Heather J. Saunders

Regulation of insulin-like growth factor-binding protein-1 in rat serum.

Previous studies support a role for insulin-like growth factor–binding protein-1 (IGFBP-1) in modulating insulin-like growth factor (IGF) availability for glucose homeostasis. We have developed a radioimmunoassay (RIA) for rat IGFBP-1 (rIGFBP-1) and have examined the regulation of circulating levels by nutritional and hormonal status. Rabbit antisera were raised against pure rIGFBP-1, and an assay was established with a sensitivity of 50 pg. In the rat, serum IGFBP-1 concentrations decrease with increasing developmental age. They were highest in fetal rat serum, exceeding 4 mg/L, and decreased to <0.1 mg/L in adult animals. Serum rIGFBP-1 levels increased during fasting, 6-fold after 24 h and 18-fold after 48 h, and were suppressed to levels identical to ad libitum–fed control rats within 2 h of refeeding. Fasting levels were >2-fold higher in female than male animals. IGFBP-1 concentrations were suppressed by >50% in two rat models of insulin resistance. Levels increased in STZ-induced (streptozotocin) diabetes and were suppressed to normal with insulin treatment. Exercise stimulated rIGFBP-1 concentrations in fasting animals. On immunoblotting after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), rIGFBP-1 in serum appeared as a doublet with molecular masses at 31 and 33 kD. The components of this doublet did not vary across the range of experimental conditions. These observations indicate that the pattern of regulation of rIGFBP-1 is similar to that seen in previous studies of human IGFBP-1, with age, sex, and nutritional status being important regulators. The availability of appropriate rat models and a sensitive ria will allow additional study of the regulatory mechanisms and the physiologucal role of IGFBP-1.

Fibrogenic Effects of Cyclosporin A on the Tubulointerstitium: Role of Cytokines and Growth Factors

The clinical utility of cyclosporin A (CyA) as an immunosuppressive agent has been significantly limited by the frequent occurrence of chronic nephrotoxicity, characterised by tubular atrophy, interstitial fibrosis and progressive renal impairment. The pathogenesis of this condition remains poorly understood, but has been postulated to be due to either direct cytotoxicity or indirect injury secondary to chronic renal vasoconstriction. Using primary cultures of human proximal tubule cells (PTCs) and renal cortical fibroblasts (CFs) as an in vitro model of the tubulointerstitium, we have been able to demonstrate that clinically relevant concentrations of CyA are directly toxic to these cells and promote fibrogenesis by a combination of suppressed matrix metalloproteinase activity and augmented fibroblast collagen synthesis. The latter effect occurs secondary to the ability of CyA to stimulate autocrine secretion of insulin-like growth factor-I by CFs and paracrine secretion of transforming growth factor-β1 by PTCs. Many of these pro-fibrotic mechanisms are completely reversed by concurrent administration of the angiotensin-converting enzyme inhibitor, enalaprilat, which has proven efficacy in preventing chronic CyA nephropathy in vivo. These studies highlight the unique potential that human renal cell cultures offer for studying the role of local cytokine networks in tubulointerstitial disease and for developing more effective treatment strategies which specifically target fibrogenic growth factor activity following nephrotoxic injuries.

Enalaprilat Directly Ameliorates in vitro Cyclosporin Nephrotoxicity in Human Tubulo-Interstitial Cells

