Linagliptin Mitigates TGF-β1 Mediated Epithelial–Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways

Abstract

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin’s (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial–mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

INTRODUCTION

Renal fibrosis is a direct result of the kidney’s limited ability to regenerate after injury, resulting in a progressive loss of renal function and, eventually, the need for dialysis or kidney transplantation.¹⁾

Tacrolimus is the most commonly used immunosuppressive medication in organ transplantation and autoimmune diseases.²⁾ Yet, chronic nephropathy, a major cause of chronic allograft failure in renal transplant recipients, limits its use.³⁾ The nephrotoxic effect is primarily characterized by tubular atrophy and tubulointerstitial fibrosis.⁴⁾

The epithelial–mesenchymal transition (EMT) has been proposed as an important step in tissue repair following injury.⁵⁾ During EMT, stationary epithelial cells lose epithelial features such as cell-cell adhesion, apical to basolateral polarity, and epithelial markers (e.g. E-cadherin) and gain mesenchymal morphology and features such as asymmetry of the trailing edge and migratory ability, expressing myofibroblasts markers such as α-smooth muscle actin (α-SMA) and increasing expression of mesenchymal markers like vimentin and the transcription factors e.g. Snail and Twist leading to the deposition of extracellular matrix (ECM) proteins.^6,7)

The transforming growth factor β1 (TGF-β1), a cytokine produced by injured parenchymal cells and macrophages has been proven to initiate EMT and fibrosis.¹⁾ The TGF-β1 exerts its effects on target genes, mainly through downstream phosphorylation of Smad 2/3 proteins, and their role in the transcriptional regulation of ECM genes such as collagen type 1 (COL1A1), fibronectin, and tissue inhibitor of metalloproteinase 1 (TIMP1), which ultimately result in the development and progression of fibrosis.^5,8) In addition, TGF-β1 can signal through Smad-independent signaling cascades.⁹⁾

Several studies confirmed that the mitogen-activated protein kinase (MAPK) signaling pathways are directly activated by TGF-β1.¹⁰⁾ Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK) and P38 has been shown to directly phosphorylate the linker region of Smad2/3 and increase their duration of transcriptional activity.^11,12) Furthermore, activated ERK has been demonstrated to increase the level of Snail, thereby reducing the expression of E-cadherin and stimulating the incidence of EMT.¹³⁾ Furthermore, p38 phosphorylates target transcription factors, such as c-Jun exhibiting a profibrotic effect consequently, the crosstalk amongst TGF-β, Smad, and MAPK pathways are involved in the EMT process.¹⁴⁾

Previous studies have presented that hypoxia-inducible factor (HIF)-1α mediates TGF-β1-induced signaling pathway,¹⁵⁾ besides HIF-1α promotes renal fibrosis by regulating EMT through directly binding to the proximal hypoxia response element of Twist and modulating its expression.¹⁶⁾ Lysyl oxidase-like 2 (LOXL2), the amine oxidase enzyme that catalyzes the crosslinking of collagens in the extracellular matrix has been proven as a crucial mediator of ECM remodeling during fibrotic processes and as a major profibrogenic target of HIF-1α.¹⁷⁾ LOXL2 has been reported to mediate EMT by silencing E-cadherin expression, or through upregulation of Snail transcription activity.^18,19)

Linagliptin is unique among gliptins in that it is the only such drug that is primarily excreted via a non-renal pathway, so decreased glomerular filtration rate does not necessitate dose adjustment. Additionally, to date, Lina is the only known DPP-4 inhibitor to be assessed in a randomized clinical trial designed to evaluate renal outcomes rigorously.^20,21) Moreover, Lina has provoked multiple renoprotective effects in animal models of acute and chronic kidney disease (CKD) and in clinical studies, including reducing albuminuria, inflammation, glomerulosclerosis, and tubulointerstitial fibrosis, independent of its glucose-lowering effect.^22,23) Nevertheless, no previous studies have reported its anti-fibrotic effects against calcineurin inhibitors (CIs) nephropathy. In the present study, we investigated the potential protective effects of Lina on TAC-induced renal fibrosis and elucidated the possible mechanisms by focusing on the TGF-β1 and HIF-1α mediated EMT pathways.

MATERIALS AND METHODS

Animal Care and Ethical Approval

Thirty-two adult male Sprague Dawley rats initially weighing 200–220 g at the start of the experiment were obtained from the Laboratory Animal Colony, Ministry of Health and Population, Helwan, Cairo. Rats were kept for two weeks for acclimatization in controlled temperature housing conditions (room temperature 25±2 °C, humidity (50–70%) and 12/12 h dark–light cycles and kept Free on a low salt diet (0.05% sodium, Raw Bright International Company, Menoufia, Egypt), allowing free access to tap water. A low-salt diet was used to increase tacrolimus nephropathy.²⁴⁾ Ethical clearance was obtained from the Institutional Animal Ethics Committee of the Faculty of Pharmacy, Al-Azhar University. All procedures performed in this study were in accordance with the ethical guidelines of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.²⁵⁾

Chemicals and Reagents

Tacrolimus (Prograf^®, Astellas Toyama Co., Ltd., Japan) was diluted in olive oil to a final concentration of 1 mg/mL. Linagliptin was provided by Boehringer Ingelheim Pharma GmbH & Co. (Ingelheim, Germany). Any other chemicals or reagents used were obtained from locally certified sources and were of the highest analytical grades.

