Markers associated with sex differences in methamphetamine-induced striatal dopamine neurotoxicity.

Three different approaches were employed to assess various markers associated with sex differences in responses to methamphetamine (MA). Bioassay measures reveal that MA treatment results in significantly greater reductions in body weight and increases in body temperature in male mice. Protein and mRNA determinations show significant increases in Bcl-2 and PAI-1 in male mice, while females show significant increases in GFAP and decreases in IGF-1R following treatment with MA. In mice with a heterozygous mutation of their dopamine transporter (+/- DAT), only female mice show significant differences in dopamine transporter binding and mRNA and associated reductions in striatal dopamine content along with increases in MA-evoked striatal dopamine output. The identification of these sex-dependent differences in markers provides a foundation for more exhaustive evaluation of their impact upon, and treatment of, disorders/neurotoxicity of the nigrostriatal dopaminergic system and the bases for the differences that exist between females and males.

Dual modulation of urinary bladder activity and urine flow by prostanoid EP3 receptors in the conscious rat.

BACKGROUND AND PURPOSE: Cyclooxygenase inhibitors function to reduce levels of prostaglandin E(2) (PGE(2)) and are broadly efficacious in models of bladder overactivity. We therefore investigated a regulation of urinary bladder function in conscious rats by modulation of the EP(3) receptor for PGE(2). EXPERIMENTAL APPROACH: The activity of the EP(3) receptor agonist GR63799X, and EP(3) receptor antagonists, CM9 and DG041, at recombinant EP(3) receptors was evaluated in vitro. In vivo, intraduodenal dosing during conscious, continuous-filling cystometry of spontaneously hypertensive rats was utilized to determine the urodynamic effect of EP(3) receptor modulation. KEY RESULTS: GR63799X dose-dependently (0.001-1 mg x kg(-1)) reduced bladder capacity, as indicated by a reduction in both the micturition interval and volume of urine per void. In contrast, CM9 (10 and 30 mg x kg(-1)) and DG041 (30 mg x kg(-1)) enhanced bladder capacity, as indicated by significantly longer micturition intervals and larger void volumes. CM9 and DG041 inhibited the responses to GR63799X supporting the in vivo activity of these pharmacological agents at the EP(3) receptor. In addition to its effect on bladder capacity, GR63799X increased endogenous urine production. Intra-arterial infusion of saline mimicked the enhancement of urine flow observed with GR63799X, and the response was inhibited by CM9. CONCLUSIONS AND IMPLICATIONS: These data support the EP(3) receptor as a modulator of urinary bladder activity in the conscious rat, and in addition, indicate a role for EP(3) receptor activity in regulating urine flow.

Oral administration of GW788388, an inhibitor of TGF-beta type I and II receptor kinases, decreases renal fibrosis.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).

Renoprotective effects of carvedilol in hypertensive-stroke prone rats may involve inhibition of TGF beta expression.

1. The effect of carvedilol on renal function, structure and expression of TGF beta and the matrix proteins fibronectin, collagen I and collagen III, was evaluated in spontaneously hypertensive stroke-prone (SHR-SP) rats fed a high fat, high salt diet. 2. Carvedilol treatment for 11 to 18 weeks did not alter systolic blood pressure in SHR-SP rats, however, it resulted in a significant reduction in heart rate. 3. Carvedilol treatment reduced renal fibrosis and total, active and chronic renal damage to levels approaching those of WKY rats on a normal diet. 4. Urinary protein excretion was higher in SHR-SP rats (51+/-10 mg day(-1)) than WKY rats (18+/-2 mg day(-1)) and this was further increased when SHR-SP rats were fed a high fat, high salt diet (251+/-120 mg day(-1)). Treatment with carvedilol resulted in significantly lower urinary protein excretion (37+/-15 mg day(-1)). 5. The expression of TGF beta mRNA was significantly higher in SHR-SP rats compared to WKY rats and a further increase was observed when rats were fed a high fat, high salt diet. Renal TGF beta expression was significantly reduced by treatment with carvedilol. The expression of fibronectin and collagen I and collagen III mRNA showed a pattern similar to that observed with TGF beta mRNA expression. Collagen I mRNA expression followed a pattern similar to renal fibrosis. 6. These data indicate that carvedilol can provide significant renal protection in the absence of any antihypertensive activity and that the mechanisms involved in this action may include reduced expression of profibrotic factors such as TGF beta.

