Protein kinase C ß inhibition ameliorates experimental mesangial proliferative glomerulonephritis.

AIM: Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the ß isoform of this enzyme has been examined. However, PKC-ß is also increased in various forms of human glomerulonephritis, including IgA nephropathy. Accordingly, we sought to examine the effects of PKC-ß inhibition in the Thy1.1 model of mesangial proliferative glomerulonephritis. METHODS: Following administration of monoclonal OX-7, anti-rat Thy-1.1 antibody, Male Wistar rats were randomized to receive either the PKC-ß inhibitor, ruboxistaurin (10 mg/kg per day in chow) or vehicle. Animals were then examined 6 days later. RESULTS: PKC-ß inhibition was associated with reductions in mesangial cellularity and extracellular matrix deposition. Proteinuria was, however, unaffected. In vitro, PKC-ß inhibition showed modest, dose-dependent reductions in mesangial cell (3) H-thymidine and (3) H-proline incorporations, indices of cell proliferation and collagen synthesis, respectively. CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by PKC-ß inhibition suggests the potential clinical utility of this approach as a therapeutic strategy in non-diabetic glomerular disease.

Laser-capture microdissection and pressure catapulting for the analysis of gene expression in the renal glomerulus.

Investigation into the molecular mechanisms regulating normal renal physiology and pathophysiology has benefited from the development of microdissection techniques enabling sampling of specific cell populations or structures within the kidney. Laser-capture microdissection and pressure catapulting is a relatively new, entirely non-contact microdissection technique that facilitates the assay of mRNA and protein expression in single nephron segments or populations. Herein, we describe methods for sample preparation, microdissection and collection of glomeruli from archival renal biopsies for later analysis of gene expression using real-time PCR. Microdissection of glomeruli from archival renal biopsy sections was carried out using the PALM Microbeam UV laser system from P.A.L.M. Technologies.

Evaluation and optimization of antifibrotic activity of cinnamoyl anthranilates.

Tranilast is an anti-inflammatory drug in use for asthma and atopic dermatitis. In studies over the last decade it has been revealed that tranilast can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. We report a structure-activity study aimed at optimizing the antifibrotic activity of tranilast. A series of cinnamoyl anthranilates were prepared and assessed for their ability to prevent TGF-beta-stimulated production of collagen in cultured renal mesangial cells. We reveal derivatives with improved potency and reduced cellular toxicity relative to tranilast. 3-Methoxy-4-propargyloxycinnamoyl anthranilate reduces albuminuria in a rat model of progressive diabetes, and thus has potential as an innovative treatment for diabetic nephropathy.

Expression, localization, and function of the thioredoxin system in diabetic nephropathy.

Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.

Role of VEGF in maintaining renal structure and function under normotensive and hypertensive conditions.

Inhibiting the actions of VEGF is a new therapeutic paradigm in cancer management with antiangiogenic therapy also under intensive investigation in a range of nonmalignant diseases characterized by pathological angiogenesis. However, the effects of VEGF inhibition on organs that constitutively express it in adulthood, such as the kidney, are mostly unknown. Accordingly, we examined the effect of VEGF inhibition on renal structure and function under physiological conditions and in the setting of the common renal stressors: hypertension and activation of the renin-angiotensin system. When compared with normotensive Sprague-Dawley (SD) rats, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats that overexpress renin with spontaneously hypertensive rat (SHR) kidneys showing VEGF expression levels that were intermediate between them. Administration of either an orally active inhibitor of the type 2 VEGF receptor (VEGFR-2) tyrosine kinase or a VEGF neutralizing antibody to TGR(mRen-2)27 rats resulted in loss of glomerular endothelial cells and transformation to a malignant hypertensive phenotype with severe glomerulosclerosis. VEGFR-2 kinase inhibition treatment was well tolerated in SDs and SHRs; although even in these animals there was detectable endothelial cell loss and rise in albuminuria. Mild mesangial expansion was also noted in hypertensive SHR, but not in SD rats. These studies illustrate: (i) VEGF has a role in the maintenance of glomerular endothelial integrity under physiological circumstances, (ii) glomerular VEGF is increased in response to hypertension and activation of the renin-angiotensin system, and (iii) VEGF signaling plays a protective role in the setting of these renal stressors.