Background/Aims: Several recent studies have suggested that angiotensin-converting enzyme (ACE) inhibitors ameliorate chronic cyclosporin A (CyA) tubulo-interstitial disease by mechanisms independent of their antihypertensive effects. The aim of the present study was to determine whether ACE inhibition exerts a direct beneficial effect on the tubulo-interstitium in an in vitro model of chronic CyA nephropathy. Methods: Primary cultures of human proximal tubular cells (PTC) and renal cortical fibroblasts (CF) were exposed for 24 h to CyA in the presence or absence of enalaprilat. Parameters of tubulo-interstitial nephrotoxicity were then measured including collagen synthesis (proline incorporation), tubular viability and function (thymidine incorporation, lactate dehydrogenase release, and apical sodium-hydrogen exchange), and secretion of insulin-like growth factor I, transforming growth factor beta 1 (TGFβ1), and platelet-derived growth factor. Results: CyA promoted CF collagen synthesis, PTC cytotoxicity (suppressed viability, growth and sodium transport), and tubulo-interstitial fibrogenic cytokine release (CF secretion of insulin-like growth factor I and PTC secretion of TGFβ1 and platelet-derived growth factor). Enalaprilat completely reversed the stimulatory effects of CyA on CF collagen synthesis (CyA + enalaprilat 6.40 ± 0.50% vs. CyA alone 8.33 ± 0.56% vs. control 6.57 ± 0.62% vs. enalaprilat alone 5.55 ± 0.93%, p < 0.05) and PTC secretion of TGFβ1 (0.71 ± 0.11, 1.13 ± 0.09, 0.89 ± 0.07, and 0.67 ± 0.09 ng/mg protein/day, respectively, p < 0.05). However, the other manifestations of CyA toxicity were not significantly reversed by concomitant enalaprilat administration. Conclusions: ACE inhibition directly prevents CyA-induced interstitial fibrosis, but not proximal tubule cytotoxicity, independently of haemodynamic and systemic renin-angiotensin system effects. Renoprotection may be partially afforded by directly preventing the tubular secretion of TGFβ1.

Role of insulin-like growth factor binding proteins in human post-nephrectomy proximal tubule cells

1 In order to determine the role of the insulin‐like growth factor‐I (IGF‐I)/IGF binding protein (IGFBP) axis in the augmentation of tubule growth and function following reductions in nephron mass, primary cultures of human proximal tubule cells (PTCs) were generated from the histologically normal sections of ten surgically removed kidneys. 2 PTC hypertrophy (cellular protein content), DNA synthesis (thymidine incorporation) and apical sodium‐hydrogen exchange (NHE) activity (ethylisopropylamiloride‐sensitive apical 22Na+ uptake) were measured following 24 h incubation in media supplemented with 10 % pre‐ or post‐nephrectomy sera obtained from these patients. The results were compared with the effects of pre‐ and post‐operative control sera collected from seven patients undergoing retroperitoneal operations not involving removal of renal tissue. 3 Day 1 post‐nephrectomy sera promoted a significant 73 % increase in apical NHE activity, which was accompanied by a significant increase in PTC binding of 125I‐IGF‐I (post‐ vs. pre‐nephrectomy, 163 ± 6 vs. 142 ± 4 fmol (mg protein)−1; P < 0.05). Subsequent post‐nephrectomy sera significantly stimulated PTC protein content and thymidine incorporation, peaking at day 7 (127.7 ± 14.0 and 118.4 ± 9.0 % of pre‐nephrectomy values, respectively; P < 0.05). The growth effects were cell specific, as they were not observed with renal cortical fibroblasts. No change was detected in any of these measured variables following exposure to control sera. 4 Serum IGF‐I and IGFBP‐1 levels did not significantly change over time or between groups. IGFBP‐3 levels progressively decreased in both control and nephrectomized sera from pre‐operative values of 3580 ± 305 and 3360 ± 217 ng ml−1, respectively, to 2670 ± 341 and 2600 ± 347 ng ml−1 at 1 week post‐operation. Serum IGFBP‐2 levels increased to a comparable extent in both controls (day 0 vs. day 7, 2940 ± 1024 vs. 7010 ± 2520 ng ml−1; P < 0.01) and nephrectomized patients (day 0 vs. day 7, 3070 ± 656 vs. 9130 ± 2010 ng ml−1; P < 0.01). 5 The results indicate that nephrectomy engenders the elaboration of one or more humoral factor(s), which promotes increased binding of IGF‐I to PTCs and which may in turn specifically stimulate PTC Na+ transport and growth.