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: leflunomide, mice, nephrotoxicity, p53/Smad2/3 pathway, TGFβ
   fibrosis

1. Introduction

   Leflunomide (LEFN) is an anti-cellular proliferation and
   immunomodulator drug that is used in rheumatoid arthritis treatment, as
   a disease-modifying anti-rheumatic drug (DMARD), and in numerous
   autoimmune diseases [[48]1]. LEFN is completely metabolized into
   teriflunomide (A-771726): an active metabolite in the liver and gut
   mucosa [[49]2]. LEFN has been proven to induce hepatotoxicity,
   anaphylaxis, toxic epidermal necrolysis, Stevens–Johnson syndrome, and
   pulmonary effects [[50]3,[51]4,[52]5,[53]6,[54]7]. Additionally,
   hepatotoxicity induced by LEFN was linked to mitochondrial dysfunction
   at the transcript level [[55]8], oxidative stress [[56]9,[57]10], and
   unfolded proteins accumulation [[58]11]. In a recent study, LEFN
   induced inflammatory reactions and pulmonary toxicity in mice, together
   with architectural structural distortion [[59]12].

   LEFN is not known as a nephrotoxic agent; however, emerging evidence
   from numerous case reports proved that LEFN induces nephrotoxicity
   (chronic interstitial nephritis), even with the recommended dose of
   leflunomide [[60]13,[61]14,[62]15,[63]16,[64]17]. The kidney is a
   vulnerable vital organ in drug elimination; therefore, chemical
   exposure to drugs and their metabolites will jeopardize the urinary
   tract system [[65]18,[66]19]. Toxic damage (either acute or chronic) to
   the kidney is attributed to various substances, such as hydrocarbons,
   heavy metals, and proven nephrotoxic drugs, as well as aflatoxins that
   might result in renal cancer or failure [[67]20,[68]21,[69]22,[70]23].

   The pathogenic processes of LEFN-induced nephrotoxicity are still
   unknown. In fact, drug-induced renal injury can be attributed to
   numerous pathogenic pathways [[71]24]. Transforming growth factor-β
   (TGFβ) is a pleiotropic cytokine accountable for the regulation of
   several important cellular pathways and activities throughout the human
   body. Ordinarily, TGFβ exerts a pivotal function in maintaining tissue
   homeostasis largely via gene transcriptional regulation, which is
   responsible for cell proliferation, cytostasis, and survival
   [[72]25,[73]26,[74]27,[75]28]. The binding of a TGFβ isoform to a
   heterotetrameric complex of the serine/threonine kinase receptors is
   the key factor in TGFβ signaling propagation [[76]29]. Activating the
   TGFβ receptors induces transcription factors Smad2/3 phosphorylation
   downstream [[77]30,[78]31,[79]32]. Consequently, the activated Smad
   complex storage in the nucleus increases, which plays an essential role
   in the regulation of transcription of several target genes
   [[80]33,[81]34].

   TGFβ is a key factor in triggering renal fibrosis in a variety of
   chronic kidney diseases. Moreover, TGFβ overexpression has been
   observed in the advanced stages of renal disease [[82]35,[83]36]. The
   activation of the TGFβ–Smad complex signal results in stimulation of
   myofibroblasts, extreme generation of extracellular matrix (ECM), and
   inhibition of ECM degradation [[84]35].

   The tumor suppressor gene (p53), known as the “Guardian of the Genome”,
   plays a significant role in the regulation of nuclear protein gene
   transcription and encoding. These genes are responsible for cellular
   apoptosis and cell-cycle arrest [[85]37,[86]38,[87]39]. The p53
   response may be prompted by numerous stress triggers, such as oncogene
   activation, genotoxic agents, hypoxia, and ribosomal stress [[88]40].
   The last four decades have seen a growing knowledge of p53 function as
   an oncogene targeting cancer cells. Furthermore, mutation of TP53, the
   gene encoding for p53, is the most frequently mutated gene among cancer
   patients [[89]41,[90]42,[91]43]. Indeed, p53 gene overexpression is
   noted in several cancer types [[92]39].

