S. Oldroyd

Transforming Growth Factor-β1 and Myofibroblasts: A Potential Pathway towards Renal Scarring in Human Glomerular Disease

Background/Aims: The cellular and humoral factors involved in the development and progression of renal scarring have not been fully investigated. Transforming growth factor-β (TGF-β1) is considered to be the main fibrogenic growth factor and it is implicated in the pathogenesis of renal fibrosis in experimental and clinical nephropathies. On the other hand, collagen III is an important component of the extracellular matrix. In this study we attempted to identify any possible links between TGF-β1 and collagen III synthesis and expression with the expression of myofibroblasts in the evolution of renal scarring in human glomerular diseases. Methods: We studied retrospectively 40 patients with various types of primary and secondary glomerulonephritis (GN), with either proliferative or nonproliferative pattern, with emphasis on the renal synthesis of TGF-β1 and collagen III (detected by in situ hybridization) and their expression (detected by immunohistochemistry) as well as myofibroblast expression. The possible links of TGF-β1 expression with myofibroblast distribution (α-smooth muscle actin, α-SMA(+) cells) and with conventional histopathology and renal function was also examined. Results: TGF-β1 protein and mRNA were detected in the renal tubular epithelial cells and interstitium and to a lesser extent within glomeruli of patients with GN. Collagen III was mainly detected in the interstitium (peritubular and periglomerular areas) and to a lesser extent in the glomeruli. Messenger RNA for collagen III followed a similar peritubular and periglomerular distribution to that of TGF-β1 and α-SMA(+) interstitial cells. The intensity of interstitial TGF-β1 protein expression was significantly related to the degree of interstitial fibrosis (r = 0.628, p < 0.01), tubular atrophy (r = 0.612, p < 0.01), interstitial collagen III expression (r = 0.478, p < 0.05), and serum creatinine values (r = 0.722, p < 0.001). Also there was a close positive correlation between the severity of interstitial myofibroblast expression and interstitial TGF-β1 (r = 0.412, p < 0.05), as well as collagen III (r = 0.409, p < 0.05). In addition, a significant correlation was found between glomerular TGF-β1 expression and severity of glomerulosclerosis (r = 0.620, p < 0.01). Conclusion: The results of this study suggest that TGF-β1 plays an important role in the pathogenesis of fibrosis developing in human kidney, during the evolution of glomerular disease. Interstitial myofibroblasts may contribute to interstitial fibrosis through the synthesis and release of both TGF-β1 and collagen III.

Expression of cytoskeletal proteins during the course of experimental diabetic nephropathy

Aims/hypothesis: Diabetic nephropathy is characterised by structural changes known to be associated in non-diabetic nephropathies with the expression of the cytoskeletal proteins a-smooth muscle actin and vimentin. We aimed to investigate the expression of cytoskeletal proteins in experimental diabetic nephropathy.¶Methods. Rats were made diabetic by an injection of streptozotocin (45 mg/kg). Groups of rats (n = 6) and their respective controls (n = 4) were killed at different time intervals. (days 7, 15, 30, 60, 90 and 120). We also studied two groups of diabetic rats treated with a long-acting insulin; the first (n = 8) was treated from the induction of diabetes and the second (n = 8) received insulin from day 15 onward. At each time-point, kidney function, proteinuria and histology were evaluated. Cytoskeletal proteins and collagens III and IV deposition was determined by immunohistochemistry. Changes in the transcription of the cytoskeletal proteins was determined by northern blot analysis.¶Results. Although normal glomeruli did not express α-smooth muscle actin until late in the time course, it was detected in diabetic mesangium from day 7 onward. In the interstitium, it appeared in a perivascular and peritubular distribution. Vimentin was detectable within normal glomerular epithelial cells and increased rapidly (days 7 and 15) in diabetic rats. Vimentin also appeared early within the lining of the peritubular capillaries and damaged diabetic tubules. These changes were associated with a delayed increased transcription of α-smooth muscle actin and vimentin. Treatment with insulin (early or late) attenuated and reversed respectively the expression of cytoskeletal proteins and collagens within diabetic kidneys. Close correlations were noted between the number of α-smooth muscle actin positive cells within diabetic glomeruli and mesangial expansion (r = 0.46, p < 0.02) as well as interstitial α-smooth muscle actin positive cells and interstitial fibrosis (r = 0.51, p < 0.002).¶Conclusion/interpretation. Changes in the expression of cytoskeletal proteins within the kidneys of diabetic rats suggest a role for α-smooth muscle actin and vimentin in the pathogenesis of diabetic kidney disease. [Diabetologia (2000) 43: 91–100]

Role of apoptosis and Bcl-2/Bax in the development of tubulointerstitial fibrosis during experimental obstructive nephropathy.

