Wei-Zhong Ying

Dietary salt modulates renal production of transforming growth factor-β in rats

Transforming growth factors (TGF) are potent multifunctional polypeptides that are involved in renal function and glomerular sclerosis. We postulated that dietary salt modified renal production of TGF-β. An increase in dietary salt produced sustained increases in steady-state levels of mRNA for TGF-β1, -β2, and -β3 in the rat kidney. While serum concentration of TGF-β1 did not change, the 8.0% NaCl diet increased urinary excretion of TGF-β1, indicating enhanced renal production was the source of TGF-β1. Increasing urinary flow rates with diuretics did not further increase synthesis of TGF-β1 in animals receiving the 8.0% NaCl diet. The 8.0% NaCl diet increased production of TGF-β1 in both glomeruli and tubules, although active TGF-β1 was secreted in greater amounts only from glomeruli. Enhanced glomerular production of both inactive and active TGF-β1 induced by the 8.0% NaCl diet was inhibited by tetraethylammonium (TEA) and not glybenclamide. Cardiac production of TGF-β1 also increased on the 8.0% NaCl diet but was not affected by TEA. The results demonstrated that increased dietary salt augmented glomerular TGF-β production by a mechanism that included a TEA-sensitive potassium channel. Dietary salt, by facilitating glomerular expression of TGF-β, may directly promote development of glomerulosclerosis.

Mapping the Binding Domain of Immunoglobulin Light Chains for Tamm-Horsfall Protein

Cast nephropathy, or myeloma kidney, is a potentially reversible cause of chronic renal failure. In this condition, filtered light chains bind to a common site on Tamm-Horsfall protein (THP), which is produced by cells of the thick ascending limb of the loop of HENLE: Subsequent aggregation of these proteins produces casts that obstruct tubule fluid flow and results in renal failure. In the present study, we used the yeast two-hybrid system to determine the site of interaction of light chains with THP. The third complementarity-determining region (CDR3) of both kappa and lambda light chains interacted with THP. These findings were confirmed in a series of competition studies using a synthetic peptide that corresponded to the CDR3 region and purified THP and light chains. Variations in the CDR3 sequence of the light chain affected binding. Thus, the current studies increase our understanding of the process of cast formation and provide an opportunity to develop strategies that may inhibit this interaction and prevent the clinical manifestations of myeloma kidney.

Dietary salt increases endothelial nitric oxide synthase and TGF-β1 in rat aortic endothelium

The amount of NaCl in the diet plays an important role in modulating nitric oxide (NO) synthesis in vivo. In the glomerulus, dietary NaCl also regulates transforming growth factor-β1 (TGF-β1) production. We hypothesized that dietary NaCl intake regulated expression of the endothelial isoform of nitric oxide synthase (NOS3) and TGF-β1 in the aorta. Administration of 8.0% NaCl diet to rats for 7 days did not affect blood pressure but increased steady-state mRNA and protein levels of NOS3 in the arterial wall compared with animals on 0.3% NaCl diet. Northern analysis demonstrated increased steady-state amounts of mRNA of TGF-β1 in aortas of rats on 8.0% NaCl diet. By ELISA, both total and active TGF-β1 were increased in these vessel segments. Endothelial denudation of aortic rings reduced active TGF-β1 secretion to undetectable levels. Addition of a neutralizing antibody to TGF-β to aortic ring segments attenuated NO production but not to that observed in animals on the 0.3% NaCl diet. The data showed that dietary NaCl intake modulated NOS3 and TGF-β1 expression in the arterial wall; NOS3 expression was at least partially regulated by endothelial cell production of TGF-β1.

Dietary salt enhances glomerular endothelial nitric oxide synthase through TGF-β1

Dietary salt controls production of nitric oxide (NO), a potent paracrine relaxation factor involved in glomerular filtration and salt excretion. We hypothesized that glomerular NO production was enhanced through endothelial nitric oxide synthase (NOS3). Rats in metabolic cages were studied after 4 days on 0.3% (Lo-salt) or 8.0% (Hi-salt) NaCl diet. Steady-state mRNA and protein levels of NOS3 and calcium-dependent NO production of isolated glomeruli from Hi-salt animals were greater than those values observed in glomeruli from Lo-salt rats. Because dietary salt enhanced glomerular production of transforming growth factor-β1 (TGF-β1) [W.-Z. Ying and P. W. Sanders. Am. J. Physiol. 274 ( Renal Physiol. 43): F635-F641, 1998], studies were then conducted to examine the interaction between NOS3 and TGF-β1. Glomerular steady-state levels of mRNA of NOS3 and TGF-β1 directly correlated ( r 2 = 0.946; P < 0.0001). A neutralizing antibody to TGF-β reduced NOS3 protein and NO production in cultured glomeruli from Hi-salt animals to levels seen in the Lo-salt glomeruli. Thus dietary salt increased glomerular expression of TGF-β1, which in turn augmented NO production through NOS3.

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 IKKβ 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.

Mechanism of dietary salt-mediated increase in intravascular production of TGF-β1

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.

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.

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.

Potassium Inhibits Dietary Salt-Induced Transforming Growth Factor-β 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)-&bgr;, 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-&bgr;. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-&bgr; demonstrated increased (35.2%) amounts of active TGF-&bgr; 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-&bgr; but did not affect production of TGF-&bgr; by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the &agr; 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-&bgr; but did not alter TGF-&bgr; production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-&bgr; in animals receiving the high-salt diet but did not change urinary active TGF-&bgr; 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 pp60c-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.