Experimental Design

Animals were randomly and equally divided into four groups (n = 8), and treated once daily for 28 consecutive days as follows:

• Group 1 (Control): Rats received olive oil (1 mL/kg/d, subcutaneously).
• Group 2 (Lina): Rats treated with linagliptin (5 mg/kg/d, per os (p.o.)).
• Group 3 (TAC): Rats received tacrolimus (1.5 mg/kg/d, subcutaneously).
• Group 4 (TAC+ Lina): Rats were treated concomitantly with tacrolimus (1.5 mg/kg/d, subcutaneously) + linagliptin (5 mg/kg/d, p.o.).

The doses and duration of treatment were chosen according to previous reports^26–28) and were further confirmed on the basis of our preliminary pilot study using doses of 1, 3, and 5 mg of Lina (data not shown).

Blood, Urine, and Tissue Sampling

At the end of the experimental period, a sufficient volume of blood samples was collected from the retro-orbital venous plexus under light ether anesthesia into non-heparinized micro-capillaries.²⁹⁾ The sample was left undisturbed at room temperature for 30 min to clot. The sera were separated by centrifugation at 3000 rpm for 15 min at 4 °C using a cooling centrifuge (centrifuge Z446-K, Hermle Labortechnik GmbH, Germany). Afterward, it was stored at −20 °C and maintained at 2–8 °C while handled for biochemical analysis. Animals were housed individually in metabolic cages at the end of the experimental protocol for 24-h urine sample collection. Rats were then euthanized by cervical dislocation technique according to standard animal euthanasia method guidelines developed by the Canadian Council on Animal Care. The kidneys were rapidly excised, weighted, sectioned longitudinally, divided into portions, and either preserved in 10% buffered formalin solution, for immunohistochemistry and histopathological processing examination or stored in a refrigerator at −80 °C for protein assay, ELISA, and Western blot analysis.

Assessment of Serum & Urinary Creatinine and Microalbumin

Using spinlab automated clinical chemistry analyzer (Stanbio Laboratory, Boerne, TX, U.S.A.), the blood urea nitrogen (BUN) level was determined by an enzymatic colorimetric assay kit obtained from Biodiagnostic (Catalog No. UR2110, Giza, Egypt) and the urinary and serum creatinine (Scr) levels were measured using a quantitative colorimetric kinetic method kit (Biodiagnostic, Catalog NO. CR1251). The 24 h urinary microalbumin excretion was assayed by a rat albumin ELISA kit according to the standard manufacturer’s instructions (Abnova, Catalog No. KA0501, Taipei, Taiwan).

Creatinine clearance (CL_cr) was calculated from the 24 h urine samples and the serum using the following formula:

  

ELISA Assay of TGF-β1 and Smad4

Rat TGF-β1 was assessed according to the human enzyme-linked immunosorbent assay kit manufacturer guide (Cusabio^®, Catalog No. CSB-E04727r, Houston, TX, U.S.A.), while Smad4 was assayed by a rat smad4 ELISA kit obtained from Lifespan Biosciences (Catalog No. LS-F39606, Seattle, WA, U.S.A.).

Immunohistochemical (IHC) Examination

Formalin-fixed and paraffin-embedded tissue sections were pretreated by the heat-induced epitope retrieval technique. Concentrated primary antibodies were subsequently diluted, incubated, and auto-stained with a high-sensitivity visualization system (EnVision™ FLEX, high pH, Agilent Technologies, Singapore) in the autostainer Link 48 instrument in which the software had been preprogrammed as stated by the manufacturer’s guidelines given in the concentrated primary antibodies’ package insert. Kidney sections were then incubated with horseradish peroxidase conjugated to Goat anti-Rat immunoglobulin G (IgG) secondary antibody (Catalog No. 31470; 1 : 10000; Thermo Fisher Scientific, MA, U.S.A.). When the staining procedure was completed, the specimens were dehydrated, cleared, and mounted in aqueous mounting media. The IHC sections were examined under a light microscope to determine the expression of the target antibodies. At least ten Images from non-overlapping fields from cortical regions were captured at 400× magnification for each group (by Canon EOS Rebel T3i Camera, Canon, Japan). The immunoreactivity was evaluated by estimating the area percentage of positive brown immunostaining using ImageJ software (Madison, WI, U.S.A.) and a color deconvolution plugin for the detection and separation of the brown stain layer.³⁰⁾ Unless stated otherwise, all primary antibodies used in this study including the catalog number and dilution range were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany): anti-TGFβ1 (sc-52893, 1 : 100), anti-Smad2/3 (sc-133098, 1 : 100), anti-COL1α1 (sc-293182, 1 : 100), anti-E-cadherin (sc-8426, 1 : 100), anti-Fibronectin (sc-8422, 1 : 100), anti-TIMP-1 (sc-21734, 1 : 100), anti-KIM-1 (sc-518008, 1 : 200), anti-NGAL (sc-515876, 1 : 200) and monoclonal mouse α-SMA clone 1A4 (Dako IR611, 1 : 200) was purchased from Agilent Technologies Inc. (Santa Clara, CA, U.S.A.).