   Under normal conditions, activated p53 and TGFβ act as gene-specific
   transcription factors, assisting in the control of multiple genes
   required in the generation of antitumor effects [[93]44]. Due to the
   wide alternating nature of these proteins, overlap of cellular
   functions occurs in the regulation of autophagy and apoptosis,
   indicating several possible ideas of convergence. Wyllie et al. (1991)
   suggested a link between the TGFβ and wild-type p53 pathway, stating
   that wild-type p53 deactivation by the SV40 virus was reported to
   result in losing the reaction to TGFβ treatment, indicating that p53
   loss may initiate resistance to the anticancer effects of TGFβ
   [[94]45]. Various studies have established the role of TGFβ1/p53
   signaling in the generation of renal fibrosis [[95]46]. Furthermore,
   TGFβ promotes the activation of the p53 and Smad complexes, both of
   which play important roles in renal fibrogenesis [[96]46].

   Hence, our aim was to assess LEFN-induced dose-dependent renal toxicity
   or fibrogenesis in mice, and the possible role of TGFβ-stimulated p53
   apoptosis.

2. Materials and Methods

2.1. Signaling Pathway Enrichment Analysis

   The chosen target pathway was selected utilizing online databases such
   as KEGG pathway [[97]47] and Reactome [[98]48].

2.2. Animals

   This experiment was performed using sixty male Swiss albino mice. Mice
   were housed in a normal light/dark cycle and a temperature equal to 25
   ± 5 °C, with free access to chow diet and water. Mice were acclimatized
   to the experimental conditions in the animal house for seven days prior
   to the experiment. This study protocol was approved by the
   Institutional Ethics Committee (201603A7c).

2.3. Design of the Experiment

   Four groups of mice were used, with fifteen mice each. LEFN was
   prepared as a suspension with the aid of 1% carboxymethylcellulose
   (CMC) solution. Oral gavaging of LEFN continued for 8 weeks according
   to the study design: vehicle group: mice received 1% CMC solution; LEFN
   2.5 mg/kg group: mice received a dose equal to 2.5 mg per kg; LEFN 5
   mg/kg group: mice received a dose equal to 5 mg per kg; and LEFN 10
   mg/kg group: mice received a dose equal to 10 mg per kg. For all
   groups, drug or vehicle administration by oral gavage was performed
   every 48 h and continued for 8 weeks.

2.4. Collection of Blood Samples and Kidneys

   After finishing the course of LEFN, blood was collected under ketamine
   anesthesia (80 mg per kg of ketamine HCL) by intraperitoneal injection
   [[99]49,[100]50]. Blood was collected in tubes, and after standing for
   25 min at room temperature, samples were centrifuged at 1600× g for 10
   min. Serum samples were then kept at −20 °C until used for renal
   estimation of function parameters. The right kidneys were frozen
   immediately at −80 °C.

2.5. Estimation of Renal Function Parameters

   The quantification of serum creatinine was performed applying the
   alkaline-picric acid method according to the method described in the
   kit instructions. The quantification of urea was achieved via applying
   the reaction with salicylate-hypochlorite in the presence of
   nitroprusside; this reaction yield forms a green indophenol product.
   Kits were purchased from Diamond Diagnostics Ltd. (Cairo, Egypt). A
   UV1601-PC spectrophotometer (Schimadzu, Kyoto, Japan) was used to
   quantify the optical density of the reaction products.

2.6. Western Blot Analysis for p-p53 and SMAD2/3 in the Renal Homogenate

   As a basic step, we determined the total protein concentration in 5-µL
   samples of the kidney homogenates. We used a Bio-Rad Quick StartTM
   Bradford Protein Assay kit (Hercules, western Contra Costa, CA, USA).
   Equal protein amounts from the kidney homogenates were denaturated
   using 4× Laemmli sample buffer from Bio-Rad, and then subjected to SDS
   polyacrylamide gel electrophoresis for separation of the proteins.
   Then, we transferred the proteins to a nitrocellulose membrane and
   blocked the free sites on the membranes by incubation for 1 h in
   Bio-Rad non-fat dried milk. After that, we washed the membranes and
   incubated them with primary antibodies for p-p53 (ab33889), smad2/3
   (202445), and β-actin (Ab8226) (Abcam, Waltham, MA, USA) overnight.
   Then, we washed the blots and added secondary antibodies conjugated
   with horseradish peroxidase. An Amersham BioSciences chemiluminescence
   kit (Buckinghamshire, UK) was used to detect the concentration of each
   protein. Then, the blots were imaged, and the band densities were
   measured by ImageJ software (NIH, Bethesda, MD, USA).

