The Anti-Rheumatic Drug, Leflunomide, Induces Nephrotoxicity in Mice via Upregulation of TGFβ-Mediated p53/Smad2/3 Signaling

Abstract

Recent studies indicated renal toxicity and interstitial nephritis in patients receiving leflunomide (LEFN), but the exact mechanism is still unknown. The transforming growth factor β (TGFβ)/p53/Smad2/3 pathway crucially mediates renal fibrosis. We aimed to assess the nephrotoxic effect of LEFN in mice and the possible role of TGFβ-stimulated p53/SMAD2/3 signaling. The study design involved distributing sixty male albino mice into four groups: (i) vehicle-treated mice, (ii) LEFN (2.5 mg/kg), (iii) LEFN (5 mg/kg), and (iv) LEFN (10 mg/kg). The drug was given orally every 48 h and continued for 8 weeks. Blood samples were then taken from mice for the determination of kidney function parameters. Right kidneys were used for histopathologic staining and immunohistochemistry, whereas left kidneys were frozen and used for Western blot analysis of the target proteins, p-p53 and Smad2/3. Results indicated that chronic administration of LEFN in mice resulted in a four- and nine-fold increase in serum urea and creatinine levels, respectively. Kidney specimens stained with hematoxylin and eosin or periodic acid-Schiff showed significant histopathological manifestations, such as cellular irregularity, interstitial congestion, and moderate lymphocytic inflammatory infiltrate in mice treated with LEFN. Western blotting indicated upregulation of the p-p53/Smad2/3 proteins. LEFN, especially in the highest dose (10 mg/kg), produced prominent nephrotoxicity in mice. This toxicity is mediated through stimulating fibrotic changes through TGFβ-stimulated p53/Smad2/3 signaling and induction of glomerular and tubular apoptosis. An improved understanding of LEFN-induced nephrotoxicity would have great implications in the prediction, prevention, and management of leflunomide-treated rheumatic patients, and may warrant further clinical studies for following up these toxidromes.

Keywords: TGFβ fibrosis; leflunomide; mice; nephrotoxicity; p53/Smad2/3 pathway.

Figure 1

Network protein–protein interaction analysis for TGFβ and Smad proteins as presented by the STRING database.

Figure 2

TGFβ-induced Smad signaling, created via Reactome and KEGG bioinformatic data bases.

Figure 3

Effect of LEFN on kidney function parameters in adult mice. (A) Serum creatinine, (B) BUN. Data are mean ± SD, and analysis was performed by applying one-way ANOVA and Bonferroni’s test at p < 0.05. †: versus the vehicle group, ‡: versus the LEFN 2.5 mg/kg group, and Σ: versus the LEFN 5 mg/kg group. BUN: blood urea nitrogen.

Figure 4

Histopathologic scores for hematoxylin and eosin-stained kidney specimens in mice treated with LEFN. Panel (A1,A2) show sections of kidney tissue in the vehicle group, showing regular tubules (dashed arrow) lined by epithelial cells with intact eosinophilic cytoplasm and regular nuclei, and glomeruli (arrowhead) showing capillary tuft with a thin wall and thin patent Bowman’s space and mesangial cells. Interstitium showed thin blood vessels (curved arrow) and loose intervening stroma. Panel (B1,B2) are kidneys from the LEFN 2.5 mg/kg group showing mild focal tubular (dashed arrow) hydropic degeneration of tubular epithelial cells, glomeruli (arrowhead) showing average normal cellularity, and stroma showing focal minimal lymphocytic inflammatory infiltrate (arrow). Panel (C1,C2) are for kidneys in the LEFN 5 mg/kg group showing moderate tubular (dashed arrow) hydropic and vacuolar degeneration of tubular epithelial cells, glomeruli (arrowhead) showing a slight increase in cellularity, and stroma showing focal mild congestion (curved arrow) and focal mild lymphocytic inflammatory infiltrate (arrow). Panel (D1,D2), the LEFN 10 mg/kg group showed moderate hydropic degeneration of tubular epithelial cells (dashed arrow), and glomeruli showed a moderate increase in cellularity with irregularity and focal shrinkage (arrowhead). There is interstitial congestion and focal hemorrhage (curved arrow) with moderate lymphocytic inflammatory infiltrate (arrow), ×100 and ×400, respectively.

Figure 5

Histopathologic scores for hematoxylin and eosin-stained kidney specimens in mice treated with LEFN. Scores for (A) lymphocytic infiltrate, (B) congestion and hemorrhage, (C) glomerular degeneration, and (D) tubular degeneration. A score from 0–5 was given to each sample, and scores are presented as medians and quartiles, and compared by Kruskal–Wallis ANOVA and Dunn’s test at p < 0.05. *: versus the vehicle group.

