comprehensive health care to: 1) identify conditions that may have detrimental effects on the mother or fetus, and 2) recommend necessary medical, behavioral, and educational interventions for increasing the likelihood of achieving optimal pregnancy outcomes. A major goal of preconception care for women with diabetes is to reduce the risk of diabetes-related complications by obtaining the lowest possible glycated hemoglobin (HbA1C [a measure of glucose control]) without significant episodes of hypoglycemia [15].
Women with diabetes who receive preconception care obtain intensive treatment to assist them with developing diabetes self-management skills, and obtaining nutritional, physical activity, and medical support needed to promote optimal glucose control and health status before becoming pregnant. During interconception periods, diabetes education, postpartum glucose testing, and ongoing support to reduce postpartum weight retention and maintain a healthy weight and glucose control may also help reduce risk factors for subsequent morbidity [3, 19, 24].
Previous studies have found that women with diabetes who received preconception care demonstrated improved glucose control during pregnancy, their offspring had fewer congenital anomalies, and the women’s hospital stays were shorter in comparison with women who did not receive preconception care [25–27]. Although these findings are very positive and they support the importance of preconception care for women with diabetes, other studies have indicated that many women who could benefit from preconception care are not receiving this intervention [25, 28].
Barriers to receiving preconception care
At every health care encounter, a woman of childbearing age should be informed about the importance of preconception care and, if she has diabetes, the steps required to maintain appropriate blood glucose control [15, 28, 29]. Unfortunately, however, reaching women who may be in need of preconception care has proven to be difficult, with only a quarter to a third of women with diagnosed diabetes receiving this care [25, 28].
There are many barriers to providing and/or receiving preconception care. Among them are: 1) Many women with diabetes do not know that they have the illness and, thus, they are undiagnosed [1], 2) Approximately 50% of all pregnancies are unintended [28, 30, 31], 3) Even among women planning to become pregnant, lack of health insurance or a regular care primary care or obstetric provider reduces contact with the health care system [32–34], 4) Many primary care practices do not have or use established guidelines for providing preconception care, or identifying women with risk factors [32, 34], 5) Some women and health care providers may not know about about the existence or the importance of preconception care or do not see it as a high priority [15, 32, 34, 35], and 6) Women with incomplete health care coverage, lack of child care or transportation, geographic isolation, distrust of health care providers or other social and economic challenges have additional barriers to receiving preconception care [15, 32, 34–36].
Women are more likely to receive preconception care if they are married or in a stable relationship, are comparatively older, are nonsmokers, are non-Hispanic whites, are more educated, have annual incomes above $20,000, have private medical insurance, and have a positive bond with their prepregnancy care provider [25, 26, 28, 33, 35]. Younger women with diabetes and those who are single, have low income, or are less educated may be particularly vulnerable to unplanned pregnancies, which greatly reduces the chances that they will receive any form of diabetes-related preconception counseling [16, 32, 36].
Interventions to reach women of childbearing age with diabetes
Since the establishment of preconception care programs, health care centers have employed numerous marketing approaches geared toward physicians, and for patients in need of preconception care intervention. Janz et al. [28] identified marketing approaches which were employed in five Michigan medical centers to encourage the use of preconception care programs in treating high-risk women. These included journal advertisements, newsletters, brochures, flyers, posters, and patient education programs. However, the results of these outreach efforts were “generally disappointing [28].”
In a Maine study, a diabetes registry was developed to promote the availability of preconception care programs [27]. In addition, health care providers formed a network to enhance the care of pregnant women using locally developed patient care guidelines. From 1987–1990, there were a total of 185 pregnancies in women with diagnosed diabetes. Of the total pregnancies, only 34% of these women received preconception care. These findings support those of Janz et al. [28], which demonstrate that very few women with diabetes are receiving preconception care. Additional innovative strategies are needed to increase awareness of the availability of preconception care.
