Matrix metalloproteinase-9 regulates afferent arteriolar remodeling and function in hypertension-induced kidney disease.

This study tested if matrix metalloproteinase (MMP)-9 promoted microvascular pathology that initiates hypertensive (HT) kidney disease in salt-sensitive (SS) Dahl rats. SS rats lacking Mmp9 (Mmp9-/-) and littermate control SS rats were studied after one week on a normotensive 0.3% sodium chloride (Pre-HT SS and Pre-HT Mmp9-/-) or a hypertension-inducing diet containing 4.0% sodium chloride (HT SS and HT Mmp9-/-). Telemetry-monitored blood pressure of both the HT SS and HT Mmp9-/- rats increased and did not differ. Kidney microvessel transforming growth factor-beta 1 (Tgfb1) mRNA did not differ between Pre-HT SS and Pre-HT Mmp9-/- rats, but with hypertension and expression of Mmp9 and Tgfb1 increased in HT SS rats, along with phospho-Smad2 labeling of nuclei of vascular smooth muscle cells, and with peri-arteriolar fibronectin deposition. Loss of MMP-9 prevented hypertension-induced phenotypic transformation of microvascular smooth muscle cells and the expected increased microvascular expression of pro-inflammatory molecules. Loss of MMP-9 in vascular smooth muscle cells in vitro prevented cyclic strain-induced production of active TGF-ß1 and phospho-Smad2/3 stimulation. Afferent arteriolar autoregulation was impaired in HT SS rats but not in HT Mmp9-/- rats or the HT SS rats treated with doxycycline, an MMP inhibitor. HT SS but not HT Mmp9-/- rats showed decreased glomerular Wilms Tumor 1 protein-positive cells (a marker of podocytes) along with increased urinary podocin and nephrin mRNA excretion, all indicative of glomerular damage. Thus, our findings support an active role for MMP-9 in a hypertension-induced kidney microvascular remodeling process that promotes glomerular epithelial cell injury in SS rats.

Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

Prevention of phenotype switching of vascular smooth muscle cells is an important determinant of normal vascular physiology. Hydrogen peroxide (H2O2) promotes osteogenic differentiation of vascular smooth muscle cells through expression of Runt related transcription factor 2 (Runx2). In this study, an increase in dietary NaCl increased endothelial H2O2 generation through NOX4, a NAD(P)H oxidase. The production of H2O2 was sufficient to increase Runx2, osteopontin and osteocalcin in adjacent vascular smooth muscle cells from control littermate mice but was inhibited in mice lacking endothelial Nox4. A vascular smooth muscle cell culture model confirmed the direct involvement of the activation of protein kinase B (Akt) with inactivation of FoxO1 and FoxO3a observed in the control mice on the high NaCl diet. The present study also showed a reduction of catalase activity in aortas during high NaCl intake. The findings demonstrated an interesting cell-cell communication in the vascular wall that was initiated with H2O2 production by endothelium and was regulated by dietary NaCl intake. A better understanding of how dietary salt intake alters vascular biology may improve treatment of vascular disease that involves activation of Runx2.

Cellular antioxidant mechanisms control immunoglobulin light chain-mediated proximal tubule injury.

A major cause of morbidity and mortality in multiple myeloma is kidney injury from overproduction of monoclonal immunoglobulin light chains (FLC). FLC can induce damage through the production of hydrogen peroxide, which activates pro-inflammatory and pro-apoptotic pathways. The present study focused on catalase, a highly conserved antioxidant enzyme that degrades hydrogen peroxide. Initial findings were that FLC increased hydrogen peroxide levels but also decreased catalase levels and activity in proximal tubule epithelium. In order to clarify, we showed that the phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited FLC-induced Akt-mediated deactivation of Forkhead box O class 3a (FoxO3a) and increased catalase activity in proximal tubule cells. Augmented catalase activity decreased FLC-mediated production of hydrogen peroxide as well as the associated increase in High Mobility Group Box 1 (HMGB1) protein release and caspase-3 activity. Coincubation of cells with FLC and an allosteric activator of Sirtuin 1 (SIRT1) was also sufficient to increase catalase activity and promote similar cytoprotective effects. Our studies confirmed that the mechanism of downregulation of catalase by FLC involved deactivation of FoxO3a and inhibition of SIRT1. Mechanistic understanding of catalase regulation allows for future treatments that target pathways that increase catalase in the setting of proximal tubule injury from FLC.