Histological Analysis

Tissue sections (5 µm) were cleared in xylene and rehydrated in serial concentrations of ethanol and then embedded in paraffin for 24 h at 56 °C in a hot air oven. On glass slides, the obtained tissue sections were collected, deparaffinized, and stained with Hematoxylin–Eosin (H&E) to assess renal injury.³¹⁾ A semi-quantitative evaluation was carried out on six photomicrographs chosen at random from non-overlapping cortical fields at ×200 and ×400 magnifications using a light microscope (Olympus IX71, Japan). A pathologist blinded to the identity of the treatment group assessed the severity of glomerular and tubulointerstitial injury in the kidney tissues of the tested groups and assigned the following scores: (1); normal histology, alterations affecting less than 25% of the cross-section, (2); changes affecting 25–50% of the section, (3); changes affecting 50–75% of the section, and (4); changes affecting more than 75% of the section.

Masson’s Trichrome and Periodic Acid Schiff (PAS) Staining

The kidney specimens from experimental groups were flushed and fixed in 10% neutral buffered formalin for 72 h. Samples were trimmed and processed by dehydration in alcohols, clearing in Xylene, synthetic wax infiltration, and blocking out into Paraplast tissue embedding media. Sections of 5 µm thick were cut by rotatory microtome and stained with either PAS staining kit (Sigma-Aldrich, Germany, Catalog No. 395B) for evaluation of basement membrane thickness or Masson’s trichrome staining kit to assess the level of collagen fibers deposition according to manufacturer’s instruction (Sigma-Aldrich, Catalog No. HT15). Briefly, the tissue sections were stained in Weigert’s iron hematoxylin working solution for 10 min followed by rinsing in running warm tap water for 10 min and washing in distilled water. Afterward, they stained in biebrich scarlet-acid fuchsin solution for 15 min and differentiated in the phosphomolybdic–phosphotungstic acid solution for 10 min. The sections were then transferred without rinse to aniline blue solution and stained for 5–10 min and differentiated in 1% acetic acid solution for 5 min (Drury and Wallington; 1983).³²⁾

Morphometric Evaluation of Renal Fibrosis

A finding of tubulointerstitial fibrosis was defined as a matrix-rich expansion of the interstitium shown in blue in Masson’s staining assessed at ×100 magnification using Leica DM500 microscope (Leica Biosystems, Germany); at least ten random non-overlapping fields from the cortical area were selected per tissue section per sample and measured using computed image analyzer (Leica LMD Software, Germany) and the degree of tubulointerstitial fibrosis in each group was expressed as a ratio relative to the total area.

For determination of tubular and glomerular basement membrane thickness photomicrographs of PAS-stained sections were taken at a 1000× magnification from non-overlapping fields by using a Leica ICC50 full HD microscopic camera (Leica Biosystems). At least twenty glomeruli and tubules were assessed in each group and the thickness was determined by a manual trace of the perimeter and automatically measured by the Leica application module for tissue section analysis (Leica LMD Software).

Western Blotting

Total protein samples were extracted from kidney tissues using the ReadyPrep™ protein extraction kit (Bio-Rad Laboratories, Catalog No. 163-2086, CA, U.S.A.), according to the manufacturer’s recommendations. The protein concentration was measured by Bradford assay (Bio-Rad Laboratories, Catalog No. 500-0201) as previously described.³³⁾ Twenty microgram of protein samples were loaded onto gradient polyacrylamide gels (4–12%; Thermo Fisher Scientific) and transferred to nitrocellulose membranes. After transfer, the membranes were blocked in 5% bovine serum albumin for 1 h. The membranes were probed with a primary antibody overnight at 4 °C. The next day, the membranes were washed using Tris-buffered saline with 0.1% Tween 20 for 7 min on the shaker. Then, the membranes were incubated with horseradish peroxidase-conjugated to goat anti-rat-IgG-AP secondary antibody (Cat. No. A18868, Thermo Fisher Scientific) for 2 h. The signals were detected using an enhanced chemiluminescence detection reagent (Novex™ AP chemiluminescent substrate, Catalog No. WP20002, Thermo Fisher Scientific). Signal intensity was analyzed using ChemiDoc Imaging System (Bio-Rad Laboratories) and quantified using either the Image Lab Software (Bio-Rad Laboratories) or ImageJ Software. The protein expression levels were normalized to β-actin. The primary antibodies used were as follows: those obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) are anti-ERK 1/2 (sc-514302,1 : 500), anti-p-ERK 1/2 (sc-81492, 1 : 500), anti-P38 (sc-7972, 1 : 500), anti-p-P38 (sc-166182, 1 : 500), HIF-1α (sc-13515, 1 : 500), LOXL2 (sc-373995, 1 : 500), anti-Twist (sc-81417, 1 : 1000), and anti-β-actin (sc-47778, 1 : 5000), the anti-Snail antibody obtained from Cell Signaling Technology (Catalog No. 3895, MA, U.S.A., 1 : 1000) and the anti-Vimentin antibody obtained from BD Biosciences (Catalog No. 550513, CA, U.S.A., 1 : 500).

Statistical Analysis

The results are presented as means ± standard deviation (S.D.). All analyses were conducted using GraphPad Prism software version 8.0.2 (San Diego, CA, U.S.A.). Comparisons between groups were statistically analyzed by parametric one-way ANOVA followed by Tukey–Kramer post hoc test. Data regarding histopathological injury scores were statistically analyzed using non-parametric one-way ANOVA (Kruskal–Wallis test) followed by post-hoc Dunn’s test. Statistical significance was set at p < 0.05.

