Kristen J. Kelynack

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 α1(I) mRNA; (2) change in the surface area of collagen I lattices; (3) mRNA transcription of the collagen binding protein β1 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 α1(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 β1 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.

Pentoxifylline Reduces in vitro Renal Myofibroblast Proliferation and Collagen Secretion

Interstitial myofibroblasts (MF) are cells with features of both smooth muscle cells and fibroblasts. They have been 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 if functions of MF relevant to fibrogenesis can be modified in vitro by the phosphodiesterase inhibitor pentoxifylline (PTX). MF were obtained from sub-culture of normal rat kidney explant outgrowths maintained in DMEM + 20% fetal calf serum (FCS), supplemented with antibiotics. Cells were characterized on the basis of growth characteristics and immunohistochemistry. MF constituted >95% of cells at passage 3. Cell culture media was supplemented with the potential antagonist PTX alone (0, 1, 10, 100 μg/ml) and in combination with TGFβ1 (5 ng/ml). Population kinetics, proliferation and collagen production were determined from cell growth, [3H]thymidine incorporation and [3H]proline incorporation in collagenous proteins, respectively. Both serum-stimulated population growth and proliferation were reduced in a linear fashion by 1, 10 and 100 μg/ml PTX (all p < 0.05 versus 0 μg/ml). Effect of PTX on cell population growth was however reversible when PTX was removed. Basal collagen secretion was decreased by PTX at 10 and 100 μg/ml (p < 0.05 versus 0 μg/ml), although cell layer collagen remained unchanged. Collagen production (secreted and cell layer) was augmented by 5 ng/ml TGFβ1. These effects on collagen production were partially reduced when 100 μg/ml PTX was added. The authors conclude that myofibroblast function can be altered with agonists/antagonists. Attempts to down-regulate fibrogenic functions of MF may therefore offer a valuable therapeutic strategy.

Intracellular Cyclic Nucleotide Analogues Inhibit in vitro Mitogenesis and Activation of Fibroblasts Derived from Obstructed Rat Kidneys

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10– 1,000 µM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for α-smooth muscle actin; α-SMA) and expression of CTGF (Northern blotting), a TGF-β1-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 ± 4% by 100 µM 8br-cAMP (p < 0.01), 49 ± 6% by 1,000 µM 8br-cAMP (p < 0.001) and 43 ± 7% by 1,000 µM 8br-cGMP (p < 0.01). 1,000 µM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 ± 5 and 60 ± 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker α-SMA by 43 ± 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 ± 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.

Renal Myofibroblasts Contract Collagen I Matrix Lattices in vitro

Myofibroblasts, cells with both fibroblastic and smooth muscle cell features, have been implicated in renal scarring. In addition to synthetic properties, contractile features and integrin expression may allow myofibroblasts to rearrange and contract interstitial collagenous proteins. Myofibroblasts from normal rat kidneys were grown in cell-populated collagen lattices to measure cell generated contraction. Following detachment of cell populated collagen lattices, myofibroblasts progressively contracted collagen lattices, reducing lattice diameter by 42% at 24 h. Alignment of myofibroblasts, rearrangement of fibrils and β1 integrin expression were observed within lattices. We postulate that interstitial myofibroblasts contribute to renal scarring through manipulation of collagenous proteins.

Thrombin is a pro-fibrotic factor for rat renal fibroblasts in vitro.

Background: Generation of thrombin occurs in response to parenchymal injury. Thrombin not only converts plasma fibrinogen into an insoluble fibrin clot, but also potentially augments inflammation through receptor-mediated activity. This study examines whether thrombin may potentially exacerbate fibrosis by upregulating the function of interstitial fibroblasts in vitro. Methods: Fibroblasts were isolated by explant outgrowth culture of rat kidneys. Subcultured cells were grown in DMEM+10% FCS supplemented with 0.1–0.5 U/ml thrombin. Functional parameters examined included kinetics (thymidine incorporation and change in cell number), differentiation (Western blotting for α-smooth muscle actin; αSMA), expression of procollagen α1(I) (Northern blotting) and contraction of collagen I lattices. RT-PCR was used to characterise expression of protease-activated receptors (PAR) previously implicated in thrombin’s cellular effects. Results: Cell population growth was increased 66 ± 41 and 47 ± 41% by 0.1 and 0.5 U/ml thrombin respectively (both p < 0.05 vs. basal). Likewise, 0.5 U/ml thrombin increased corrected procollagen α1(I) expression 2.4-fold (p < 0.05 vs. basal) and exacerbated the ability of fibroblasts to contract collagen matrix (p < 0.05 vs. basal). These effects were not associated with any change in expression of the myofibroblast marker αSMA. Effects on cell number were inhibited by treatment with (D)-Phe-Pro-Arg-chloromethylketone HCl (PPACK) suggesting that functional effects were mediated by serine protease activity. PAR-1 was the only fully functional known thrombin receptor expressed by these cells. Conclusion: Thrombin is a potential unrecognised fibroblast agonist in renal disease. Further studies of thrombin and its receptors may yield valuable insights into the pathogenesis of interstitial fibrosis.

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 α1(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 α1(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.

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 µM) 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 µM. 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.

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.

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 kinetics and tissue contraction following renal parenchymal cell death

The kinetics of interstitial extracellular matrix remodelling in the kidney following parenchymal cell death were studied after injury was produced by a single application of liquid nitrogen to the surface of the kidney. The size of the injury decreased to 30% of the original size at 4 days and to 5% at 16 days after injury. The primary response to injury consisted of an acute polymorphonuclear infiltration followed by monocyte/macrophage and myofibroblast accumulation from day 2 to 8. Collagen III accumulation increased progressively until day 16 and was inversely related to lesion size. These results demonstrate the time course of cellular events and collagen synthesis in renal tissue remodelling following renal parenchymal cell death.