Renoprotective effect of Stat1 deletion in murine aristolochic acid nephropathy.

Injured tubule epithelium stimulates a profibrotic milieu that accelerates loss of function in chronic kidney disease (CKD). This study tested the role of signal transducer and activator of transcription 1 (STAT1) in the progressive loss of kidney function in aristolochic acid (AA) nephropathy, a model of CKD. Mean serum creatinine concentration increased in wild-type (WT) littermates treated with AA, whereas Stat1-/- mice were protected. Focal increases in the apical expression of kidney injury molecule (KIM)-1 were observed in the proximal tubules of WT mice with AA treatment but were absent in Stat1-/- mice in the treatment group as well as in both control groups. A composite injury score, an indicator of proximal tubule injury, was reduced in Stat1-/- mice treated with AA. Increased expression of integrin-ß6 and phosphorylated Smad2/3 in proximal tubules as well as interstitial collagen and fibronectin were observed in WT mice following AA treatment but were all decreased in AA-treated Stat1-/- mice. The data indicated that STAT1 activation facilitated the development of progressive kidney injury and interstitial fibrosis in AA nephropathy.

Free light chains injure proximal tubule cells through the STAT1/HMGB1/TLR axis.

Free light chains (FLCs) induce inflammatory pathways in proximal tubule cells (PTCs). The role of TLRs in these responses is unknown. Here we present findings on the role of TLRs in FLC-induced PTC injury. We exposed human kidney PTC cultures to κ and λ FLCs and used cell supernatants and pellets for ELISA and gene expression studies. We also analyzed tissues from Stat1-/- and littermate control mice treated with daily i.p. injections of a κ FLC for 10 days. FLCs increased the expression of TLR2, TLR4, and TLR6 via HMGB1, a damage-associated molecular pattern. Countering TLR2, TLR4, and TLR6 through GIT-27 or specific TLR siRNAs reduced downstream cytokine responses. Blocking HMGB1 through siRNA or pharmacologic inhibition, or via STAT1 inhibition, reduced FLC-induced TLR2, TLR4, and TLR6 expression. Blocking endocytosis of FLCs through silencing of megalin/cubilin, with bafilomycin A1 or hypertonic sucrose, attenuated FLC-induced cytokine responses in PTCs. IHC showed decreased TLR4 and TLR6 expression in kidney sections from Stat1-/- mice compared with their littermate controls. PTCs exposed to FLCs released HMGB1, which induced expression of TLR2, TLR4, and TLR6 and downstream inflammation. Blocking FLCs' endocytosis, Stat1 knockdown, HMGB1 inhibition, and TLR knockdown each rescued PTCs from FLC-induced injury.

Avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) plays a role in renal microvascular pathophysiology in the Dahl salt-sensitive rat.