RESULTS

Effects of Lina Treatment on Physical and Biochemical Indices in TAC-Treated Rats

Table 1 lists the changes in the functional and physical parameters in the experimental groups as a result of TAC and Lina treatment for 4 weeks. Our results showed that the kidney function biomarkers including Scr, BUN, urine volume, and microalbumin in urine were significantly increased in TAC-treated groups amounting to 83, 226, 38, and 90%, respectively, while the kidney weight/body weight ratio and CL_cr were decreased to 17 and 44%, respectively as compared to the control group. Conversely, Lina treatment attenuated all mentioned parameters compared to the TAC group.

Table 1. Effect of Lina Treatment on Physical and Biochemical Parameters of TAC-Treated Rats

Physical and biochemical parameters	Control	Lina	TAC	TAC+ Lina
Scr (mg/dL)	0.42 ± 0.07	0.41 ± 0.05	0.77 ± 0.1a)	0.5 ± 0.07b)
BUN (mg/dL)	46.8 ± 4.9	50.4 ± 5.7	152.8 ± 13.9a)	71.3 ± 11.8a,b)
Microalbumin in urine (mg/L/24 h)	20 ± 3.6	22 ± 4.0	38 ± 3.6a)	30.1 ± 3.9a,b)
CLCr (mL/min/100 g body weight)	0.52 ± 0.05	0.5 ± 0.04	0.29 ± 0.03a)	0.43 ± 0.07a,b)
Urinary output (mL/24 h)	8.3 ± 1.2	8.7 ± 1.0	10.8 ± 1.1a)	9.1 ± 1.6
Final body weight (g)	253 ± 19	219 ± 11a)	238 ± 9.7	225 ± 9.1a)
Kidney-body weight index	7.0 ± 0.72	7.5 ± 0.76	5.8 ± 0.19a)	6.3 ± 0.56

Data are presented as mean ± S.D. Data were statistically analyzed by one-way ANOVA test followed by a post hoc Tukey–Kramer test for multiple comparison between groups. a) Significantly different from the Control group; b) Significantly different from the TAC group at p < 0.05.

Effect of Lina Administration on Tubular Injury Markers

Immunoreactivity is represented as the area percentage of the positively immunostained cells of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) antibodies of different groups (Fig. 1). Kidney tissues of TAC treated group showed moderate membrane reactivity in glomeruli and marked reactivity in tubules for KIM-1 and marked cytoplasmic reactivity in glomeruli (Fig. 1C) and marked reactivity in tubules for NGAL (Fig. 1G) with a statistically significant increase by 646 and 175%, respectively comparable to the control group. Administration of Lina with TAC showed a significant improvement of 41% for KIM-1 and 28.5% for NGAL as compared to the TAC group (Figs. 1D, H).

Fig. 1. Effect of Lina Treatment on Kidney Injury Markers in TAC-Treated Rats

Immunohistochemistry photographs of KIM-1 (A–D) and NGAL (E–H). Original magnification 400×. (I) and (J) are quantitative analysis of expression in kidney sections using the ImageJ software. (A, E): kidney sections of control showing negative membrane reactivity for KIM-1 in glomeruli and in tubules, moderate cytoplasmic reactivity for NGAL in glomeruli; (B, F): kidney sections of Lina showing mild membrane reactivity for KIM-1 in tubules and mild cytoplasmic reactivity for NGAL in glomeruli; (C, G): kidney sections of TAC showing moderate membrane reactivity for KIM-1 in glomeruli and marked reactivity in tubules and marked cytoplasmic reactivity for NGAL in glomeruli and moderate reactivity in tubules; (D, H): kidney sections of TAC + Lina showing mild membrane reactivity for KIM-1 in glomeruli and mild reactivity in tubules and moderate cytoplasmic reactivity for NGAL in glomeruli and mild reactivity in tubules. Data are represented as percentage of positively immunostained cells, results are expressed as mean ± S.D. and are a representative of at least 10 photographs for each group. Scale bars represent 50 µm. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer multiple comparison test. a: Significantly different from Control group and b: Significantly different from TAC group at p < 0.05.

Linagliptin Attenuated Histopathological Alterations Induced by TAC

Histopathological changes in kidney tissues are demonstrated in Fig. 2. The histopathological investigation of kidney damage induced by TAC treatment and the effects of co-treatment with Lina was evaluated by H&E. Compared to control animals (Fig. 2A), renal sections from animals treated with TAC alone (Figs. 2C, D) showed evidence of glomerular and tubulo-interstitial injury including irregular renal capsule, scattered small-sized and atrophied glomeruli with widened Bowman’s spaces, proximal tubules with complete loss of brush borders and marked apoptotic epithelial lining, thick interstitial fibrous bands with marked inflammatory infiltrate, and renal medulla showed collecting tubules with atrophied epithelial lining and hyaline casts formation and marked interstitial inflammatory infiltrate. Co-treatment with Lina clearly prevented these pathological deteriorations (Figs. 2E, F). Furthermore, Lina administration obviously alleviated all kidney injury scores induced by TAC including (glomerular, tubular, fibrous bands, and interstitial inflammatory infiltrate) (Figs. 2G, J).