The role of transforming growth factor-beta and its receptors in human prostate smooth muscle cell fibronectin production.

In the present study, the role of transforming growth factor-beta (TGFbeta) on the production of the extracellular matrix component, fibronectin, in the prostate has been studied. The mRNA levels of fibronectin, TGFbeta and the two TGFbeta receptors, ALK5 (activin like kinase) and type II, were measured using reverse-transcription polymerase chain reaction (RT-PCR). TGFbeta increased fibronectin mRNA and protein (7-fold) in a concentration-dependent fashion. An interesting relationship between the two TGFbeta receptors was found in that TGFbeta caused an upregulation of its type I receptor mRNA (5-6-fold) and a downregulation of the type II receptor mRNA (5-fold). Time-course experiments revealed that the change in expression of the TGFbeta receptors reached maximum at 24 h with an early increase at 4-5 h, whereas the fibronectin gene expression was not significantly stimulated until about 24 h. These data provide evidence that TGFbeta stimulates extracellular matrix production in prostate cells.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Gene expression in rats with renal disease treated with the angiotensin II receptor antagonist, eprosartan.

The role of ANG II on renal and cardiac gene expression of matrix proteins was studied in rats with progressive renal disease. Induction of renal failure by five-sixths nephrectomy of Sprague-Dawley rats resulted in hypertension (163 +/- 19 vs. control pressures of 108 +/- 6 mmHg), proteinuria (83 +/- 47 vs. 14 +/- 2 mg/day), and increased renal expression of fibronectin, thrombospondin, collagen I and III, transforming growth factor-beta (TGF-beta), and plasminogen activator inhibitor-1 (PAI-1) mRNA. Treatment with the ANG II receptor antagonist, eprosartan (60 mg. kg(-1).day(-1)), lowered blood pressure (95 +/- 5 mmHg) and proteinuria (19 +/- 8 mg/d) and abrogated the increased TGF-beta, fibronectin, thrombospondin, collagens I and III, and PAI-1 mRNA expression. An increase in left ventricular weight was observed in five-sixths nephrectomized rats (0.13 +/- 0.01 vs. 0.08 +/- 0.01 g/100 g body wt), a response that was inhibited by eprosartan treatment (0.10 +/- 0.01 g/100 g). Left ventricular expression of TGF-beta and fibronectin was also increased in rats with renal disease; however, the small decreases in expression observed in eprosartan-treated rats did not reach statistical significance. These data suggest that eprosartan may be beneficial in progressive renal disease and that the mechanism of action includes inhibition of cytokine production in addition to antihypertensive activity.

Transforming growth factor-beta1 induces transforming growth factor-beta1 and transforming growth factor-beta receptor messenger RNAs and reduces complement C1qB messenger RNA in rat brain microglia.

Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth factor-beta1 in the rodent brain support a major role of microglia in neural activities of transforming growth factor-beta1 and give a new link between transforming growth factor-beta1 and the complement system. The auto-induction of the transforming growth factor-beta1 system has implications for transgenic mice that overexpress transforming growth factor-beta1 in brain cells and for its potential role in amyloidogenesis.

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.

Hepatocyte growth factor: a regulator of extracellular matrix genes in mouse mesangial cells.

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

Sgk, a putative serine/threonine kinase, is differentially expressed in the kidney of diabetic mice and humans.

Differential display PCR was used to identify alternate expression of serum glucocorticoid-regulated kinase (Sgk) mRNA in diabetes-induced renal disease. Differential expression of Sgk mRNA was identified in the kidneys of normal and obese db/db mice, a model of select aspects of human diabetic nephropathy. Sgk mRNA was selectively increased in diabetic mouse kidneys. The Sgk mRNA levels remained constant in other tissues from obese db/db mice. An increase in Sgk mRNA was also observed in the human diabetic kidney. In addition, thrombin, which may play a role in the progression of renal disease, increased Sgk message in cell culture. Because the diabetes-induced increase in Sgk was only observed in the kidney, which is particularly susceptible to diabetes-induced damage, Sgk may play a role in diabetic nephropathy.

