A bstract : Progressive fibrosis due to excess extracellular matrix (primarily collagen) is the final common pathway in all forms of chronic renal disease, regardless of etiology, and leads to tissue dysfunction, when normal tissue is replaced by scar tissue. Emerging work from ourselves and others suggests that the naturally occurring hormone relaxin has the potential to limit renal collagen production and reorganization, while increasing its degradation. The outlined studies demonstrate relaxin's potential as an antifibrotic agent against experimental progressive renal disease.
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 α<sub>1</sub>(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 α<sub>1</sub>(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.
Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine transforming growth factor-β1 (TGF-β1) on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Fibroblasts propagated from rat kidneys after ureteric obstruction were treated with recombinant TGF-β1 or vehicle for 48 h. TGF-β1-induced myofibroblast activation was accompanied by a net decrease in total H3 acetylation, although changes in individual marks were variable. This was paralleled by a generalized reduction in histone acetyltransferases (HAT) and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally, this was manifest in a reduction in total HAT activity and increase in HDAC activity. TGF-β1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis, resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate or alternative sources of acetyl-CoA without ameliorating changes in HAT/HDAC activity. In conclusion, TGF-β1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetylation, increased deacetylation, and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-β1.
Quantitative analysis of messenger RNA (mRNA) from tissue homogenates, cell extracts, or fixed tissue sections is vital for studies involving gene regulation and expression. Quantitative analysis of mRNA allows the investigator to establish the transcription level of particular genes either in relative or absolute terms. Traditionally Northern blot analysis is a comparative technique that detects the amount of mRNA of the gene of interest normalized to the amount of a housekeeping gene (,). This is generally performed by densitometry of band intensities from an autoradiograph. The method has been employed for more than two decades, and its relative simplicity has made it the first port of call when examining RNA expression. It is particularly useful for examining mRNA transcription in tissue/cells exposed to various treatments. While Northern analysis will inform the investigator of the size and relative abundance of the mRNA of interest, it is a limited technique. Some of the limitations include: inefficient transfer of the RNA to the filter, its relative insensitivity for examining smaller quantities of mRNA and the saturable nature of autoradiography when film is used (). The last limitation has been overcome by phos-phorimaging, which allows measurement of band intensity in a linear fashion. Therefore, Northern blotting is best used as a semiquantitative method of examining relatively abundant mRNA.
SUMMARY 1. Animals and histology from two previous investigations were used to test the hypothesis that a similar elevation in blood pressure may result in a different sequence of pathological changes in different experimental models of hypertension, DOCA‐salt and aortic‐ligature hypertension. 2. To assess differences in morphological parameters, individual animals in the two groups were paired ( n = 12) for the same level of blood pressure at sacrifice. 3. Vascular damage was significantly less in the DOCA‐salt group ( P < 0.01). Glomerular lesions however were more severe in the DOCA‐salt group. In rats with aortic‐ligature hypertension significantly fewer glomeruli had fibrinoid and/or crescents than in DOCA‐salt rats ( P < 0.001). There were fewer glomeruli with epithelial cell droplets (ECD) and fewer ECD per glomerulus in aortic‐ligature when compared with DOCA‐salt hypertension ( P < 0.001, P < 0.001, respectively). 4. This study highlights the significance of factors other than blood pressure per se in producing vessel and glomerular lesions in experimental hypertension.
Chronic Kidney Disease (CKD) is characterized by organ remodeling and fibrosis due to failed wound repair after on-going or severe injury. Key to this process is the continued activation and presence of matrix-producing renal fibroblasts. In cancer, metabolic alterations help cells to acquire and maintain a malignant phenotype. More recent evidence suggests that something similar occurs in the fibroblast during activation. To support these functions, pro-fibrotic signals released in response to injury induce metabolic reprograming to meet the high bioenergetic and biosynthetic demands of the (myo)fibroblastic phenotype. Fibrogenic signals such as TGF-β1 trigger a rewiring of cellular metabolism with a shift toward glycolysis, uncoupling from mitochondrial oxidative phosphorylation, and enhanced glutamine metabolism. These adaptations may also have more widespread implications with redirection of acetyl-CoA directly linking changes in cellular metabolism and regulatory protein acetylation. Evidence also suggests that injury primes cells to these metabolic responses. In this review we discuss the key metabolic events that have led to a reappraisal of the regulation of fibroblast differentiation and function in CKD.
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 TGFβ<sub>1</sub> (5 ng/ml). Population kinetics, proliferation and collagen production were determined from cell growth, [<sup>3</sup>H]thymidine incorporation and [<sup>3</sup>H]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 TGFβ<sub>1</sub>. 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.
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