Long-term evolution of the acute tubular necrosis (ATN) induced by glycerol: role of myofibroblasts and macrophages

Abstract

Late structural changes such as interstitial fibrosis in the renal cortex and tubular atrophy have been detected after severe acute tubular necrosis (ATN). The aim of this study was to investigate the expression of fibronectin, alpha-smooth muscle actin and macrophages during the evolution of the ATN induced by glycerol and their relationship with the late structural changes observed in the kidneys of these animals. Forty-nine male Wistar rats were injected with a 50% glycerol solution, 8 mL/kg (4 mL/kg applied i.m. to each hind leg) and 14 with 0.15 m NaCl solution. Before glycerol injection on day 1, water was removed for 17 h. Blood and urine samples were collected 1 day after the injection to quantify sodium and creatinine. The animals were killed 5, 30 and 60 days after the injections and the kidneys removed for histological and immunohistochemical studies. The results of the histological and immunohistochemical studies were scored according to the extent of lesion or staining in the cortical tubulointerstitium, respectively. The percentage of tubulointerstitial lesions was determined by morphometry. Glycerol-injected rats presented a transitory increase in plasma creatinine levels and in fractional sodium excretion. The immunohistochemical studies showed increased fibronectin, alpha-smooth muscle actin (alpha-SM-actin), TGF-beta and ED-1 (macrophages) staining in the renal cortex from rats killed 5, 30 and 60 days after glycerol injection (P < 0.05) compared to control. The animals killed on day 30 and 60 also presented chronic lesions (fibrosis, tubular dilatation and atrophy) in the renal cortex, despite the recovery of renal function. Macrophages, TGF-beta and myofibroblasts may have contributed to the development of renal fibrosis in these rats.

Figure 1

Masson trichrome-stained histological sections from renal cortex (a, b, c and d) of a control rat (a) and of rats killed 5 (c) and 30 days (b and d) after glycerol injection. Note the presence of tubular dilatation, swelling and flattening of proximal tubular cells with brush border loss, diffuse interstitial oedema and interstitial inflammatory cellular infiltrates in (c) and of fibrosis, interstitial inflammatory cellular infiltrate and tubular dilatation and atrophy in (b) and (d) X120, (a) and (b); X280, (c) and (d).

Figure 2

Immunolocalization of fibronectin in the renal cortex from a control rat (a) and from rats killed 5 (b) and 30 days (c) after glycerol injection. Observe that the immunoreaction for fibronectin is more intense in glycerol-injected rats (b and c) than in the control animal (a). X280.

Figure 3

Immunolocalization of α-SM-actin (a, c and e) and ED1 (b, d and f) in the renal cortex from a control rat (a and b) and from rats killed 5 (c and d) and 30 days (e and f) after glycerol injection. Note that the staining for α-SM-actin is more intense in c and e than in a and the increase of ED1 + cells (monocytes/macrophages) is more marked in d and f compared to b. X280.

Figure 4

Immunolocalization of TGF-β in the renal cortex from a control rat (a) and from rats killed 5 (b) and 30 days (c) after glycerol injection using a polyclonal anti-TGF-β antibody. Note that the staning for TGF-β in tubular and interstitial cells is more intense in b and c than in a. d, Immunolocalization of TGF-β in the renal cortex from rats killed 30 days after glycerol injection using a normal rabbit IgG; the staining was abolished by this treatment. X280.