2.7. Kidney Histopathology and Immunohistochemistry

   The left kidneys were fixed in 10% phosphate-buffered formalin. After
   standing overnight, tissues were dehydrated and embedded in paraffin.
   Then, serial sections were cut to 4-μm and stained with hematoxylin and
   eosin (H&E), and periodic acid–Schiff (PAS) stain for the detection of
   mucopolysaccharides at the glomerular capillary basement membranes
   [[101]51]. Slides were also stained with Masson’s trichrome for the
   detection of collagen deposition. A blinded experienced pathologist
   examined the stained sections under a light microscope (Olympus^® CX21)
   and captured several representing images. The pathologist captured
   images using a calibrated digital microscope camera (Tucsen ISH1000
   digital microscope camera) with 10 megapixels resolution (3656 × 2740
   pixel each image) at original magnifications equal to 100× and 400×
   (objective lens 10× and 40×), and a UIS optical system (Universal
   Infinity System, Olympus^®, Tokyo, Japan). We also used the “IS
   Capture” software to enhance the images. Kidney slides were captured.

2.7.1. Morphometric Analysis of H&E-Stained Sections

   Slides were examined for the pathological changes described
   (lymphocytic infiltrate, congestion, and hemorrhage, and tubular and
   glomerular degeneration), and the severity of changes was assigned
   using scores on a scale of 0–5 (score (0) denotes no change, score (1)
   is changes affecting < 20%, score (2) is changes affecting 20 −< 40%,
   score (3) is changes affecting 40 −< 60%, score (4) is changes
   affecting 60 −< 80%, score (5) is changes affecting 80–100%) and
   applied for five readings per group [[102]52].

2.7.2. Examination of PAS-Stained Sections

   For the quantification of integrated intensities (pixels) and area
   percentages (%) of mucopolysaccharides using periodic acid–Schiff (PAS)
   stain, twenty-five captured microscopic images at 400× magnification
   fields were evaluated using ImageJ software. All positive orange-to-red
   reactions were captured in different groups. Random fields were
   selected in the control non-treated group using ImageJ software
   [[103]53]. Slides were examined for pathological changes, described as:
   lymphocytic infiltrate, congestion and hemorrhage, and tubular and
   glomerular degeneration. These changes were assigned on a scale of none
   = 0, mild (changes affecting < 25%) = 1, moderate (changes affecting
   25–50%) = 2, and severe damage (changes affecting > 50%) = 3 [[104]54].

2.8. Immunohistochemistry and Image Analysis of TGFβ and p53 in Renal Tissues

   Serial sections were deparaffinized, hydrated, and then immersed in
   EDTA solution (pH = 8) for antigen retrieval. Hydrogen peroxide (0.3%)
   was then added to the slides for 12 min, and then antibodies to p53
   (GTX100629, 1:500, Irvine, GeneTex, CA, USA) and TGFβ1 (GTX45121,
   1:100, Irvine, GeneTex, CA, USA) were applied at 4 °C for 12 h.
   Phosphate buffered saline (PBS) was used to wash the slides three
   times, and then the secondary antibodies were applied to the slides for
   1 h. A Power-StainTM 1.0 poly HRP-3,3-diaminobenzidine tetrachloride
   (DAB) kit (Genemed Biotechnologies, South San Francisco, CA, USA) was
   used to detect the color of the reaction, and Mayer’s hematoxylin was
   applied as a final stain. Multiple fields were imaged and analyzed
   using the ImageJ program (NIH, Bethesda, MD, USA).

2.9. Statistical Analysis

   We tabulated and averaged the whole data. In case of quantitative data
   with normal distribution, we applied one-way ANOVA, whereas post hoc
   analysis was performed by Bonferroni’s test. The scoring data were
   presented as medians and quartiles, and analyzed by the Kruskal–Wallis
   ANOVA and Dunn’s test. The bilateral significance was set at α = 0.05.