Prior studies reported that haploinsufficiency of the transcription factor ETS-1 is renoprotective in Dahl salt-sensitive rats, but the mechanism is unclear. Here, we tested whether ETS-1 is involved in hypertension-induced renal microvascular pathology and autoregulatory impairment. Hypertension was induced in salt-sensitive rats and salt-sensitive rats that are heterozygous with 1 wild-type or reference allele of Ets1 (SSEts1+/-) by feeding a diet containing 4% sodium chloride for 1 week. Increases in blood pressure did not differ. However, phosphorylated ETS-1 increased in afferent arterioles of hypertensive salt-sensitive rats, but not in hypertensive SSEts1+/- rats. Afferent arterioles of hypertensive salt-sensitive rats showed increased monocyte chemotactic protein-1 expression and infiltration of CD68 positive monocytes/macrophages. Isolated kidney microvessels showed increased mRNA expression of vascular cell adhesion molecule, intercellular adhesion molecule, P-selectin, fibronectin, transforming growth factor-ß, and collagen I in hypertensive salt-sensitive rats compared with hypertensive SSEts1+/- rats. Using the in vitro blood-perfused juxtamedullary nephron preparation, pressure-mediated afferent arteriolar responses were significantly blunted in hypertensive salt-sensitive rats compared to hypertensive SSEts1+/- rats. Over a 65-170 mm Hg pressure range tested baseline arteriolar diameters averaged 15.1 µm and remained between 107% and 89% of baseline diameter in hypertensive salt-sensitive rats vs. 114% and 73% in hypertensive SSEts1+/- rats (significantly different). Thus, ETS-1 participates in renal arteriolar pathology and autoregulation and thereby is involved in hypertension-mediated kidney injury in salt-sensitive rats.

Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury.

Because of the less-than-robust response to therapy and impact on choice of optimal chemotherapy and prognosis, chronic kidney disease has drawn attention in the treatment of multiple myeloma, a malignant hematologic disorder that can produce significant amounts of monoclonal immunoglobulin free light chains (FLCs). These low-molecular-weight proteins are relatively freely filtered through the glomerulus and are reabsorbed by the proximal tubule. The present study demonstrated that during the process of metabolism of immunoglobulin FLCs, ROS activated the STAT1 pathway in proximal tubule epithelium. STAT1 activation served as the seminal signaling molecule that produced the proinflammatory molecule IL-1ß, as well as the profibrotic agent TGF-ß by this portion of the nephron. These effects occurred in vivo and were produced specifically by the generation of hydrogen peroxide by the VL domain of the light chain. To the extent that the experiments reflect the human condition, these studies offer insights into the pathogenesis of progressive kidney failure in the setting of lymphoproliferative disorders, such as multiple myeloma, that feature increased circulating levels of monoclonal immunoglobulin fragments that require metabolism by the kidney.

Transforming growth factor-ß mediates endothelial dysfunction in rats during high salt intake.

Endothelial dysfunction has been shown to be predictive of subsequent cardiovascular events and death. Through a mechanism that is incompletely understood, increased dietary salt intake promotes endothelial dysfunction in healthy, salt-resistant humans. The present study tested the hypothesis that dietary salt-induced transforming growth factor (TGF)-ß promoted endothelial dysfunction and salt-dependent changes in blood pressure (BP). Sprague-Dawley rats that received diets containing 0.3% NaCl [low salt (LS)] or 8.0% NaCl [high salt (HS)] were treated with vehicle or SB-525334, a specific inhibitor of TGF-ß receptor I/activin receptor-like kinase 5, beginning on day 5. BP was monitored using radiotelemetry in four groups of rats (LS, LS + SB-525334, HS, and HS + SB-525334) for up to 14 days. By day 14 of the study, mean daytime systolic BP and mean pulse pressure of the HS group treated with vehicle was greater than those in the other three groups; mean daytime systolic BP and pulse pressure of the HS + SB-525334 group did not differ from the LS and LS + SB-525334-treated groups. Whereas mean systolic BP, mean diastolic BP, and mean arterial pressure did not differ among the groups on the seventh day of the study, endothelium-dependent vasorelaxation was impaired specifically in the HS group; treatment with the activin receptor-like kinase 5 inhibitor prevented the dietary HS intake-induced increases in phospho-Smad2 (Ser(465/467)) and NADPH oxidase-4 in endothelial lysates and normalized endothelial function. These findings suggest that HS-induced endothelial dysfunction and the development of salt-dependent increases in BP were related to endothelial TGF-ß signaling.