Age-related expression of renal thrombospondin 1 mRNA in F344 rats: resemblance to diabetes-induced expression in obese Zucker rats.

Age-related increases occurred in renal thrombospondin 1 (TSP1) mRNA in F344 rats, resembling diabetes-induced TSP1 mRNA in the obese Zucker rat. TSP1 mRNA was 3.5-fold higher in 24-month-old than in 3-month-old F344 rats. TSP1 mRNA increased similarly in 5-month-old obese Zucker rats as compared with lean littermates and correlated positively with the extent of proteinuria (r = 0.71). In situ hybridization identified elevated TSP1 mRNA levels in epithelial cells of distended tubules as well as in interstitium near dilated tubules of both 24-month-old F344 rats and 5-month-old obese Zucker rats. Furthermore, thrombin increased TSP1 mRNA in mesangial and epithelial cells in culture, indicating that thrombin may contribute to elevated TSP1 expression in renal disease. Thrombin increased TSP1 mRNA within 30 min after treatment which required de novo synthesis of protein. The thrombin receptor tethered ligand peptide, SFLLRN, increased TSP1 mRNA, indicating that the thrombin-induced increase in TSP1 mRNA was due to direct thrombin receptor (PAR1) stimulation. These results show that increased TSP1 mRNA levels are a component of interstitial fibrosis seen in aged and diabetic kidneys and suggest that similar pathological changes occur in kidneys of aging and diabetic rats.

Hepatocyte growth factor in renal disease: cause or cure?

Hepatocyte growth factor (HGF) is an injury-released growth factor with diverse effects on epithelial and endothelial cells. These effects include proliferation, migration, extracellular matrix production and tubulogenesis. These activities allow HGF to function as an organizer of repair processes that bring about restoration of tubular function following renal injury. However, while HGF has been demonstrated to accelerate recovery of renal function after an acute insult, prolonged exposure to elevated levels of HGF can reduce renal function and may contribute to progressive renal disease. This review will describe the cellular activities of HGF, how they pertain to renal repair and the therapeutic application of regulating HGF activity in acute versus chronic renal disease.

Therapeutic uses of smad protein inhibitors: Selective inhibition of specific TGF-beta activities.

Recent advances in TGF-beta signaling biology have identified smad proteins as cytoplasmic mediators of TGF-beta receptor activation. Current evidence suggests that smad proteins are transcription factors whose activity is regulated through phosphorylation by TGF-beta type I receptors. Several isoforms of the smad proteins have been identified which confer in part selectivity for different members of the TGF-beta family of receptors as well as some selectivity for downstream effects of TGF-beta activation. Therefore, smad proteins provide an attractive means for targeting select activities of TGF-beta by either inhibiting their phosphorylation or specific DNA binding modes. Because TGF-beta is a central player in the development of fibrosis, selective inhibition of smad proteins would provide an important therapeutic benefit in many acute and chronic fibrotic diseases.

Angiotensin-converting enzyme inhibition alters clusterin mRNA expression in the kidney following renal mass reduction.

The effect of the angiotensin-converting enzyme inhibitor captopril on clusterin mRNA was examined in partially nephrectomized male rats. Urine protein excretion was measured 3, 7, and 28 days after removal of five sixths of the renal mass. Nephrectomy caused a progressive increase in clusterin mRNA levels in the remnant kidney. Maximal clusterin mRNA levels occurred 7 days after nephrectomy and declined 28 days after nephrectomy. Captopril, 250 mg/ml in drinking water, prevented the injury-induced increase in clusterin mRNA at 7 and 28 days. Captopril had no effect on clusterin in sham-operated rats. As expected, the urine protein excretion increased progressively after nephrectomy, and this was attenuated by administration of captopril in the drinking water. Therefore, clusterin is a marker of renal injury which, along with proteinuria, is modulated by angiotensin-converting enzyme inhibition.

Angiotensin-converting enzyme inhibition attenuates proteinuria and renal TGF-beta 1 mRNA expression in rats with chronic renal disease.