3. Results

3.1. The Bioinformatic Study

   For highlighting the role of the TGFβ signaling pathways and
   demonstrating the key players in its signal transduction, bioinformatic
   analysis was performed utilizing the STRING database, which is a
   comprehensive database. It shows the predicted interaction between
   proteins, and reveals whether this is a physical (direct) or indirect
   interaction. In the STRING database, protein–protein interactions are
   given a “score” individually; this is done according to seven evidence
   channels which are marked by colored lines or edges in [105]Figure 1,
   representing experimental evidence, database citations, gene
   neighborhooding, fusions, as well as co-occurrence, appearance in
   literature text, co-expression, and protein h. The bioinformatic tools
   demonstrated the ability of TGFβ to activate Smad proteins. TGFβ
   activates a transcriptional cascade, which leads, finally, to enhancing
   the production of Smad proteins ([106]Figure 2).

Figure 1.

   [107]Figure 1
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   Network protein–protein interaction analysis for TGFβ and Smad proteins
   as presented by the STRING database.

Figure 2.

   [109]Figure 2
   [110]Open in a new tab

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

3.2. Assessment of Renal Function

   [111]Figure 3A,B shows the effects of eight weeks oral administration
   of LEFN (2.5, 5, and 10 mg/kg) on serum creatinine and BUN levels of
   adult mice. The LEFN (10 mg/kg) group (iv) demonstrated a statistically
   significant rise in serum creatinine and BUN levels compared with the
   vehicle group and mice who received LEFN (2.5 or 5 mg/kg) ([112]Figure
   3A,B). The LEFN (5 mg/kg) group (iii) displayed a statistically
   significant high level of BUN compared to groups i and ii [113]Figure
   3B.

Figure 3.

   [114]Figure 3
   [115]Open in a new tab

   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.

3.3. Kidney Histopathology for H&E-Stained Sections and Scoring

   In [116]Figure 4, sections of kidney tissues in the vehicle group
   showed regular tubules lined by epithelial cells with intact
   eosinophilic cytoplasm and regular nuclei, and glomeruli showed
   capillary tuft with a thin wall and thin patent Bowman’s space and
   mesangial cells. The interstitium showed thin blood vessels and loose
   intervening stroma (Panel A1,A2). The LEFN 2.5 mg/kg group showed mild
   focal tubular hydropic degeneration of tubular epithelial cells,
   glomeruli showed average normal cellularity, and stroma showed focal
   minimal lymphocytic inflammatory infiltrate (Panel B1,B2). The LEFN 5
   mg/kg group showed moderate tubular hydropic and vacuolar degeneration
   of tubular epithelial cells, glomeruli showed a slight increase in
   cellularity, and stroma showed focal mild congestion and focal mild
   lymphocytic inflammatory infiltrate (Panel C1,C2). Finally, the LEFN 10
   mg/kg group showed moderate hydropic degeneration of tubular epithelial
   cells, and glomeruli showed a moderate increase in cellularity with
   irregularity and focal shrinkage. There is interstitial congestion and
   focal hemorrhage with moderate lymphocytic inflammatory infiltrate
   (Panel D1,D2).

Figure 4.

   [117]Figure 4
   [118]Open in a new tab

   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.

   [119]Figure 5 shows the four panels that indicate the histopathologic
   scores for the H&E-stained kidney specimens. These panels show
   lymphocytic infiltration ([120]Figure 5A), congestion and hemorrhage
   ([121]Figure 5B), glomerular degeneration ([122]Figure 5C), and tubular
   degeneration ([123]Figure 5D). It is shown that the LEFN 10 mg/kg group
   showed the highest scores, which were significantly greater than the
   score of the vehicle group every time.

Figure 5.

   [124]Figure 5
   [125]Open in a new tab

   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.