Nitric oxide and carbon monoxide antagonize TGF-ß through ligand-independent internalization of TßR1/ALK5.

Transforming growth factor (TGF)-ß plays a central role in vascular homeostasis and in the pathology of vascular disease. There is a growing appreciation for the role of nitric oxide (NO) and carbon monoxide (CO) as highly diffusible, bioactive signaling molecules in the vasculature. We hypothesized that both NO and CO increase endocytosis of TGF-ß receptor type 1 (TßR1) in vascular smooth muscle cells (VSMCs) through activation of dynamin-2, shielding cells from the effects of circulating TGF-ß. In this study, primary cultures of VSMCs from Sprague-Dawley rats were treated with NO-releasing molecule 3 (a NO chemical donor), CO-releasing molecule 2 (a CO chemical donor), or control. NO and CO stimulated dynamin-2 activation in VSMCs. NO and CO promoted time- and dose-dependent endocytosis of TßR1. By decreasing TßR1 surface expression through this dynamin-2-dependent process, NO and CO diminished the effects of TGF-ß on VSMCs. These findings help explain an important mechanism by which NO and CO signal in the vasculature by decreasing surface expression of TßR1 and the cellular response to TGF-ß.

Sodium and potassium regulate endothelial phospholipase C-γ and Bmx.

The amount of Na(+) and K(+) in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of Na(+) and K(+) in the regulation of two pleckstrin homology domain-containing intracellular signaling molecules, phospholipase C (PLC)-γ1 and epithelial and endothelial tyrosine kinase/bone marrow tyrosine kinase on chromosome X (Bmx), and agonist-generated Ca(2+) signaling in the endothelium. Extracellular K(+) concentration regulated the levels of activated PLC-γ1, Bmx, and carbachol-stimulated intracellular Ca(2+) mobilization in human endothelial cells. Additional experiments confirmed that high-conductance Ca(2+)-activated K(+) channels and phosphatidylinositol 3-kinase mediated these effects. The content of Na(+) and K(+) in the diet also regulated Bmx levels in endothelial cells and activated PLC-γ1 levels in rats in vivo. The effects of dietary K(+) on Bmx were more pronounced in rats fed a high-salt diet compared with rats fed a low-salt diet. These experiments elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary Na(+) and K(+) content.

Transforming growth factor-ß regulates endothelial function during high salt intake in rats.

Previous studies have demonstrated that an increase in dietary NaCl (salt) intake stimulated endothelial cells to produce transforming growth factor-ß (TGF-ß). The intent of the present study was to determine the functional significance of increased TGF-ß on endothelial cell function. Young Sprague-Dawley rats were fed diets containing 0.3 or 8.0% NaCl for 2 days before treatment with a specific inhibitor of the TGF-ß receptor I/activin receptor-like kinase 5 kinase, or vehicle for another 2 days. At day 4 of study, endothelial phosphorylated Smad2 (S465/467) increased and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) levels decreased in the high-salt-treated rats. In addition, phosphorylated Akt (S473) and phosphorylation of the endothelial isoform of NO synthase (NOS3) at S1177 increased. Treatment with the TGF-ß receptor I/activin receptor-like kinase 5 inhibitor reduced Smad2 phosphorylation to levels observed in rats on the low-salt diet and prevented the downstream signaling events induced by the high-salt diet. In human umbilical vein endothelial cells, reduction in PTEN levels increased phosphorylated Akt and NOS3. Treatment of macrovascular endothelial cells with TGF-ß1 increased phosphorylated NOS3 and the concentration of NO metabolites in the medium but had no effect on either of these variables in cells pretreated with small interfering RNA directed against PTEN. Thus, during high salt intake, an increase in TGF-ß directly promoted a reduction in endothelial PTEN levels, which in turn regulated Akt activation and NOS3 phosphorylation. This effect closes a feedback loop that potentially mitigates the effect of TGF-ß on the vasculature.

