Becker Gj

Clinical features of Fabry's disease in Australian patients

Abstract

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.

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.

Extracellular matrix components in Kimmelstiel Wilson nodules

Summary: The distribution of collagen types I, III, IV, V, VI, fibronectin laminin, tenascin, heparan sulfate proteoglycan and chondroitin sulphate proteoglycan was examined by light microscopy immunocytochemistry (ICC) in paraffin‐embedded renal biopsies with nodular diabetic glomeruloscierosis. Immunoglobulin A, G and M, complement (C3c and C1q), fibrinogen and k and γ light chain deposition was also sought by ICC in the same tissue.

Pregnancy-Related Complications in Women With Reflux Nephropathy

Three hundred and forty-five pregnancies in 137 women with reflux nephropathy have been studied. All pregnancies took place after 1971. Overall foetal loss was 48 (14%) of which only 6 (2%) were therapeutic abortions. Maternal complications (urine infection, hypertension, proteinuria, oedema, deterioration in renal function, hematuria or renal stones) occurred alone or in combination in 39% of pregnancies. Fifty-two pregnancies took place in women with plasma creatinine (P.Cr > 0.11 mmol/l) prior to conception. Foetal loss after 12 weeks gestation (excluding therapeutic abortions) was 18% compared with 8% in the 104 pregnancies where maternal P.Cr was known to be < or = o.11 per/l at conception (p < 0.05). Maternal complications were also more common in the impaired renal function group (p < 0.001). Comparison of pregnancies in women with unilateral versus bilateral renal scarring revealed no significant difference in foetal loss but an increased incidence of over 50% maternal complications in the bilateral renal scar group (p < 0.01). The incidence of pre-eclampsia was higher in women with bilateral renal scars, 50 (24%) than in women with unilateral scars 8 (7%) (p < 0.001). Persistent vesicoureteric reflux was not associated with increased foetal loss or maternal risk. Impaired renal function prior to conception is associated with increased foetal and maternal complications in pregnancy. Bilateral renal scarring is associated with increased maternal complications during pregnancy.