3.4. Kidney Histopathology in PAS Stained Sections

   Results of periodic acid–Schiff staining are shown in [126]Figure 6.
   Kidney specimens in the vehicle group sowed glomerulus with thin
   basement membranes and patent vascular lumen, and tubules showed (right
   panel) a preserved brush border of tubular epithelial cells
   ([127]Figure 6A). Sections in kidney tissue from the LEFN 2.5 mg/kg
   group showed glomerulus with thin basement membranes and patent
   vascular lumen, and tubules showed focal disruption and an effaced
   brush border of tubular epithelial cells ([128]Figure 6B). Sections in
   kidney tissue from the LEFN 5 mg/kg group showed glomerular
   irregularities of vascular lumens with focal increased cellularity, but
   thin basement membranes showed a disrupted and effaced brush border of
   tubular epithelial cells with moderate cytoplasmic vacuolation
   ([129]Figure 6C). Sections in kidney tissue from the LEFN 10 mg/kg
   group showed a mild glomerular increase in cellularity with thin
   basement membranes, and showed multiple intratubular eosinophilic
   hyaline casts filling tubules, with a marked disruption of lining
   epithelial cells’ brush borders ([130]Figure 6D).

Figure 6.

   [131]Figure 6
   [132]Open in a new tab

   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.

3.5. Kidney Histopathology in Masson’s Trichrome Stained Sections

   [133]Figure 7 shows the Masson’s trichrome staining for the kidney
   specimens. Sections in kidney tissue from the vehicle group showed
   ([134]Figure 7A, left panel) glomerulus and proximal tubules with
   interstitial tissue showing no deposition of green staining fibers in
   between and no fibrosis; [135]Figure 7A (right panel) shows distal
   tubules and a vessel in the center with no perivascular or peritubular
   deposition. Sections from the kidneys of the LEFN 2.5 mg/kg group
   showed ([136]Figure 7B, left panel) no deposition of collagen fibers,
   as indicated by faint green staining in peritubular and interstitial
   areas (arrow). The right panel shows areas of congested vessels with no
   or faint perivascular green staining, indicating no fibrosis
   ([137]Figure 7B). Sections in kidney tissues from the LEFN 5 mg/kg
   group showed ([138]Figure 7C, left panel) peritubular and interstitial
   mild focal deposition of thin green staining collagen fibers. The right
   panel shows areas of perivascular green-stained collagen fibers
   deposition, indicating more localization to perivascular areas
   ([139]Figure 7C, right panel). Sections in kidney tissue from the LEFN
   10 mg/kg group (left panel) showed peritubular and interstitial
   inflammatory infiltrate with deposition of thin green staining collagen
   fibers ([140]Figure 7D, left panel). Right panel shows wider areas of
   perivascular green-stained collagen fibers deposition, indicating more
   localization to perivascular areas ([141]Figure 7D, right panel).

Figure 7.

   [142]Figure 7
   [143]Open in a new tab

   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.

3.6. Immunohistochemical and Image Analysis of TGFβ and p53 Expression in
Renal Specimen

   [144]Figure 8 shows the immunohistochemistry for TGFβ in the kidney
   specimens. Sections from the vehicle group show focal weak TGFβ
   staining in the normal kidneys, localized to periglomerular and
   peritubular areas (A, left panel) and focal minimal staining in
   perivascular areas (A, right panel). Sections from the LEFN 2.5 mg/kg
   group show faint focal staining of TGFβ in periglomerular areas (B,
   left panel) and minimal focal weak staining in peritubular areas (B,
   arrow in right panel). Sections from the LEFN 5 mg/kg group show
   moderate staining of TGFβ encircling periglomerular areas (C, left
   panel), and this is also seen in perivascular areas or focally in
   peritubular areas (C, right panel). Sections from the LEFN 10 mg/kg
   group show moderate to strong staining of TGFβ, especially in areas
   surrounded by inflammatory cells infiltrate (D, left panel), and
   perivascular and peritubular areas (D, right panel).

Figure 8.

   [145]Figure 8
   [146]Open in a new tab

   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.

   [147]Figure 9 shows the immunohistochemistry for p53 in the kidney
   specimens. Sections from the vehicle group show focal weak p53
   staining, localized to periglomerular cells (A, left panel), and focal
   minimal staining in tubular cells (A, right panel). The LEFN 2.5 mg/kg
   group showed a very faint focal staining of p53 in glomerular areas (B,
   left panel), and weak staining in peritubular areas (B, arrow in right
   panel). The LEFN 5 mg/kg group showed focal moderate or weaker staining
   of p53 encircling periglomerular areas (C, left panel), and this was
   also seen in peritubular and perivascular areas (C, right panel). The
   LEFN 10 mg/kg group revealed moderate to strong staining of p53, with
   most staining in periglomerular tubules ([148]Figure 9D, left panel)
   and in areas surrounded by inflammatory cells infiltrate ([149]Figure
   9D, right panel).

