An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization.

Vascular calcification (VC) is seen ubiquitously in aging blood vessels and prematurely in disease states like renal failure. It is thought to be driven by a number of systemic and local factors that lead to extra-osseous deposition of mineral in the vascular wall and valves as a common endpoint. The response of resident vascular smooth muscle cell to these dystrophic signals appears to be important in this process. Whilst in vivo models allow the observation of global changes in a pro-calcific environment, identifying the specific cells and mechanisms involved has been largely garnered from in vitro experiments, which provide added benefits in terms of reproducibility, cost, and convenience. Here we describe a 7-21 day cell culture model of calcification developed using immortalized murine vascular smooth muscle cells (MOVAS-1). This model provides a method by which vascular smooth muscle cell involvement and manipulation within a mineralizing domain can be studied.

Explanting is an ex vivo model of renal epithelial-mesenchymal transition.

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.

Cell-populated floating collagen lattices: an in vitro model of parenchymal contraction.

The pathology of progressive renal disease is characterized by glomerular and interstitial inflammation, glomerulosclerosis, and tubulointerstitial fibrosis. This is a consequence of excessive matrix synthesis, reduced matrix degradation, and contraction (reorganization) of extracellular matrix. Fibroblasts, and to a lesser degree, other mesenchymal cells, are known to contribute to renal scar formation through local proliferation, synthesis, and reorganization of matrix proteins. Although much work has focused on the balance between collagen synthesis and degradation, the mechanisms of parenchymal collapse and contraction are becoming increasingly important. Like their counterparts in the skin, the contractile properties of renal fibroblasts are now well recognized. This chapter details an in vitro method for studying the contraction of collagens by homogeneous populations of cultured cells. The method can be altered so that reagents influencing this process may also be studied.

Histochemical localization of cell proliferation using in situ hybridization for histone mRNA.

Monoclonal antibodies to proliferation associated antigens have long been used to histologically localize mitogenesis. However, techniques that distinguish cells in the synthetic or S phase have tended to rely on the in vivo incorporation of tritiated thymidine or thymidine analogs such as bromodeoxyuridine. The necessity to pulse with these labels before retrieving tissue means that they cannot be used in humans and are not available retrospectively. Measuring expression of histones serves as a useful adjunct to these techniques. As expression of histone proteins (H2A, H2B, H3, H4) are restricted to the synthetic phase of the cell cycle, hybridization for histone mRNA precisely distinguishes those cells in the S phase. Measuring their expression can easily be applied to the histological localization of proliferation, and can be used both prospectively and with archived tissue specimens. Several histone in situ hybridization probes and nonradioactive detection systems are now available commercially. A generalized protocol for their use in measuring in situ proliferation is provided in this chapter.

Effect of inhibition of farnesylation and geranylgeranylation on renal fibrogenesis in vitro.

BACKGROUND: The Ras and Rho family of GTPases serve as essential molecular switches in the downstream signalling of many cytokines involved in the regulation of renal fibroblast activity. Prenylation is a post-translational process critical to the membrane localization and function of these GTPases. We studied the effects of a farnesyltransferase inhibitor BMS-191563 and geranylgeranyltransferase inhibitor GGTI-298 on renal fibrogenesis in vitro. METHODS: Functional studies examined the effects of BMS-191563 and GGTI-298 on rat renal fibroblast kinetics, collagen synthesis and collagen gel contraction. Pro-collagen alpha1(I) mRNA expression was measured by Northern analysis and CTGF expression by Western blotting. RESULTS: Fibroblast proliferation was significantly reduced by both agents. Exposure of fibroblasts to BMS-191563 resulted in a significant reduction in total collagen production and pro-collagen alpha1(I) mRNA expression, an effect also observed but to a lesser degree with GGTI-298. Both agents significantly reduced CTGF protein expression. Fibroblast-mediated collagen I lattice contraction was decreased at 48 h by GGTI-298, an effect not observed with BMS-191563. Consistent with this finding, marked actin filament disassembly was evident by phalloidin staining of fibroblasts exposed to GGTI-298. CONCLUSION: BMS-191563 and GGTI-298 exhibit different effects on renal fibroblast function reflecting their predominant roles in inhibiting prenylation of Ras or Rho proteins respectively. Further studies are warranted to establish their potential therapeutic application in the treatment of progressive renal disease.

Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro.

BACKGROUND: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS: Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS: H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS: The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.

Parathyroid hormone has a prosclerotic effect on vascular smooth muscle cells.