Macrophage infiltration and cellular proliferation in the non-ischemic kidney and heart following prolonged unilateral renal ischemia.

BACKGROUND/AIMS: Although ischemic renal failure remains a major cause of morbidity and mortality, whether ischemic changes within a kidney might also have adverse effects on other organs has not been examined. Furthermore, given the protective effects of angiotensin II receptor (AT1) antagonism in renal ischemia, we considered whether a similar strategy might also modulate the response to acute renal insult. METHODS: Unilateral renal artery ligation was performed in Sprague-Dawley rats, treated with or without the AT1 antagonist losartan (30 mg/kg/day). After 24 h of renal ischemia, changes in the contralateral kidney and heart were examined. RESULTS: Contralateral non-ischemic kidneys displayed increased expression of platelet-derived growth factor-B (PDGF-B) in association with increased tubular cell proliferation. Gene expression for the macrophage chemokine osteopontin (OPN) was similarly increased along with substantial macrophage infiltration. In the heart, expression of OPN and macrophage numbers were increased. All of these changes, in both the heart and kidney were attenuated by losartan. CONCLUSION: Rather than affecting a single organ, the present study demonstrates that after prolonged renal ischemia, the contralateral kidney and heart undergo changes in growth factor and chemokine expression, resulting in pathological proliferation and inflammation that can be modulated by blockade of the AT1 receptor.

Transforming growth factor-beta in human diabetic nephropathy: effects of ACE inhibition.

OBJECTIVE: Studies in rodent models have suggested that reduction in renal transforming growth factor (TGF)-beta1 may underlie the renoprotective effects of the renin-angiotensin system (RAS) blockade. However, the role of the RAS blockade in abrogating TGF-beta in human disease is unknown. Accordingly, we sought to examine TGF-beta gene expression and biological activity in human renal biopsies, before and after ACE inhibition. RESEARCH DESIGN AND METHODS: RNA was extracted from renal biopsies taken from participants in the Diabiopsies study, a randomized controlled 2-year trial of 4 mg/day perindopril versus placebo that reported a reduction in proteinuria and cortical matrix expansion in type 2 diabetic nephropathy. Biopsies taken at study entry and at 2 years were obtained in 12 patients (6 placebo and 6 taking perindopril). TGF-beta1 and its receptor mRNA were quantified by real-time PCR, and its biological activity was assessed by examining the activation of its intracellular signaling pathway (phosphorylated Smad2) and the expression TGF-beta-inducible gene H3 (betaig-H3). RESULTS: At baseline, TGF-beta1 expression was similar in both placebo- and perindopril-treated groups and was unchanged over a 2-year period in biopsies of placebo-treated subjects. In contrast, perindopril treatment led to a substantial diminution in TGF-beta1 mRNA (mean 83% reduction, P < 0.05). Phosphorylated Smad2 immunolabeling and betaig-H3 mRNA were similarly reduced with ACE inhibition (P < 0.05) but unchanged in the placebo group. No differences were noted in the gene expression of TGF-beta receptor II in biopsies of either placebo- or perindopril-treated subjects. CONCLUSIONS: This study demonstrates that over a 2-year period, treatment with perindopril in patients with type 2 diabetes and nephropathy leads to a reduction in both renal TGF-beta1 gene expression and its downstream activation.

Cells expressing the stem cell factor receptor, c-kit, contribute to neoangiogenesis in diabetes.