Effect of aging and dietary salt and potassium intake on endothelial PTEN (Phosphatase and tensin homolog on chromosome 10) function.

Aging promotes endothelial dysfunction, defined as a reduction in bioavailable nitric oxide (NO) produced by the endothelial isoform of nitric oxide synthase (NOS3). This enzyme is critically regulated by phosphorylation by protein kinase B (Akt), which in turn is regulated by the lipid phosphatase, PTEN. The present series of studies demonstrated a reduction in bioavailable NO as the age of rats increased from 1 to 12 months. At 12 months of age, rats no longer demonstrated increases in phosphorylated NOS3 in response to high dietary salt intake. Endothelial cell levels of PTEN increased with age and became refractory to change with increased salt intake. In contrast to the reduction in NO production, endothelial cell production of transforming growth factor-ß (TGF-ß) relative to NO increased progressively with age. In macrovascular endothelial cells, PTEN was regulated in a dose-dependent fashion by TGF-ß, which was further regulated by extracellular [KCl]. When combined with prior studies, the present series of experiments suggested an integral role for PTEN in endothelial cell pathobiology of aging and an important mitigating function of TGF-ß in endothelial PTEN regulation. The findings further supported a role for diet in affecting vascular function through the production of TGF-ß and NO.

Mechanism and prevention of acute kidney injury from cast nephropathy in a rodent model.

A common renal complication of multiple myeloma is "myeloma kidney," a condition also known as cast nephropathy. The renal lesions (casts) are directly related to the production of monoclonal immunoglobulin free light chains (FLCs), which coprecipitate with Tamm-Horsfall glycoprotein (THP) in the lumen of the distal nephron, obstructing tubular fluid flow. Here, we report that analysis of the binding interaction between FLCs and THP demonstrates that the secondary structure and key amino acid residues on the complementarity-determining region 3 (CDR3) of FLCs are critically important determinants of the molecular interaction with THP. The findings permitted development of a cyclized competitor peptide that demonstrated strong inhibitory capability in the binding of FLCs to THP in vitro. When used in a rodent model of cast nephropathy, this cyclized peptide construct served as an effective inhibitor of intraluminal cast formation and prevented the functional manifestations of acute kidney injury in vivo. These experiments provide proof of concept that intraluminal cast formation is integrally involved in the pathogenesis of acute kidney injury from cast nephropathy. Further, the data support a clinically relevant approach to the management of renal failure in the setting of multiple myeloma.

Pivotal role of apoptosis signal-regulating kinase 1 in monoclonal free light chain-mediated apoptosis.

Renal failure, a major complication associated with multiple myeloma, is usually related to deposition of monoclonal immunoglobulin free light chains (FLCs) and directly contributes to morbidity and mortality in this disease. The present study focused on the cytotoxic effects of monoclonal FLCs. Human proximal tubular epithelial cells (HK-2) were examined after incubation with two human monoclonal FLCs (termed κ2 and λ3). Incubation of HK-2 cells for 24 and 48 hours with either FLCs at 1 mg/mL promoted activation of caspase-9 and caspase-3 and increased the rate of apoptosis. Because prior studies demonstrated that FLCs generated intracellular oxidative stress, our studies focused on the redox-sensitive mitogen-activated protein kinase kinase kinase known as apoptosis signal-regulating kinase 1 (ASK1). A time-dependent increase in phosphorylation of ASK1 at T845, indicating activation of this enzyme, was observed. Small interfering RNA designed to reduce ASK1 expression in HK-2 cells successfully decreased ASK1, which was confirmed by Western blot analysis. Incubation of ASK1-depleted HK-2 cells with the two FLCs prevented the increase in apoptosis while pretreating HK-2 cell with nontargeting small interfering RNA did not prevent FLCs-mediated apoptosis. The combined data demonstrate that monoclonal FLCs activated the intrinsic apoptotic pathway in renal epithelial cells by activation of ASK1.