Background/Aims: To examine the role of apoptosis in experimental unilateral ureteral obstruction (UUO). Methods: Rat kidneys were examined 3, 7 and 11 days following UUO or sham operation (SO). Tissue was immunohistochemically stained for α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), Bcl-2 and Bax proteins. Apoptotic analysis was carried out in kidney sections using in situ end labelling of endonuclease cleaved DNA. Results: The relative volume (Vv) of cortical interstitium and interstitial α-SMA increased progressively following UUO. ED1-positive monocytes/macrophages peaked at day 7 and significantly decreased at day 11. PCNA-positive cells in tubulointerstitium were significantly increased on day 3. Staining returned to the level of the SO group by day 11, meanwhile those in the interstitium remained much higher than baseline. TUNEL-positive cells were persistently raised following UUO. Transient tubular cell proliferation seemed unable to counteract the apoptosis since tubular atrophy was apparently present by day 11 of UUO. However, interstitial cell proliferation was high enough to overwhelm apoptosis, particularly with respect ot myofibroblasts, since α-SMA immunostaining and Vv remained elevated. The ratio of the number of PCNA-positive cells to apoptotic cells formed a predictive pattern for the staining score of interstitial α-SMA (R2 = 47.23%, p < 0.05) and Vv (R2 = 49.93%, p < 0.05). Tubular Bcl-2 immunostaining peaked on day 3, and then gradually decreased to baseline by day 11. The expression of Bax protein was inhibited on day 3 when compared with that of the SO group, but increased with time following UUO. Conclusion: These findings suggest an important role for apoptosis and its regulatory proteins in the processes of tubular atrophy and fibrogenesis following UUO.

The acute effect of ioversol on kidney function: role of endothelin

The effect of ioversol, a non-ionic monomer with high hydrophilicity, on renal function was studied using the isolated perfused rat kidney (IPRK). The involvement of endothelin in the renal effect of ioversol was established pharmacologically using the selective endothelin ETA receptor antagonist BQ123. Ioversol 20 mgI/ml produced a sustained fall in both renal perfusate flow (RPF) and the glomerular filtration rate (GFR) together with a fall in sodium reabsorption (FRNa) and increase in urine flow (n = 6). In the presence of BQ123 (10 microM), the effect of ioversol 20 mgI/ml on GFR was completely abolished and the fall in RPF and FRNa markedly reduced (n = 6). These results suggest that effect of ioversol on renal haemodynamics in the IPRK is mediated by endothelin. Ioversol produced a significantly smaller decrease in GFR than iopromide, a contrast media with similar osmolality but lower hydrophilicity, when compared to a previous study using an identical experimental technique. Increased hydrophilicity may therefore present an advantage for ioversol, reducing its effects on renal function.

Relationship between the diuretic effect of radiocontrast media and their ability to increase renal vascular resistance

The relationship between diuresis and natriuresis induced by radiocontrast media (RCM) and their renal haemodynamic effects were investigated. The effects of the iso-osmolar iotrolan and the hyperosmolar diatrizoate on the renal vascular resistance (RVR) were studied in the filtering and non-filtering variants of the isolated perfused rat kidney (IPRK) preparation. In the non-filtering model, no tubular regulatory process can be activated. The effect of diatrizoate on the RVR of the filtering IPRK in the presence of fursemide (0.3 mmol l-1) an inhibitor of the tubuloglomerular feedback (TGF) was also investigated. There was no significant difference (p > 0.05) in the response of the filtering (n = 6) and non-filtering (n = 6) IPRK to iotrolan. The induced reduction in the renal perfusate flow (RPF) by iotrolan was 20.5 +/- 3.05% and 22.9 +/- 3.03%, respectively. The reduction in the RPF which was observed with diatrizoate in the non-filtering IPRK (n = 5, 17.5 +/- 3.04%) was significantly less (p < 0.05) in comparison to that of the filtering IPRK (n = 6, 26.9 +/- 4.28%). In the frusemide experiments, a reduction in the RPF comparable to that of the non-filtering kidney was observed (n = 5, 13.7 +/- 4.34%). This study demonstrates that the renal vascular effect of diatrizoate is partially dependent on the TGF response. No tubular regulatory mechanism was accountable for the haemodynamic effect of iotrolan. The activation of the tubular response is osmolarity dependent.

Reduced depression of renal function by iotrolan in the isolated rat kidney

The direct effects of iotrolan, a non-ionic dimer, on renal function were compared to iopromide, a non-ionic monomer and diatrizoate, an ionic monomer using the isolated perfused rat kidney. Kidneys were perfused ex vivo at 100 mmHg in a recirculating perfusion system with an albumin-based perfusate containing angiotensin II. All contrast media were studied at starting concentration of 20 mg Iodine/ml of renal perfusate. Each contrast agent produced a biphasic effect on the glomerular filtration rate (GFR) characterised by a transient increase followed by a sustained fall. However, the sustained fall in GFR induced by iotrolan (-24.7 +/- 2.1%) was significantly smaller than that produced by diatrizoate (-40.6 +/- 3.5%, P < 0.05) but there was no significant difference in comparison to the fall induced by iopromide (-34.2 +/- 3.7%). Each contrast agent produced a sustained decrease in renal perfusate flow (RPF) with iotrolan exerting a significantly smaller response (-21.7 +/- 2.0%) than either diatrizoate (-29.4 +/- 2.6%, P < 0.05) or iopromide (-32.2 +/- 2.9%, P < 0.05). The results indicate that at an equivalent iodine concentration iotrolan produces a smaller reduction of renal function in comparison to either iopromide or diatrizoate.