While neoangiogenesis in diabetes is of greatest clinical significance in the retina, the pathological formation of new blood vesselsalso develops in other vascular beds in diabetes, including the mesentery of the streptozotocin-induced diabetic rat. However, the contribution of bone marrow-derived cells to this process of vasculogenesis is unknown. In this study, male Sprague-Dawley rats were randomised to receive either streptozotocin or vehicle, and their mesenteric vasculature was examined after three weeks. Origins from bone marrow and endothelial cell differentiation were identified by immunolabelling for the stem cell factor receptor, c-kit and von Willebrand factor (vWF), respectively. Expression for the angiogenic chemokine, stromal derived factor-1 (SDF-1) was assessed by quantitative real-time polymerase chain reaction (PCR). At three weeks, rats with diabetes had a dramatic (190-fold) increase in lectin-labelled blood vessel profiles in the mesenteric bed (p < 0.0001) in association with a five-fold increase in SDF-1 mRNA (p < 0.01). Immunohistochemical studies identified abundant large, ovoid, lumen-forming, c-kit+ cells in the mesentery of diabetic, but not control, rats. Many of these c-kit+ cells also showed positive immunolabelling for vWF, consistent with endothelial differentiation. In conclusion, cells of bone marrow origin contribute to new vessel formation in the diabetic mesentery. This phenomenon may also apply to the neovascularisation that develops at clinically important sites such as in the retina.

Inhibition of protein kinase C reduces left ventricular fibrosis and dysfunction following myocardial infarction.

Despite current therapies, chronic heart failure (CHF) remains a major complication of myocardial infarction (MI). The pathological changes that follow MI extend to regions remote from the site of infarction (non-infarct zone, NIZ) where fibrosis is a prominent finding. Although the mechanisms underlying this adverse remodeling are incompletely understood, activation of protein kinase C has recently been implicated in its pathogenesis. MI was induced in Sprague-Dawley rats by ligation of the left anterior descending coronary artery. One week post-MI, animals were randomized to receive the PKC-inhibitor, ruboxistaurin (LY333531) for 4 weeks, or no treatment. When compared with sham-operated animals, post-MI rats showed a 33+/-7% reduction in fractional shortening over a 4 weeks period, that was attenuated by treatment with ruboxistaurin (6+/-11%, P<0.05). Increased matrix deposition was noted in the NIZ, particularly in the subendocardial region of post-MI rats, in association with elevated expression of the profibrotic growth factor, transforming growth factor-beta. These findings were also significantly reduced by ruboxistaurin. PKC-inhibition with ruboxistaurin led to attenuation in both the pathological fibrosis and impaired cardiac function that follow experimental MI, suggesting a possible role for this agent in preventing post-infarction heart failure.

Protein kinase Cbeta inhibition attenuates osteopontin expression, macrophage recruitment, and tubulointerstitial injury in advanced experimental diabetic nephropathy.

Tubulointerstitial macrophage accumulation is an important marker of prognosis that correlates closely with declining renal function in a range of human and experimental diseases, including diabetic nephropathy. These inflammatory cells are rich in the profibrotic growth factor TGF-beta such that their presence in areas of injury is frequently associated with tissue fibrosis. The migration of macrophages occurs in response to the site-specific production of chemokines, with osteopontin closely associated with their trafficking into the tubulointerstitium of the kidney. Although cell culture studies indicate that protein kinase C (PKC) mediates the expression of osteopontin, its role in the in vivo setting is unknown. Accordingly, Ren-2 control and diabetic rats that were treated with or without the specific PKC-beta isoform inhibitor ruboxistaurin (10 mg/kg per d) were examined. After 12 wk, diabetic rats showed increases in osteopontin expression in tubular epithelial cells of the cortex in association with macrophage infiltration, interstitial fibrosis, and activity of TGF-beta as indicated by the expression of its receptor activated protein phospho-Smad2 (P < 0.05 for all parameters). Ruboxistaurin treatment significantly attenuated these parameters (P < 0.05) in diabetic rats without affecting either BP or glycemic control. These findings suggest that osteopontin and macrophage accumulation may play a role in the tubulointerstitial injury in diabetic nephropathy and that inhibition of osteopontin expression may be one of the mechanisms by which inhibition of the beta-isoform of PKC confers a renoprotective effect.