Fig. 2. Histopathological Findings Showing the Effect of Lina Administration on Tacrolimus-Induced Renal Injury in Rats

(A–F) Representative H&E staining in tissue sections. (G–J) Representative semi-quantitative renal injury scores (n = 6 per group). The images were captured under a light microscope at 200x magnification (400× for D and F). The scale bar represents 100 µm (50 µm for high power). (A; Control) Kidney showing average renal capsule (black arrow), average glomeruli (blue arrows), and average tubules (red arrow); (B; Lina) Kidney showing average renal capsule (black arrow), average glomeruli (blue arrows), and mildly dilated congested interstitial blood vessels (yellow arrow). (C, D; TAC) Kidney showing scattered small-sized glomeruli (blue arrows), and thick striped fibrous bands with marked inflammatory infiltrate (yellow arrows), high power view showing small-sized glomerulus (G) with widened Bowman’s spaces (BS), proximal tubules (P) with partial loss of brush borders (black arrows) and apoptotic epithelial lining (blue arrow), and marked interstitial inflammatory infiltrate (yellow arrow). (E, F; TAC + Lina) Kidney showing average glomeruli (black arrows), mildly dilated congested interstitial blood vessels (blue arrows), and mild inflammatory infiltrate (yellow arrow), high power view showing average collecting tubules (CT) with mildly congested peri-tubular capillaries (black arrow) and mild interstitial inflammatory infiltrate (blue arrow). Data are expressed as mean ± S.D. ^aSignificantly different from control group; ^bSignificantly different from TAC group, using Kruskal–Wallis test (non-parametric one-way ANOVA), followed by Dunn’s post-hoc test for the multiple comparisons between the groups at p < 0.05.

Effect of Lina on TAC-Induced Collagen Deposition and Basement Membrane Thickening

The effect of Lina on tubulointerstitial fibrosis induced by TAC was evaluated by Masson’s trichrome staining. Compared to the control group (Figs. 3I, K), treatment with TAC resulted in increased deposition of collagen around the tubules and in the interstitium as evidenced by intense blue staining (about 600% increase comparable to the control group). On the other hand, tissue sections obtained from the TAC+ Lina treated group displayed a lower deposition of extracellular matrix (about 55% reduction compared to the TAC group). Moreover, the PAS staining assessment (Figs. 3J, L, M) of kidney sections of the TAC group showed distinct glomerulopathy along with tubulointerstitial injury as proven by thickening of the glomerular and tubular basement membrane (The mean glomerular and tubular perimeter 1.95 ± 0.29 and 1.5 ± 0.12 µm, respectively compared to 0.9 ± 0.18 and 0.71 ± 0.27 µm, respectively for control group). Conversely, Lina treatment significantly ameliorated these deteriorations (the mean perimeter was reduced to 1.24 ± 0.29 and 1.2 ± 0.18 µm compared to that of the TAC group).

Fig. 3. Effect of Lina Treatment on TAC-Induced Collagen Deposition, Glomerulosclerosis, and Tubular Basement Membrane Thickening

(I) Representative photographs of kidney tissue sections stained with Masson’s trichrome captured under a Leica microscope at 100 × magnification, scale bar represents 200 µm. (K) Representative of the quantitative score of tubulointerstitial collagen content in renal cortex (n = 10 per group). (J) Representative photographs of PAS staining sections taken at 1000 × magnification, scale bars represent 20 µm. (L, M) Representative of the quantitative analysis of glomerular and tubular basement membrane thickness (n = 20 per group). The data are represented as mean ± S.D. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer test for multiple comparisons. a: Significantly different from the control group; b: Significantly different from TAC group at p < 0.05.

Lina Treatment Mitigated the Expression of ECM Proteins in TAC-Induced Renal Fibrosis

In the present study, we examined the effect of Lina on the expression of important ECM proteins including collagen I (COL1A1), fibronectin, and TIMP1 by immunohistochemical staining to determine the localization and intensity of proteins. Kidney sections of TAC-treated rats showed moderate expression for fibronectin in glomeruli and strong reactivity in tubules, marked reactivity for TIMP1 in tubules, and intense COL1A1 reactivity in tubules with statistical significance increment to 250, 340, and 435%, respectively as compared to the control group (Figs. 4C, G, K). Lina treatment significantly decreased the expression of fibronectin, TIMP1, and COL1A1 to 36, 41, and 44%, respectively compared to the TAC-treated group (Figs. 4D, H, L).

Fig. 4. Effect of Lina Treatment on Extracellular Matrix Proteins Deposition in a Rat Model of TAC-Induced Renal Fibrosis

Representative immunohistochemical staining of fibronectin (A–D), TIMP1 (E–H), and type I collagen (I–L) and their quantitative analysis (M–O). Original magnification 400×. (A, E, I) Kidney sections of control showing negative cytoplasmic reactivity in glomeruli and tubules. (B, F, J) Kidney sections of Lina showing negative reactivity for TIMP1 and weak cytoplasmic reactivity for fibronectin and COL1A1 around tubules. (C, G, K) Kidney sections of TAC showing moderate cytoplasmic reactivity for fibronectin in glomeruli and strong reactivity in tubules, marked reactivity for TIMP1 and intensive COL1A1 reactivity in tubules. (D, H, L) Kidney sections of TAC + Lina showing moderate tubular reactivity for fibronectin and COL1A1 and pale staining in tubules for TIMP1. Data are represented as percentage of positively immunostained cells. Results are expressed as mean ± S.D. and are representative of at least 10 photographs for each group. Scale bars represent 50 µm. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer for multiple comparison test. a, b indicates means for all markers are significantly different from Control group, and TAC group, respectively at p < 0.05.