Figure 9.

   [150]Figure 9
   [151]Open in a new tab

   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.

3.7. Western Blot Analysis of p-p53 and SMAD2/3 in Renal Homogenates

   The Western blot gels are shown in [152]Figure 10A for p-p53 and
   SMAD2/3. The mean values of p-p53 protein density are shown in
   [153]Figure 10B, whereas the mean values for SMAD2/3 protein are shown
   in [154]Figure 10C. Both proteins were significantly upregulated in the
   LEFN 10 mg/kg group compared to the other experimental groups.

Figure 10.

   [155]Figure 10
   [156]Open in a new tab

   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.

4. Discussion

   LEFN exhibits immunosuppressive and anti-inflammatory activity
   [[157]55]. It is used as DMARD, since it inhibits the biosynthesis of
   pyrimidine and, in turn, inhibits the progression of disease [[158]56].
   Even though LEFN utility has increased during the past years, it is
   associated with many serious adverse effects affecting the immune
   system, hematological system, and hepatic system [[159]57]. Moreover,
   this drug’s long half-life of 2 weeks delays the treatment of adverse
   reactions.

   Drug-induced nephrotoxicity is a significant clinical challenge,
   particularly in immunocompromised patients. Although LEFN was approved
   to treat rheumatoid arthritis in 1998, renal impairment and severe
   interstitial nephritis were often reported as fatal side effects during
   the recommended therapeutic regimen [[160]13,[161]14,[162]15,[163]16].
   Surprisingly, renal function is not monitored when receiving the
   recommended dose [[164]2], and much uncertainty still exists about the
   exact mechanisms of renal injury induced by LEFN.

   The present study confirms that LEFN induces dose-dependent kidney
   affection, which is identified in the form of elevations in renal
   function parameters (blood urea and BUN) and established
   histopathological abnormalities.

   The results revealed that LEFN at a dose of 10 mg/kg markedly induced
   renal dysfunction, as indicated by higher levels of serum creatinine
   and BUN, and marked histopathological changes in the renal tissues. The
   second LEFN dose (5 mg/kg) induced moderate histopathological changes,
   with significant elevation in the BUN level only. Finally, the 2.5
   mg/kg dose of LEFN was found to produce minimal histopathological
   abnormalities, with non-significant changes in serum creatinine or BUN.

   The results are in agreement with the findings of
   [[165]13,[166]15,[167]16,[168]17], which showed the potential
   nephrotoxicity of LEFN. Hurtado et al. (2016) demonstrated that rapid
   deterioration of kidney function with severe tubulointerstitial
   nephritis confirmed by renal biopsy was proved in patients who received
   LEFN [[169]16]. Chronic LEFN dosage was linked to IgA
   glomerulonephritis [[170]58]. Furthermore, the combined therapy of
   methotrexate with LEFN was documented to cause acute kidney failure
   [[171]59]. The elimination half-life of LEFN is about 2 weeks, and may
   be prolonged with renal impairment, which exerts more stress on kidneys
   [[172]60]. To explain the relatively good response to 2.5 mg, the
   starting point of the injury may be the mitochondrial model of
   idiosyncrasy. The doses that saturate the capacity of the mitochondria
   end in drastic effects, whereas lower toxic doses produce off/on-like
   effects. A similar model has been seen in liver toxicity, as the
   toxicity is clearer in females and older age mice. Future studies can
   track such a hypothesis in Sod2 (+/−) mice to see if direct and
   mitochondrial causes can contribute to the pathology [[173]61].

   LEFN-induced hepatotoxicity has been reported in various studies
   [[174]62,[175]63,[176]64]. The underlying pathogenesis involved in
   hepatotoxicity induced by LEFN is not fully elucidated. A liver
   signaling molecule, TGFβ, is involved in apoptosis, differentiation,
   and maturation [[177]65]. Continuous inflammation of the liver and
   unrelieved injury leads to hepatic fibrosis. Hepatic stellate cell
   activation causes extracellular matrix deposition [[178]66]. Even
   though a study conducted before has reported LEFN’s inhibitory effect
   on fibrosis of the liver, its cumulative dose has been reported to
   cause hepatic fibrosis in patients with rheumatoid arthritis [[179]67].
   TGFβ, by its paracrine and autocrine mechanisms, is involved in causing
   hepatic and lung fibrosis [[180]68]. A review was conducted on renal
   toxicity associated with DMARDs used for the treatment of rheumatoid
   arthritis [[181]69], and it was reported that LEFN exhibits kidney
   toxicity. Similarly, the current results showed an increment in renal
   TGFβ in mice who received LEFN, indicating that LEFN induces
   nephrotoxicity.