Increased expression of urotensin II and urotensin II receptor in human diabetic nephropathy.

BACKGROUND: Urotensin II (UII) is an 11-amino acid vasoactive peptide, recently identified as the ligand for a novel G protein-coupled receptor, GPR-14 (renamed urotensin receptor [UT]). In addition to its potent vasoconstrictive actions, UII also has trophic and profibrotic effects, leading to its implication in the pathogenesis of heart failure. However, elevated plasma UII levels also were reported in association with renal impairment and diabetes. Accordingly, the present study sought to examine the expression and localization of UII and its receptor in kidney tissue from patients with diabetic nephropathy. METHODS: We quantified UII and UT gene expression in renal biopsy tissue samples from patients with diabetic nephropathy by using quantitative real-time polymerase chain reaction and determined the intrarenal distribution of their peptides by means of immunohistochemistry. RESULTS: In human diabetic tissue, gene expression of UII and UT were increased 45- and almost 2,000-fold in comparison to control nephrectomy tissue, respectively (P < 0.0001). Immunohistochemical studies showed intense UII peptide staining in diabetic tissue localized predominantly to tubular epithelial cells, and fluorescein-labeled ligand binding studies showed a similar tubular pattern of distribution. CONCLUSION: In the context of its known biological actions, the dramatic overexpression of UII and its receptor implicate this vasoactive peptide as a possible novel factor in the pathogenesis of diabetic nephropathy.

Plasmin is not protective in experimental renal interstitial fibrosis.

BACKGROUND: The plasminogen-plasmin system has potential beneficial or deleterious effects in the context of renal fibrosis. Recent studies have implicated plasminogen activators or their inhibitors in this process. METHODS: The development of renal interstitial fibrosis was studied in mice genetically deficient in plasminogen (plg-/- mice) and littermate controls (plg+/+ mice) by inducing unilateral ureteric obstruction (UUO) by ligating the left ureter. RESULTS: Collagen accumulation in the kidney was decreased in plg-/- mice at 21 days compared with plg+/+ mice by hydroxyproline assay (plg+/+ 19.0 +/- 1.2 microg collagen/mg tissue, plg-/- 15.6 +/- 0.5 microg collagen/mg tissue, P= 0.04). Macrophage accumulation in plg-/- mice was reduced at 21 days, consistent with a role for plasmin in macrophage recruitment in this model. Myofibroblast accumulation, assessed by the expression of alpha-smooth muscle actin (alpha-SMA), was similar in both groups at both time points. Endogenous plasmin played a role in the activation of transforming growth factor-beta (TGF-beta), as plg-/- mice had lower ratios of betaig-h3:TGF-beta1 mRNA than plg+/+ mice. Matrix metalloproteinase (MMP)-9 activity was unchanged in the absence of plasmin, but MMP-2 activity was decreased. CONCLUSION: Plasminogen, the key proenzyme in the plasminogen-plasmin system, does not protect mice from experimental interstitial fibrosis and may have significant pathogenetic effects. These findings, together with other recently published studies in the biology of renal fibrosis, imply that effects of proteins such as plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator receptor (uPAR) on renal fibrosis occur independently from the generation of plasmin.

Platelet-derived growth factor receptor transactivation mediates the trophic effects of angiotensin II in vivo.