Lina Alleviated EMT Progression Induced by TAC

EMT is distinguished by the loss of intracellular epithelial adhesion molecule (E-cadherin) expression and the acquisition of mesenchymal phenotyping (α-SMA, Vimentin). Thus, we used Western blotting and immunohistochemical staining techniques to examine their protein expression levels in kidney tissues of different groups as shown in Figs. 5-І and -ІІ. Our results showed that the TAC group exhibited remarkable upregulation of α-SMA and Vimentin (340 and 130%, respectively compared to the control group) and downregulation of E-cadherin expression (52% compared to the control group). In contrast, Lina administration significantly normalized E-cadherin expression to 75% compared to the TAC group. Moreover, the Lina treatment significantly decreased the corresponding expression levels of α-SMA and Vimentin (40 and 38%, respectively compared to the TAC group).

Fig. 5. Effect of Lina on EMT Protein Expression Induced by Tacrolimus in Kidney Tissue

Protein content and localization of epithelial and mesenchymal markers were assayed using the Western blot and immunohistochemistry methods. (І) Immunostaining of E-cadherin (A–D, I) and α-SMA (E–H, J). Micrographs shown were captured at a magnification of 400× (scale bar = 50 µm). Data were quantified by using ImageJ software and are represented as percent intensity of the areas of positive immunostained cells of at least 10 photographs taken from non-overlapping fields for each group. (ІІ) Representative western blots for vimentin (A, D), snail (B, E) and twist (C, F). The data were quantified by using ImageJ software and are represented as mean optical density of bands in each lane relative to β-actin band from the same gel ± S.D. of three individual experiments. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer multiple comparison test. a: significantly different from control group; b: significantly different from TAC group at p < 0.05

Further, we assessed the effect of tacrolimus and Lina administration on the key transcription factors (Snail and Twist) protein levels (Fig. 5-ІІ). Western blotting showed that the protein expression of Snail and Twist was significantly increased in the TAC group up to 249 and 41%, respectively compared with that of the control group, while following Lina treatment, their levels were notably suppressed to 55 and 29%, respectively compared to the TAC group.

Lina Mitigated the TGF-β1/Smad Signaling in TAC-Induced Renal Fibrosis

The TGF-β1/Smad pathway plays a pivotal role in excessive ECM accumulation and renal fibrosis progression. Therefore, we evaluated the protein expression of TGF-β1 and Smad2/3 as well as Smad4 and examined whether Lina modulates their expression in the kidney. Our results showed that the protein levels of TGF-β1 (Fig. 6K) and Smad4 (Fig. 6L) determined by ELISA were significantly increased in kidney tissues of the TAC group amounting to 1200 and 500%, respectively, compared with the control group. Conversely, Lina treatment significantly reduced their protein levels to 68 and 58%, respectively, comparable to the TAC group. Similarly, the immunohistochemical study revealed a robust increase of the protein expression for TGF-β1 in tubule-interstitial cells to 550% of kidney sections of the TAC group (Fig. 6C) and marked nuclear Smad2/3 expression in tubules to 250% (Fig. 6G). On the contrary, after Lina treatment, the protein expression of TGF-β1 and Smad2/3 significantly decreased to 58 and 34%, respectively comparable to the TAC group (Figs. 6D, H).

Fig. 6. Effect of Lina Treatment on TGF-β1 Signaling Molecules in TAC-Induced Renal Fibrosis in Rats

Representative immunostaining of TGFβ1 (A–D) and, Smad2/3 (E–H). (I, J) are quantitative analysis of expression in kidney sections. Data are represented as percentage of positively immunostained cells, results are expressed as mean ± S.D. and are representative of at least 10 photographs for each group. Scale bars represent 50 µm. (A, E) Kidney sections of control group showing negative membrane reactivity for TGF-β1 in glomeruli and in tubules and negative cytoplasmic reactivity for Smad2/3. (B, F) Kidney sections of Lina group showing mild membrane reactivity for TGF-β1 and mild cytoplasmic expression in tubules for Smad2/3. (C, G) Kidney sections of TAC showing marked reactivity in tubule-interstitial cells for TGF-β1 and marked nuclear Smad2/3 expression in tubules. (D, H) Kidney sections of TAC + Lina demonstrated moderate reactivity for TGF-β1 in glomeruli and mild reactivity in tubules and moderate nuclear expression for Smad2/3 in tubules. (K, L) Representative of TGF-β1 and Smad4 contents in kidney tissues determined by ELISA assay (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer multiple comparison test. a: Significantly different from control group. b: Significantly different from TAC group at p < 0.05.

Lina Suppressed the MAPK Signaling Pathway

Beside the TGF-β1 canonical signaling pathway, the non-canonical signaling pathways play critical roles in the progression of renal fibrosis. To determine whether Lina affects MAPK signaling, we assessed the protein expression of P38 MAPK, p-P38 MAPK, ERK1/2, and p-ERK1/2 in the kidneys of the TAC-treated rats by Western blotting (Fig. 7). After TAC treatment the ratio of phosphorylated protein levels of p38 to total p38 and p-ERK1/2 to total ERK1/2 were significantly increased by 120 and 130%, respectively as compared with the control. However, Lina treatment significantly attenuated the TAC-induced phosphorylation of P38, and ERK1/2 by 40 and 39%, respectively. These results indicated that Lina treatment could inhibit the MAPK signaling pathway to alleviate the progression of renal fibrosis in TAC-treated rats.