Immunoglobulin light chains activate nuclear factor-κB in renal epithelial cells through a Src-dependent mechanism.

One of the major attendant complications of multiple myeloma is renal injury, which contributes significantly to morbidity and mortality in this disease. Monoclonal immunoglobulin free light chains (FLCs) are usually directly involved, and tubulointerstitial renal injury and fibrosis are prominent histologic features observed in myeloma. The present study examined the role of monoclonal FLCs in altering the nuclear factor κ light chain enhancer of activated B cells (NF-κB) activity of renal epithelial cells. Human proximal tubule epithelial cells exposed to 3 different human monoclonal FLCs demonstrated Src kinase-dependent activation of the NF-κB pathway, which increased production of monocyte chemoattractant protein-1 (MCP-1). Tyrosine phosphorylation of inhibitor of κB kinases (IKKs) IKKα and IKKß and a concomitant increase in inhibitor of κB (IκB) kinase activity in cell lysates were observed. Time-dependent, Src kinase-dependent increases in serine and tyrosine phosphorylation of IκBα and NF-κB activity were also demonstrated. Proteasome inhibition partially blocked FLC-induced MCP-1 production. These findings fit into a paradigm characterized by FLC-induced redox-signaling events that activated the canonical and atypical (IKK-independent) NF-κB pathways to promote a proinflammatory, profibrotic renal environment.

Immunoglobulin light chains activate tubular epithelial cells through redox signaling.

The renal proximal tubule metabolizes circulating low-molecular-weight proteins such as Ig free light chains. In the setting of plasma cell dyscrasias, the burden of filtered protein can be very high. Endocytosis of certain nephrotoxic light chains induces H(2)O(2) production and monocyte chemoattractant protein-1 (MCP-1) release, leading to recruitment of inflammatory cells and interstitial fibrosis, but how these processes are linked mechanistically is not well understood. This study investigated the relationship between H(2)O(2) generated after light chain endocytosis by human proximal tubular (HK-2) cells and activation of c-Src, a redox-sensitive tyrosine kinase. HK-2 cells exposed to two different light chains upregulated c-Src activity, which increased the production of MCP-1. In parallel, we observed a time-dependent oxidation of c-Src. Inhibition of c-Src activity and silencing c-Src expression abrogated the light chain-induced MCP-1 response, but had no effect on H(2)O(2), indicating that production of H(2)O(2) is upstream of c-Src in the signaling cascade. Silencing megalin and cubilin expression inhibited the MCP-1 response, whereas extracellular catalase did not, indicating that endocytosis is required and that intracellular generation of reactive oxygen species activates c-Src. These data show that intracellular H(2)O(2) induced by endocytosis of monoclonal free light chains oxidizes and activates c-Src, which promotes release of MCP-1.

Potassium inhibits dietary salt-induced transforming growth factor-beta production.

Human and animal studies demonstrate an untoward effect of excess dietary NaCl (salt) intake on cardiovascular function and life span. The endothelium in particular augments the production of transforming growth factor (TGF)-beta, a fibrogenic growth factor, in response to excess dietary salt intake. This study explored the initiating mechanism that regulates salt-induced endothelial cell production of TGF-beta. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-beta demonstrated increased (35.2%) amounts of active TGF-beta in the medium of aortic ring segments from rats on the high-salt diet compared with rats maintained on a 0.3% NaCl diet. Inhibition of the large-conductance, calcium-activated potassium channel inhibited dietary salt-induced vascular production of TGF-beta but did not affect production of TGF-beta by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the alpha subunit of the calcium-activated potassium channel in endothelial cells. Increasing medium [K+] inhibited production of dietary salt-induced vascular production levels of total and active TGF-beta but did not alter TGF-beta production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-beta in animals receiving the high-salt diet but did not change urinary active TGF-beta in animals receiving the low-salt diet. The findings demonstrated an interesting interaction between the dietary intake of potassium and excess NaCl and further showed the fundamental role of the endothelial calcium-activated potassium channel in the vascular response to excess salt intake.