Novel interventions to reach reproductive-aged women with or at risk of diabetes
The National Public Health Initiative on Diabetes and Women’s Health (cosponsored by the American Diabetes Association, the American Public Health Association, the Association of State and Territorial Health Officials, and the Centers for Disease Control and Prevention) is a partnership devoted to increasing public and provider awareness of the importance of interventions such as preconception care in the prevention and management of diabetes for women [37, 38]. Using a life stage approach, the Initiative’s cosponsors and partners developed and disseminated a national action plan, The National Agenda for Public Health Action [39], to identify 10 priority action steps to improve the lives of women with or at risk of diabetes, from adolescence to the older years (see Table 1). The National Agenda serves as a blueprint to guide the nation in implementing strategies related to diabetes and women’s health. Given the devastating effects that diabetes can have on women’s health, the National Agenda recognizes and identifies the need for effective interventions, such as quality health care and programs, public health research and surveillance, and specific policies to address the burden of this chronic illness on the lives of women and their families [37, 38].Table 1The National Agenda’s 10 priority recommendations for action1. Encourage and support diabetes prevention and control programs   in state health departments to develop prevention programs for all   women and establish efficient links for women at risk for type 2   diabetes2. Expand community-based health promotion, education, activities,   and incentives for all ages in a wide variety of settings—schools,   workplaces, senior centers, churches, and other locations where   women live, learn, work, and play3. Strengthen advocacy on behalf of women with or at risk for   diabetes4. Fortify community programs for women with sufficient training,   tools, and materials5. Expand population-based surveillance to monitor and understand: a. Variations in the distribution of diagnosed and undiagnosed   diabetes b. The factors—cultural, racial, ethnic, geographic, demographic,   socioeconomic, and genetic factors—that influence the risk for   diabetes and complications among women at all life stages6. Educate community leaders about diabetes and its management   and about the value of healthy environments7. Encourage healthcare providers to promote risk assessment,   quality care, and self-management for diabetes and it   complications in their practice settings8. Ensure access to trained healthcare providers who offer quality   services consistent with established healthcare guidelines9. Encourage healthcare coverage and incentives for recommended   diabetes prevention management practices by: a. Promoting partnerships between insurers and workplaces or   labor communities and encouraging employers and employees to   discuss needed diabetes benefits in offered health care packages b. Working with health insurers and policymakers to expand   coverage and reimbursement policies to include prevention   services for women throughout their lives10. Conduct public health research to further our knowledge about   the epidemiological, socioenvironmental, behavioral,   translational, and biomedical factors that influence diabetes and   women’s health
Several of the National Agenda’s recommendations support the need for preconception care and follow-up care for women with or at risk for diabetes through community intervention efforts and policy development. These recommendations identify the need to: 1) expand our outreach to communities where women live, learn, work, and play by offering health promotion, education, activities and other incentives; 2) provide sufficient funding, tools, training, and materials to fortify community programs that focus on women’s health needs; 3) educate community members and leaders about diabetes prevention and control and the roles that they can play in promoting healthy environments; 4) encourage healthcare providers to promote risk assessment and quality care for women, including preconception care; and 5) conduct public health research to better understand multiple factors that influence diabetes and women’s health.
Community intervention and the involvement of community health workers
One of the community-focused strategies from the National Agenda, is the need to “identify and develop links with community agencies that interface with women at greatest risk for diabetes, including organizations in the non-health related sectors [39].” Given that preconception care programs have had difficulty reaching high-risk women, nontraditional approaches to outreach and preconception care should be explored [32, 36].
Community health organizations and community health workers (CHWs) appreciate and understand the social, political, environmental, and cultural factors that affect individuals within their own neighborhoods and have the potential to influence the consumers’ relationship with the health care system [33]. They serve as “bridges”—connecting community members who have traditionally lacked access to the appropriate services needed [40, 41]. The Institute of Medicine (IOM) recommends supporting the use of CHWs. The IOM identifies CHWs as an integral component of the health care system that help to address racial and ethnic disparities in access to care [41, 42].
Many community-based projects funded by the Centers for Disease Control and Prevention have included CHWs in community-based programs aimed at health intervention for people with diabetes [43]. Programs employing CHWs have reported that they improve access and health outcomes for persons with chronic diseases, including diabetes, particularly in minority and underserved populations [44–48]. Although, no empirical studies have been published that assess the effectiveness or impact of CHWs in providing preconception care services for women with diabetes, CHWs have also played an important role in maternal and child health care. Thus, studying the methods for, and outcomes of an expanded role for CHWs with preconception outreach and education for women with, or at risk for diabetes, is recommended.
Community health workers could play a vital role in linking women to preconception care services in the following manner: 1) increasing women’s awareness about the importance of preconception care programs, 2) providing culturally and linguistically appropriate diabetes-related health information and education, 3) reminding women about scheduled health provider visits, 4) providing a communication bridge linking women and providers in terms of patients’ needs and providers’ recommendations.
The prevalence of diabetes among women of childbearing age is rising, particularly in communities with ethnic minorities and low resources [4, 8]. There is a strong relationship between these conditions and adverse maternal, child and subsequent adult health outcomes. Assessing the processes and effects of preconception care interventions implemented by providers and CHWs, especially in those communities with varied ethnic and socioeconomic backgrounds, is a critical first step toward identifying and promoting effective preconception care [18, 49–55].