Fig. 7. Effect of Lina Treatment on MAPK Signaling Proteins in TAC-Induced Renal Fibrosis in Rats

Western blotting for (A) ERK/p-ERK, and (B) P38/p-P38. (C, D) are quantitative analysis of the relative expression in kidney sections using Image Lab software and are represented as optical density of bands in each lane of phosphorylated protein relative to total protein bands from the same gel. Results are expressed as mean ± S.D. of three individual experiments. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer multiple comparison test. a: Significantly different from control group. b: Significantly different from TAC group at p < 0.05.

Effect of Lina on Hypoxia

As shown in Fig. 8, the protein expression of HIF-1α and its downstream protein LOXL2 were evaluated by Western blot analysis. The protein expression levels of HIF-1α and LOXL2 were significantly increased in the TAC group by 175 and 500%, respectively compared with the control group (Figs. 8A, B). In contrast, these levels were markedly decreased to 35 and 69%, respectively following treatment with Lina compared with the TAC group (Figs. 8C, D). These results together with those mentioned above suggested that HIF-1α and LOXL2 can regulate EMT and ECM proteins in TAC-induced renal fibrosis.

Fig. 8. Effect of Lina on Hypoxia Related Proteins Induced by Tacrolimus in Rat Kidney Tissue

Representative western blots for (A) HIF-1α, and (B) LOXL2. (C, D) are quantitative analysis of the expression in kidney sections. Data were quantified by using Image Lab software and are represented as relative optical density of bands to β-actin band from the same gel. Results are expressed as mean ± S.D. of three randomly selected samples per group. Statistical significance was determined by one-way ANOVA followed by Tukey–Kramer multiple comparison test. a: Significantly different from control group. b: Significantly different from TAC group at p < 0.05.

DISCUSSION

Renal fibrosis is the final pathological outcome of CKD, a clinical condition defined by a progressive decline in kidney function and eventual progression to end-stage renal failure. As a result, slowing or stopping the advancement of CKD has arisen as a critical issue for the global medical community.³⁴⁾

Despite that TAC is a widely used immunosuppressive drug, its nephrotoxic effect, characterized by tubulointerstitial fibrosis, restricts its long-term administration.²⁶⁾

The DDP-4 is a multifunctional glycoprotein with serine exopeptidase activity. There are 2 isoforms of DDP-4: a type II transmembrane protein that has a short cytoplasmic tail and a circulating soluble form (sDPP-4) in the plasma which is responsible for the cleavage of GLP-1, activates intracellular signaling pathways and increases the proliferation of lymphocytes.³⁵⁾ The role of DPP-4 in fibrosis progression has been reported in many studies. Shi et al.³⁶⁾ have reported that DPP-4 is important for TGF-β receptor hetero-dimerization and signaling in human microvascular endothelial cells. In addition, the DPP-4 has been identified as a marker of a highly activated subset of myofibroblasts, and its inhibition resulted in reduced ECM deposition.³⁷⁾ Moreover, Lee et al.³⁸⁾ detected an increase of phosphorylated Smad2/3 and activation of nuclear factor-kappaB (NF-κB) pathway in murine fibroblasts upon stimulation with sDPP-4 alone. Furthermore, the extracellular portion of the DPP-4 enzyme contains a cysteine-rich domain that has been shown to bind to the ECM components.³⁵⁾

Lina is the most potent and a highly selective DPP-4 inhibitor. The protective effects of Lina on renal impairment were extensively studied in different experimental models of acute and chronic kidney (diabetic and non-diabetic) diseases independent of its glucose-lowering effect.^22,23) The present study was carried out to explore the protective role of Lina in a rat model of TAC-induced renal fibrosis and to elucidate the underlying molecular mechanisms.

In the present study, administration of TAC has resulted in a substantial increase in renal function parameters including S_cr, BUN, urinary output, and urinary albumin excretion along with a significant decrease in Cl_cr. In addition, the expression of tubular injury markers NGAL and KIM-1, sensitive and reliable predictors of early-stage renal impairment³⁹⁾ have been upregulated in our study following TAC treatment. These effects are in line with other previous reports.^40–42) On the other side, treatment with Lina significantly improved renal dysfunction and significantly decreased the tubular expression of NGAL and KIM-1 in TAC-treated group. Our findings are similar with observations made by Mayer et al.²¹⁾ In addition, the study of Oraby et al.⁴³⁾ showed that Lina significantly modulated renal tubular and glomerular injury markers (KIM-1, NGAL, vanin-1 and nephrin) in a diabetic nephropathy model.

The typical histological features associated with TAC nephropathy were observed in TAC-treated kidneys. These alterations were consistent with other previous investigations.^1,44) Contrariwise, the co-treatment with Lina significantly improved the morphological changes and reduced interstitial collagen deposition.^21,45) Subsequently, we tested our hypothesis that Lina could improve TAC-induced EMT and ECM protein deposition.

EMT is one of the key events of renal fibrosis in which epithelial cells lose their functionality and characteristics, and acquire mesenchymal cell phenotyping.⁴⁶⁾ The role of EMT process in the pathogenesis of TAC-induced interstitial fibrosis has been partially explored.^47,48) Several studies indicated that TGF-β1 can induce EMT in renal fibrosis.⁷⁾ In addition, TGF-β1 has been reported to induce the expression of Twist, Snail, and Slug downstream transcription factors of EMT.⁴⁹⁾ The current study clearly showed that Lina reversed the progression of EMT as indicated by upregulation of E-cadherin expression and a decrease in α-SMA and Vimentin overexpression accompanied by a consequent downregulation of Snail and Twist expression. Although our work is the first to report the protective effect of Lina against EMT in vivo, a recent in vitro study by Huang and his colleagues⁵⁰⁾ showed that sDPP-4 upregulated the EMT markers (actin alpha 2 and collagen type1) and increased total collagen content. In addition, the sDPP-4 activated Smad signaling through TGFBR in renal epithelial cells, whereas treatment with Lina abrogated such effects.