In addition to the modulation of vascular tone, angiotensin II (Ang II) has growth factor-like effects in vascular tissue. The mechanisms whereby Ang II mediates these trophic actions are incompletely understood but are thought to include effects on systemic blood pressure, stimulation of transforming growth factor-beta (TGF-beta) expression, and transactivation of growth factor receptor kinases. To investigate the role of platelet-derived growth factor receptor (PDGFR) transactivation in mediating the growth factor-like effects of Ang II we administered Ang II (200 ng/kg per minute) or saline to male Sprague-Dawley rats by osmotic minipump for 12 days and treated with imatinib mesylate, an inhibitor of the PDGFR tyrosine kinase. In addition to systolic blood pressure elevation, Ang II infusion led to increased vascular weight, media:lumen ratio, matrix expansion, and overexpression of TGF-beta mRNA in mesenteric arteries. Without affecting blood pressure or PDGF ligand expression, imatinib attenuated the changes in vessel morphology but further increased TGF-beta mRNA. Western blot analysis of mesenteric arterial tissue demonstrated the presence of the beta- but not the alpha-isoform of PDGFR. Phosphorylation of PDGFR-beta was increased by Ang II in vascular smooth muscle cells, and this was inhibited by imatinib. The findings of attenuation of vascular hypertrophy and matrix deposition by imatinib indicate that transactivation of the PDGFR in vivo contributes to the growth factor-like effects of Ang II, independent of its hemodynamic effects or its ability to induce TGF-beta gene expression.

Mast cell infiltration and chemokine expression in progressive renal disease.

BACKGROUND: Mast cells are growth factor-rich, bone marrow-derived cells that infiltrate injured tissue where they have been implicated in the pathogenesis of progressive fibrosis. METHODS: Mast cell infiltration and the expression of related chemoattractants was examined following 5/6 nephrectomy, a model of progressive, nonimmune-mediated renal injury. In addition, expression of the profibrotic cytokine, transforming growth factor-beta (TGF-beta) within mast cells and the effects of renoprotective therapy with angiotensin-converting enzyme (ACE) inhibition were also determined. RESULTS: Renal injury was accompanied by mast cell infiltration, in close proximity to areas of tubulointerstitial fibrosis. Mast cells displayed toluidine blue metachromasia and were immunopositive for TGF-beta1 as well as chymase and tryptase. The expression of several mast cell chemokines, including stem cell factor, interleukin-8 (IL-8), and also TGF-beta1, were increased in 5/6 nephrectomized kidneys. ACE inhibition with ramipril led to a reduction in renal injury in association with attenuation of mast cell infiltration and chemokine expression. CONCLUSION: Mast cell infiltration and related chemokine expression are prominent and early features following renal mass reduction and may contribute pathogenetically to progressive renal injury.

Protein kinase C beta inhibition attenuates the progression of experimental diabetic nephropathy in the presence of continued hypertension.

In addition to hyperglycemia, hypertension and the renin-angiotensin system have been consistently implicated in the pathogenesis of diabetic nephropathy. Each of these pathogenetic factors may induce changes in cellular function by a common intracellular signaling pathway, the activation of protein kinase C (PKC) beta. The present study thus sought to determine the in vivo effect of PKC beta inhibition in experimental diabetic nephropathy in the setting of continued hyperglycemia, hypertension, and activation of the RAS. Studies were conducted in the (mRen-2)27 rat, a rodent that is transgenic for the entire mouse renin gene (Ren-2) and develops many of the structural, functional, and molecular characteristics of human diabetic nephropathy when experimental diabetes is induced with streptozotocin (STZ). Six-week-old female Ren-2 rats received an injection of STZ or vehicle and were maintained for 6 months. Within 24 h, diabetic rats were further randomized to receive treatment with the specific PKC beta inhibitor, LY333531, admixed in diet (10 mg x kg(-1) x d(-1)) or no treatment (n = 8/group). Diabetic rats developed albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis with a concomitant increase in transforming growth factor-beta (TGF-beta). Western blot analysis demonstrated increased PKC beta in diabetic animals, localized by immunofluorescence to the glomerular mesangium. In vivo inhibition of PKC beta with LY333531 led to a reduction in albuminuria, structural injury, and TGF-beta expression, despite continued hypertension and hyperglycemia.