Signaling
Authors: Claire C. Sharpe, Bruce M. Hendry
Journal: Journal of the American Society of Nephrology (2003)

Control of glomerular blood flow is complex, crucial in renal pathophysiology, and an attractive target for therapeutic intervention. In this issue of JASN, Cavarape et al. (1) observe that inhibition of Rho-kinase leads to a decrease in basal tone of the glomerular afferent and efferent arterioles associated with a rise in glomerular blood flow in the split hydronephrotic rat kidney model. Moreover, Rho-kinase inhibition also antagonized the actions of three distinct vasoconstrictors: an endothelin B receptor agonist, a guanylyl cyclase inhibitor, and an adenosine A1 receptor agonist. This represents one of the first applications in the kidney of the seminal demonstration by Uehata et al. (2) that Rho GTPases play a vital role in smooth muscle contraction in vivo and brings attention to the very rapid advances that are being made in the whole field of cell signaling and renal disease. Cells rely on complex networks of signaling pathways to translate extracellular stimuli into cellular events such as mechanical responses, gene transcription, protein translation, and cell proliferation. Most signaling cascades are activated by the binding of a ligand to a cell surface receptor. Certain immunosuppressive therapeutics (e.g., cyclosporin, tacrolimus) target the signaling pathways downstream from the T cell receptor. In other areas of nephrology, selective targeting of signaling is not yet a reality, but the pathways of interest include TGF-β-activated signals and pathways activated through receptor tyrosine kinases (RTK) via Ras GTPases. The justification for this focus is the considerable body of evidence implicating both TGF-β and RTK ligands (such as platelet-derived growth factor [PDGF], fibroblast growth factor, etc.) in renal pathology (3–5). The extracellular ligand most often implicated in the pathogenesis of renal disease is TGF-β (6–8). This cytokine signals to the cell nucleus through two co-dependent receptors (TGF-β receptors I and II) and the Smad family of transcription factors. Activation of Smad pathways has been demonstrated to play a role in diabetic nephropathy (9), and overexpression of the inhibitory Smad 7 can ameliorate the effects of TGF-β on mesangial cells (10). One of the proteins upregulated by TGF-β is connective tissue growth factor (CTGF), which has been held responsible for many of the pro-fibrotic effects of TGF-β. Some insight has recently been gained into the signaling pathways affected by this molecule thereby opening the way for potential therapeutic intervention (11). The costs of developing a new class of therapeutic agents may be prohibitive, particularly where the target patient group is limited as in the case of renal disease. For this reason, the Ras and Rho signaling pathways are of particular interest because they also offer therapeutic potential in cancer, vascular disease, and hypertension. Strategies designed to target these molecules have been successful in animal models, and some are currently in clinical trials (12). Ras and Rho GTPases act as molecular switches directing upstream signals to a plethora of downstream effector pathways. In their quiescent state, these GTPases are bound to GDP. When activated, the GDP molecule dissociates with the help of specific guanine nucleotide exchange factors (GNEF), resulting in binding of GTP. This confers a conformational change on the molecule and allows it to bind to downstream effectors, thereby turning on the switch. The activated Ras-GTP switches off, returning to the Ras-GDP form by hydrolyzing the GTP molecule to GDP with the release of the inorganic phosphate group. This process is accelerated by GTPase activating proteins (GAP) (13). The best-studied family in this group is the classical Ras family itself, as mutated oncogenic Ras is associated with uncontrolled cell proliferation and is implicated in 20 to 30% of human cancer (14). The role of wild-type Ras in disease processes is now becoming a focus of research, and there is evidence to suggest that Ras isoforms may be potential targets in the control of renal fibroblast and mesangial cell proliferation (5). Cell proliferation is a key process in the early stages of fibrosis as interstitial fibroblasts, which are sparse in the normal kidney, rapidly increase in number. Additionally, many progressive renal diseases are associated with glomerular epithelial and mesangial cell proliferation. More recently, the Rho family has been highlighted in disease pathogenesis. Rho family proteins share about 30% of their amino acid identity with Ras and are members of signaling pathways that regulate the organization of the actin cytoskeleton. At least eleven members of this family have been identified, including Rho (A–D, E, and G), Rac and Cdc42 which share 50 to 90% homology (15). Most of the current knowledge of the Rho subgroup specifically has been gained through experiments on RhoA, although RhoA, RhoB, and RhoC share the same amino acid sequence in their effector regions and will be collectively referred to here as Rho. Rho has a number of downstream effector molecules so far identified including Rho-kinase (also known as ROKα/ROCKII and a highly homologous isoform ROKβ/ROCKI), mDia, Rhophilin, Rhotekin, citron, and protein-kinase N. Rho-kinase is a serine/threonine protein kinase, which influences both smooth muscle contraction and stress fiber formation. The other effectors are less well characterized, but they influence actin polymerization, stress fiber formation, cell migration, and cytokinesis (16). Rho has recently been implicated in the etiology of renal fibrosis, having been shown to be necessary for TGF-β-induced upregulation of CTGF (17). Rho-dependent pathways are known to be activated by a number of profibrogenic growth factors, such as angiotensin II, PDGF, and endothelin-1 (18–20), and a recent study by Nagatoya et al. has demonstrated that the Rho-kinase inhibitor Y-27632 reduces tubulointerstitial fibrosis in a mouse model of unilateral ureteral obstruction (UUO) (21). Rho may affect the progression of renal fibrosis by numerous different mechanisms. It is known to be involved