One of the most prominent pathological alterations of renal intestinal fibrosis is excessive ECM deposition. This deposition results from fibroblast differentiation into myofibroblasts that can be promoted by TGF-β⁵¹⁾ and have been regulated by metalloproteinases, lysyl oxidase enzymes, and the EMT process.⁵²⁾ Consistent with that published by Lim et al.⁵³⁾ our immunohistochemical results have shown a significant increase in tubular expression of ECM proteins including collagen type І, and fibronectin following TAC treatment. One more finding in our study which has not been published elsewhere is that TAC treatment has resulted in an increased tubular expression of TIMP1. On the contrary, Lina administration efficiently decreased the expression of collagen 1 and fibronectin and preventing accumulation of ECM in kidney interstitium. This finding is one of the proposed mechanisms through which Lina exerts its renal protective effect in our model of fibrosis and was in line with other previous studies.^45,54)

The TGF-β1cytokine is a critical regulator of renal fibrosis including TAC-nephropathy.^40,55) Accumulating evidence showed that TGF-β1 can stimulate renal fibrosis by activating both canonical and non-canonical signaling pathways.⁵⁶⁾ In the classical TGF-β/Smad signaling pathway, TGF-β1 binds to the type II TGF-β receptor and then recruits type I TGF-β receptor. The activated TGFβ receptor complex directly phosphorylates and activates Smad2/3 subsequently; the phosphorylated Smad2/3 forms a complex with Smad4. The trimeric complex translocates into the nucleus and induces transcription of fibrotic target genes.¹⁴⁾

Accordingly, we investigated whether Lina could block the TGF-β1/Smad signaling. Our results proved that Lina treatment significantly decreased the expression and protein content of TGF-β1, Smad2/3, and Smad4 in TAC kidney tissues. The current results are in accordance with the study of Hasan et al.⁵⁷⁾ Additionally, TGF-β1 could act by Smad-independent signaling, such as the MAPK pathway which is also important in the progression of renal fibrosis and EMT.⁵⁸⁾ The MAPK family involves three main kinases ERK, c-Jun N-terminal kinase (JNK), and P38 that can be directly activated upon TGF-β receptor complex activation. Furthermore, MAPK can phosphorylate Smad2/3 linker residues and alter their downstream genes transcription.^14,56) Our results showed that the protein expression of phosphorylated and total P38 MAPK and ERK1/2 were notably elevated by TAC. These conclusions are in agreement with a recent report of Azouz et al.⁵⁹⁾ who showed that the relative protein expressions of p-ERK/ERK and p-p38 MAPK/p38 MAPK were upregulated in TAC nephrotoxicity model. Our work is one of the few in the literature to address the protective effect of Lina during MAPK pathway activation.

Hypoxia has been proposed as a contributor to the pathogenesis of TAC nephrotoxicity resulting from renal afferent arteriole vasoconstriction and RAAS activation and subsequent decrease in renal blood flow and glomerular filtration.^4,60) The transcription factor, HIF-1α is a sensitive indicator of hypoxia that upregulates the expression of biosynthetic enzymes and profibrogenic proteins such as LOXL2.¹⁸⁾ The present study uniquely proves that treatment with TAC has resulted in a remarkable increase in the protein expression of HIF-1α and LOXL2. Our results are the first demonstration highlighting the significance of HIF-1α in TAC nephropathy. Likewise, limited studies have reported the role of HIF-1α downstream proteins on TAC-induced renal fibrosis.⁶¹⁾ On the other hand, Lina effectively attenuated the protein expression of HIF-1α and LOXL2 in TAC-treated rats.

CONCLUSION

In summary, the DPP-4 inhibitor, Lina, protects against TAC-induced renal impairment and tubulointerstitial fibrosis. The results of our study are the first to show that Lina could attenuate TAC-induced TGF-β1-mediated EMT in vivo by inhibiting activation of Smad/ERK/P38 phosphorylation, as well as HIF-1α/LOXL2 signaling resulting in a subsequent reduction of protein expression of epithelial, mesenchymal, and transcriptional biomarkers. These results demonstrate that Lina provides a renoprotection irrespective of its glucose-lowering action in non-diabetic kidney diseases.

Acknowledgments

We are deeply thankful to Prof. Dr. Sayed Abdel Raheem, Department of Pathology, Faculty of Medicine, Al-Azhar University for his kind help in histopathological and immunohistochemical investigation, and Prof. Dr. Dina Sabry Abdel Fatah, Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University for her kind help in conducting the Western blot analysis.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Nady ME, Abd El-Raouf OM, and El-Sayed EM designed the study, acquired, analyzed, and validated data. Nady ME provided the necessary tools and reagents and performed all experimental studies under the direct supervision of Abd El-Raouf OM, and El-Sayed EM. Nady ME and Abd-El Raouf OM wrote the first original draft of the manuscript. All authors have revised and approved the manuscript in its final form. El-Sayed EM submitted the manuscript to the journal as the corresponding author.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data and materials can be freely obtained from the authors through correspondence.

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