Although accelerated atherosclerosis and arteriosclerosis are common in patients with renal failure, the pathogenesis of these changes is poorly understood. Parathyroid hormone (PTH) levels are elevated in renal failure, and have been linked to uraemic vascular changes in some studies. We examined the in vitro effects of increasing doses of the 1-34 fragment of PTH on human aortic vascular smooth muscle cells (VSMCs). Factors examined were: (1) collagen production using tritiated hydroxyproline incorporation and transcription of procollagen alpha(1)(I) mRNA; (2) change in the surface area of collagen I lattices; (3) mRNA transcription of the collagen binding protein beta1 integrin; (4) proliferation using tritiated thymidine incorporation, and (5) methyl tetrazolium salt conversion to estimate live cell number after 5 days' exposure to PTH. PTH at a concentration of 200 pmol/l increased total collagen synthesis (188 +/- 25% of control, p < 0.01) as well as transcription of procollagen alpha(1)(I) mRNA (136 +/- 11% of control, p < 0.005). PTH also increased reorganisation of collagen I lattices (surface area 47 +/- 8% of well for control vs. 35.7 +/- 2.5 and 34.3 +/- 3.0% for PTH 100 and 200 pmol/l, respectively, p = 0.02) and upregulated beta1 integrin mRNA expression (160 +/- 20% of control at PTH concentration of 200 pmol/l, p < 0.05). PTH had no effect on VSMC proliferation or number at doses up to 200 pmol/l. In conclusion, PTH increases production and reorganisation of collagen by VSMCs in vitro. It is possible that more aggressive control of hyperparathyroidism in patients with renal failure may help to reduce the burden of cardiovascular disease in this patient population.

Dipyridamole inhibits in vitro renal fibroblast proliferation and collagen synthesis.

Fibroblasts are universally recognized in situations of tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. The objective of this study was to determine whether the functions of fibroblasts relevant to fibrogenesis can be modified in vitro with dipyridamole. Cells were obtained from obstructed rat renal tissue and characterized on the basis of immunohistochemical findings. Fibroblasts constituted all of the cells from passage 3. Functional parameters were measured in cells cultured with 1, 5, and 50 micromol/L dipyridamole and compared to basal parameters of cells grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum (control). Northern-blot analysis indicated that dipyridamole decreased procollagen alpha1(I) messenger ribonucleic acid expression (P <.05, 50 micromol/L vs control), results that were reflected in a reduction in total collagen secretion as measured on the basis of hydroxyproline incorporation (P <.001, 50 micromol/L vs control). Mitogenesis, as measured on the basis of incorporation of tritiated thymidine, was decreased in a dose-dependent fashion by dipyridamole. Likewise, 50 micromol/L dipyridamole reduced cell-population growth to 16.8% +/- 2.1% of basal growth over 3 days (P <.001 vs control). Effects of dipyridamole on population growth were prevented by coincubation with a protein kinase G inhibitor peptide (P <.001 vs 50 micromol/L dipyridamole; P = not significant vs control). No such effect was observed with inhibitors for protein kinase A (H-89) and protein kinase C (bisindolylmaleimide I). Consistent with a protein kinase G-dependent mechanism, immunofluorescence staining indicated that dipyridamole increased basal expression of the inducible form of nitric oxide synthase. In conclusion, the results of this study demonstrate that at clinically relevant concentrations, dipyridamole inhibits profibrotic activities of renal fibroblasts. Effects on mitogenesis are mediated through a cyclic guanosine monophosphate-protein kinase G effector pathway.

Lovastatin downregulates renal myofibroblast function in vitro.

Interstitial fibrosis is recognised as the best histological predictor of progressive renal disease. Myofibroblasts contribute to this process through several functions including hyperproliferation, collagen and collagenase synthesis and reorganisation of extracellular matrix. Recent limited in vitro studies suggest that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors may reduce renal injury not only through their lipid-lowering effects but also by antagonising myofibroblast function. This study therefore examined the effects of lovastatin on the above interstitial myofibroblast behaviours in vitro. Primary cultures of rat renal cortical myofibroblasts were grown by explantation and characterised by immunohistochemistry. Dose response effects of lovastatin (0, 15, 30 microM) in DMEM and 10% FCS were examined on myofibroblast kinetics, total collagen synthesis, collagen I lattice contraction and actin filament rearrangement. Lovastatin decreased myofibroblast proliferation and growth. Likewise, collagen I lattice contraction and actin filament rearrangement were partially inhibited when lovastatin was added at 30 microM. In addition, lovastatin decreased both collagen and collagenase synthesis. Our results suggest that myofibroblast function may be downregulated by lovastatin in vitro. Although a decrease in myofibroblast activity may offer potential benefit in the prevention of progressive scarring, further studies will be necessary to determine the relative importance of these functions.