Figure 6

Periodic acid–Schiff staining for the kidney specimen. (A) Sections in kidney tissue from vehicle group showing (left panel) glomerulus with thin basement membranes (black arrow) and patent vascular lumen, and tubules showing (right panel) a preserved brush border (black arrow) of tubular epithelial cells with average cytoplasm (red arrow). (B) Sections in kidney tissue from the LEFN 2.5 mg/kg group showing (left panel) glomerulus with thin basement membranes (black arrow) and patent vascular lumen, and tubules showing (right panel) a focally disrupted and effaced brush border (black arrow) of tubular epithelial cells and early cytoplasmic vacuoles (red arrow). (C) Sections in kidney tissue from the LEFN 5 mg/kg group showing (left panel) glomerular irregularities of vascular lumens with focal increased cellularity (red arrow), but with thin basement membranes (black arrow) (right panel) showing a disrupted and effaced brush border (black arrow) of tubular epithelial cells with moderate cytoplasmic vacuolation (red arrow). (D) Sections in kidney tissue from the LEFN 10 mg/kg group showing (left panel) a mild glomerular increase in cellularity (red arrow) with thin basement membranes (black arrow); the right panel shows multiple intratubular eosinophilic hyaline casts filling tubules (black arrow), with marked disruption of lining epithelial cells’ brush borders.

Figure 7

Masson’s trichrome staining for the kidneys of mice treated with LEFN. (A, left panel) Sections in kidney tissue from the vehicle group showing glomerulus and proximal tubules with interstitial tissue showing no deposition of green staining fibers in between, and no fibrosis. (A, right panel) shows distal tubules and a vessel in the center with no perivascular or peritubular collagen deposition (arrow). Sections from the kidneys of the LEFN 2.5 mg/kg group showing (B, left panel) no glomerular deposition of collagen fibers (arrow) (B, right panel) shows areas of congested vessels (arrow) with no or faint perivascular green staining, indicating no fibrosis from the kidneys of the LEFN 2.5 mg/kg group. (C, left panel) Sections in kidney tissues from the LEFN 5 mg/kg group showing peritubular and interstitial mild focal deposition of thin green staining collagen fibers (arrow). (C, right panel) shows areas of perivascular, green-stained collagen fibers deposition (arrow), indicating more localization to perivascular areas in the kidneys of the LEFN 5 mg/kg group. (D, left panel) Sections in kidney tissue from the LEFN 10 mg/kg group showing peritubular and interstitial inflammatory infiltrate with deposition of thin green staining collagen fibers (arrow). (D, right panel) shows wider areas of perivascular, green-stained collagen fibers deposition (arrow), indicating more localization to perivascular areas from the kidneys of the LEFN 10 mg/kg group. (E) Column chart for the mean ± SD of the fibrosis area % as measured in each group kidney sepecimens. †: versus the vehicle group, ‡: versus the LEFN 2.5 mg/kg group, and Σ: versus LEFN the 5 mg/kg group at p < 0.05.

Figure 8

Immunohistochemistry for TGFβ in kidney specimens from LEFN-treated mice. (A) Sections from the vehicle group show focal weak TGFβ-staining in normal kidneys, localized to periglomerular and peritubular areas (in left panel), and focal minimal staining in perivascular areas (right panel). (B) Sections from the LEFN 2.5 mg/kg group show faint focal staining of TGFβ in periglomerular areas (left panel) and minimal focal weak staining in peritubular areas (arrow in right panel). (C) Sections from the LEFN 5 mg/kg group show moderate staining of TGFβ encircling periglomerular areas (left panel), and this is also seen in perivascular areas or focally in peritubular areas (right panel). (D) Sections from the LEFN 10 mg/kg group show moderate to strong staining of TGFβ, especially in areas surrounded by inflammatory cells infiltrate (left panel), and perivascular and peritubular areas (right panel), arrows in all images indicate positive staining. (E) Column chart for mean ± SD of the stained area % in kidney specimens from the experimental groups. †: versus the vehicle group, ‡: versus the LEFN 2.5 mg/kg group, and Σ: versus the LEFN 5 mg/kg group at p < 0.05.

Figure 9

Immunohistochemistry for p53 in the kidney specimens. (A) Images from the vehicle group show focal weak p53 staining, localized to periglomerular cells (in left panel) and focal minimal staining in tubular cells (right panel). (B) Images from the LEFN 2.5 mg/kg group show a very faint focal staining of p53 in glomerular areas (left panel) and weak staining in peritubular areas (arrow in right panel). (C) The LEFN 5 mg/kg group showed focal moderate or weaker staining of p53 encircling periglomerular areas (left panel), and this is also seen in peritubular and perivascular areas (right panel). (D) The LEFN 10 mg/kg group revealed moderate to strong staining of p53, with most staining in periglomerular tubules (left panel) and in areas surrounded by inflammatory cells infiltrate (right panel), arrows in all images indicate positive staining (E) Column chart for mean ± SD of the stained area % in kidney specimens from the experimental groups and data were analyzed using one-way ANOVA. †: versus the vehicle group, ‡: versus the LEFN 2.5 mg/kg group, and Σ: versus the LEFN 5 mg/kg group at p < 0.05.

Figure 10

Western blot analysis for the target proteins. (A) The Western blot gels for p-p53, SMAD2/3, and SMA compared to β-ACTIN. (B,C) Column charts for the p-p53 and SMAD2/3 in the experimental groups. Data are mean ± SD. †: versus the vehicle group, ‡: versus the LEFN 2.5 mg/kg group, and Σ: versus the LEFN 5 mg/kg group at p < 0.05.