Dietary salt activates an endothelial proline-rich tyrosine kinase 2/c-Src/phosphatidylinositol 3-kinase complex to promote endothelial nitric oxide synthase phosphorylation.

Although many laboratories have shown that dietary NaCl (salt) intake increases NO production in rodents and humans, the mechanism has not been uncovered. In the present study, pharmacological and dominant-negative strategies were used to show that feeding a formulated diet containing increased amounts of salt to young male Sprague-Dawley rats induced the formation of an endothelial cell-signaling complex that contained proline-rich tyrosine kinase 2, c-Src (also known as pp60(c-src)), and phosphatidylinositol 3-kinase. In the setting of a high-salt diet, proline-rich tyrosine kinase 2 served as the scaffold for c-Src-mediated phosphatidylinositol 3-kinase activation. Phosphatidylinositol 3-kinase was the upstream activator of protein kinase B (Akt), which was responsible for phosphorylation of the rat endothelial isoform of NO synthase at S1176 and thereby promoted the increase in NO production. The combined findings illustrated the crucial role for a proline-rich tyrosine kinase 2-signaling complex in the endothelial response to salt intake.

Mechanism of dietary salt-mediated increase in intravascular production of TGF-beta1.

Clinical and preclinical studies have demonstrated an important effect of arterial pathobiology on the progressive loss of renal function that occurs in chronic kidney disease. Chronic kidney disease, in turn, promotes alterations in vascular function. A modulating role for dietary salt has been suggested, with the amount of salt intake regulating endothelial cell production of transforming growth factor-beta1 (TGF-beta1), a fibrogenic growth factor that promotes arteriosclerosis and glomerulosclerosis. The purpose of the present studies was to determine how the interaction between dietary salt intake and vasculature promoted the production of TGF-beta1 in rats. Two different vascular tissues, aortic rings and glomeruli, were chosen for study. Dietary salt induced, in a dose-dependent fashion, activation of proline-rich tyrosine kinase-2 (Pyk2) and further identified c-Src as an important binding partner of Pyk2 in these tissues. Use of pharmacological inhibitors and dominant negative strategies confirmed that dietary salt induced complex formation of Pyk2 and c-Src with downstream activation of p38 and p42/44 mitogen-activated protein kinases and generation of TGF-beta1. The experiments defined the molecular signaling events that promoted the production of TGF-beta1, a key growth factor involved in the vascular response to increased salt intake.

EGF receptor activity modulates apoptosis induced by inhibition of the proteasome of vascular smooth muscle cells.

The observation that intracellular protein turnover rates participate directly in cell viability led to the development and clinical use of potent proteasome inhibitors. This study determined that the mechanism of apoptosis that is induced by inhibition of the proteasome of vascular smooth muscle cells (VSMC) was related to the intracellular accumulation of Bad, a BH3-only member of the Bcl-2 family of apoptosis regulators. Experiments confirmed that the apoptotic process was mitochondria- and caspase-dependent. Ubiquitination and accumulation of Bad in VSMC followed inhibition of the proteasome, and depletion of Bad using RNA interference prevented apoptosis that was induced by proteasome inhibition with PS-341. EGF receptor (EGFR) activation produced posttranslational modifications of Bad, providing the pro-survival signals that prevented apoptosis of smooth muscle cells during proteasome inhibition. Antagonists of the EGFR potentiated the apoptotic rate. In summary, the activities of the EGFR and the proteasome focused on Bad and the intrinsic apoptotic pathway and were involved integrally in determining viability of VSMC. These findings might prove useful in the management of diseases in which proliferation of vascular smooth muscle cells plays a central role.