Evidence suggests that transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine, induces renal extracellular matrix production and glomerular hypertrophy. The aim of the present study was to investigate the effect of captopril on the expression of TGF-beta 1 mRNA in a rat model of chronic renal failure: five-sixths nephrectomy. Chronic renal disease was induced by removal of the right kidney and ligation of three blood vessels supplying the left kidney. Sham-operated animals were used as controls. RNA was extracted from the viable remnant kidney of rats 1 day and 1 and 2 weeks following five-sixths nephrectomy and from the kidneys of rats who underwent sham surgery. TGF-beta 1 mRNA was induced within 24 h of partial nephrectomy, similar to that reported for early-onset genes. Subsequently, TGF-beta 1 mRNA expression continued to increase over the next 2-4 weeks. The upregulation of TGF-beta 1 correlated with the degree of proteinuria. Both the increase in TGF-beta 1 mRNA and proteinuria were abrogated by captopril treatment. In addition, no change in expression of ALK-5 or type II TGF-beta receptors following five-sixths nephrectomy was observed. These data suggest that captopril may protect against development of glomerulosclerosis and proteinuria by reducing TGF-beta 1 expression and hence matrix production.

Activation of glomerular mesangial cells by hepatocyte growth factor through tyrosine kinase and protein kinase C.

Hepatocyte growth factor (HGF) induces mitogenesis, chemotaxis, and tubule formation in renal epithelial cells. This study examined the effects of wortmannin and protein kinase C (PKC) inhibitors on HGF-mediated changes in metabolic activity in glomerular mesangial cells and renal epithelial carcinoma A498 cells. The extracellular acidification rate of transformed mouse glomerular mesangial cells and A498 cells was measured as an index of metabolic activity with a microphysiometer. HGF increased the acidification rate of mesangial cells and A498 cells in a concentration-dependent fashion that was inhibited completely by the tyrosine kinase inhibitor tyrophostin-23 (100 microM). The PKC inhibitors RO-32-0432 and SKF-57048 also inhibited HGF-induced acidification. The IC50 values for SKF-57048 were 59 +/- 2 and 20 +/- 10 nM in mesangial cells and A498 cells, respectively (P < 0.05). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a phorbol ester that activates PKC, increased acidification in mesangial and epithelial cells similar to HGF. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase (IC50 value 1-10 nM), inhibited HGF-induced acidification with an IC50 of 93 +/- 31 and 9 +/- 1 nM in mesangial and A498 cells, respectively (P < 0.05). In contrast, there was no significant difference in the IC50 value of wortmannin for epidermal growth factor (EGF)-induced acidification between mesangial and A498 cells (23 +/- 9 vs 14 +/- 1 nM, respectively). Because the IC50 value for wortmannin in inhibiting HGF but not EGF-induced acidification was an order of magnitude higher in mesangial cells than in epithelial A498 cells, a wortmannin-sensitive PI 3-kinase pathway may not be involved in HGF-mediated acidification in mesangial cells.

The age-related increase in renal clusterin mRNA is accelerated in obese Zucker rats.

Clusterin is a multifunctional glycoprotein associated with development and tissue injury. Because renal function decreases with advancing age in the obese Zucker rat, clusterin mRNA expression was examined in the kidney of young adult Zucker rats and compared with age-related changes in renal clusterin mRNA expression in Fischer 344 (F344) rats. Renal clusterin mRNA levels in the obese Zucker rat were 2.5-fold higher by 3 mo of age and fourfold higher at 5 mo of age compared with the lean strain. In comparison, renal clusterin mRNA in 12-mo-old F344 rats was twofold higher than in 3-mo-old animals and was tenfold higher at 24 mo of age. Clusterin mRNA was positively correlated with urinary protein excretion and negatively correlated with creatinine clearance in Zucker rats. Clusterin was increased in select nephrons of the obese Zucker rat kidney and in 24-mo-old F344 rat kidney as assessed by in situ hybridization. Increased expression of clusterin mRNA occurred mostly in the tubular epithelium of dilated, convoluted proximal tubules. These data indicate that renal clusterin mRNA levels increase as a function of age and that age-related increases in renal clusterin and the associated tubular abnormalities are accelerated in obese Zucker rats.

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells.

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.