   TGFβ is a group of growth factors promoting inflammation, growth, and
   differentiation. In mammals, there are three isoforms, ranging from
   TGFβ 1, 2, and 3. It is secreted as a precursor that is activated with
   multiple signaling pathways [[182]70]. Progressive renal failure is
   characterized by renal fibrosis replacing active kidney cells. Whatever
   the cause of renal failure, the condition is associated with high TGFβ.
   The hypothesis behind that unfortunate organ failure is the activation
   of the TGFβ/Smad pathway initiating pro-fibrogenic cascade [[183]71].
   Experimental inhibition of TGF beta1 resulted in a reduction of the
   fibrosis process in the kidney. The TGFβ can activate the
   myofibroblasts and induce the extracellular matrix to proliferate via
   the Smad pathway and less common ones. The Smad track modifies the
   balance between fibrotic and anti-fibrotic situations. The TGFβ is
   controlled through epigenetic mechanisms [[184]35]. In obstructive
   kidney fibrosis, it has been shown that TGFβ promotes the transition of
   pericytes to myofibroblasts [[185]72]. The main source of this cytokine
   is macrophages actively working in the fibrotic area [[186]73].

   TGFβ1 has other functions than the induction of fibrosis; it has
   regulatory functions of apoptosis, cell proliferation, differentiation,
   and inhibition of the immune reaction. The latter function causes
   concerns about the use of TGF beta in treating progressive renal
   failure [[187]74]. TGFβ via Smad3 induces a battery of genes involved
   in the fibrosis process, and indirectly inhibits the anti-fibrotic
   genes. There are clinical hopes that the anti-Smad strategy can be an
   alternative mechanism to overcome chronic progressive renal fibrosis
   [[188]35].

   Beside nephrotoxicity, two recent studies highlighted dose-dependent
   toxicity for the liver and the lungs of adult mice. The first one
   explored the hepatotoxicity, whereas the second one explored the
   pulmonary toxicity in albino mice.

   The histopathological findings of LEFN on the liver showed that the
   pathologic score in the LEFN-treated groups was greater than the
   vehicle group. Further, results highlighted that LEFN dose-dependently
   raised the hepatic TLR4 immunostaining. The gene expression studies in
   liver tissues indicated significant over-expression of PI3K and TGFβ in
   LEFN-treated mice. An ELISA test confirmed that there was a significant
   elevation of TGFβ in mice treated with the different doses of LEFN
   [[189]3].

   A previous experimental study investigated the pulmonary toxicity of
   leflunomide in mice. The hematoxylin and eosin staining of lung
   specimens demonstrated fibrotic changes in the lungs of albino mice,
   which were dose-dependent; these results were confirmed by Masson’s
   trichrome staining as well. LEFN use for eight weeks resulted in lung
   injury, and there was a marked increase in lung injury when the dose of
   LEFN increased. Western blot analysis revealed an increase in α-SMA,
   vimentin, and collagen 1 in LEFN-treated animals; there was also a
   dose-dependent increase of the same [[190]12]. In the later study,
   elevated levels of inflammatory markers (NLRP3 and interlukin-1) were
   found in dose-dependent pulmonary toxicities, together with
   architectural structural distortion [[191]12].

5. Conclusions

   The current results demonstrated, for the first time, detailed and
   comprehensive results about the nephrotoxicity of leflunomide in mice.
   Leflunomide produced dose-dependent pathologic changes in the kidneys,
   including leukocyte infiltration, glomerular degeneration, and tubular
   regeneration. We demonstrated that this toxicity included upregulation
   of TGFβ-mediated p53/Smad2/3 signaling, and induction of fibrosis. 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.

Acknowledgments