in both the control of cell proliferation and apoptosis and can activate serum response factor (SRF) and nuclear factor (NF)-κβ, as well as transcription factors dependent on the activity of the stress signal pathways involving c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) (22,23). Evidence for a role of RhoA in cell proliferation comes mainly from studies inhibiting its posttranslational modification. Ras superfamily proteins are adapted after translation by a series of well-defined modifications to facilitate localization to the plasma membrane, a process necessary for signaling function. This modification involves the addition of a prenyl moiety, which takes the form of either a farnesyl group (C15) or a geranylgeranyl group (C20). Both these molecules are byproducts of the cholesterol synthesis pathway, and their production is therefore inhibited by the HMG CoA reductase inhibitors (statins). RhoA is specifically geranylgeranylated, and this process can be blocked by inhibitors of the enzyme geranylgeranyltransferase I (GGTI). Studies that have used these compounds or the statins have demonstrated that Rho activity is necessary to promote cell proliferation and protect against apoptosis (24–26). Although Nagatoya et al. (21) were unable to demonstrate an antiproliferative effect of Y-27632 on mouse renal fibroblasts in vitro, they did show an inhibition of fibroblast activation both in vitro and in vivo. Interestingly, they were also able to demonstrate inhibition of macrophage infiltration by the Rho-kinase inhibitor in the interstitium of UUO kidneys. Yokota et al. (27), however, who used a statin to inhibit Rho in a mouse renal ischemia-reperfusion model, did not show a difference in macrophage infiltration but were able to demonstrate protection of renal function and reduced tubular injury. The effects of Y-27632 on renal vascular smooth muscle tone as demonstrated by Cavarape et al. is an additional and novel mechanism by which Rho inhibition can protect against renal injury. Activation of smooth muscle by different agonists or by electrical depolarization results in a rapid rise in intracellular calcium concentration. This is achieved by calcium entry through voltage-gated calcium channels and by release of calcium from intracellular stores in the sarcoplasmic reticulum. The key event in the regulation of smooth muscle contraction is the phosphorylation and subsequent dephosphorylation of the regulatory light chains of myosin II (MLC). Phosphorylation is dependent upon the enzyme myosin light chain kinase (MLCK), which is activated by intracellular calcium and calmodulin, and dephosphorylation is catalyzed by type I myosin light chain phosphatase (MLCP). The force of smooth muscle contraction is dependent on the balance of activities of MLCK and MLCP (28). When activated by Rho, Rho-kinase phosphorylates a regulatory subunit on MLCP inhibiting its function, thereby allowing more MLC to remain in the phosphorylated form. This has the effect of increasing the cell's calcium sensitivity and increases contraction (29). In addition to its effects on smooth muscle contraction, Rho-kinase inhibition has also been shown to disrupt stress fiber formation in cells in tissue culture through its inhibition of the Rho-kinase/LIM -kinase/cofilin pathway (30). Interestingly, this did not occur in the model studied by Cavarape et al., implying that other factors protect against this effect in vivo. One possible mechanism is that the different Rho isoforms themselves may regulate a balance between stress fiber formation and actin depolymerization via differential activation of their numerous downstream effector pathways. The effects of Rho-kinase inhibition on vascular smooth muscle tone have been well studied in other physiologic systems. Uehata et al. (2) demonstrated that Y-27632 reduced BP in hypertensive rats but did not affect normotensive animals. Fasudil, another Rho-kinase inhibitor, reduces coronary artery spasm in a canine model of angina pectoris (31) and cerebral vasospasm after subarachnoid hemorrhage in humans (32). More recently, Rho-kinase activity has been demonstrated to be involved in the pathophysiology of erectile dysfunction (33). The microcirculation of the kidney, however, is a unique and complex system involving autoregulation of GFR and renal blood flow (RBF) across a wide range of systemic BP. This is achieved by two mechanisms (myogenic and transglomerular feedback), which the kidney employs to regulate contraction of both the afferent and efferent arterioles (34). The findings by Cavarape et al. are therefore particularly interesting because they are able to demonstrate a vasodilatory effect of Rho-kinase inhibition in both preglomerular and postglomerular systems with an associated increase in glomerular blood flow and hence GFR. Increasing GFR and RBF in this manner should have obvious beneficial effects in many models of renal injury. Interestingly, previous studies using statins have demonstrated a protective effect of these molecules on chronic allograft nephropathy (35). The pathophysiology of this disease process is characterized by recurrent inflammation and endothelial injury, microvascular obliteration and the histologic effects of chronic ischemia. Although the precise mechanism by which the HMG CoA reductase inhibitors protect against this kind of injury is unknown, it now seems possible that they do so, in part, by inhibiting Rho prenylation and hence activation of Rho-kinase and vasoconstriction as well as targeting cell proliferation and macrophage infiltration. Novel targets for renal therapy are needed to improve the precision of treatments in nephrology. Many of the drugs that are currently in use, such as angiotensin-converting enzyme inhibitors and HMG CoA reductase inhibitors, are proving to be pleiotropic in action and target pivotal cell-signaling networks as well as the pathways they were initially designed to influence. In addition, potential new drugs are being developed, such as the Rho-kinase inhibitors, Ras-Raf pathway inhibitors, and drugs that inhibit the enzymes responsible for prenylation, which may prove to be beneficial both in renal disease and in vascular disease and cancer (12,36,37).