The protocol of the experimental study was approved by the Animal Care Committee of the Yanbian University of China (SYXK [J]2020-0009; YBU-2018-045). A total of 32 male Sprague-Dawley rats (Yanbian, China) weighing 200 to 220 g were housed in individual cases in a temperature- and light-controlled environment and allowed free access to a low-salt diet (0.05% sodium; Teklad Premier, Madison, WI, USA) and tap water. TAC was diluted in olive oil (Sigma, St. Louis, MO, USA) to a final concentration of 1.5 mg/mL.

Following a 1-week acclimatization period on a low-salt diet, weight-matched rats were randomized into four subgroups and treated daily for 4 weeks with: (1) subcutaneous injection with olive oil (vehicle [VH] group, n = 8, 1 mL/kg); (2) simultaneous treatment with olive oil and NIC (NIC group, n = 8, 1.5 mg/kg; intraperitoneal, n = 8); (3) subcutaneous injection with TAC (TAC group, n = 8, 1.5 mg/kg); (4) simultaneous treatment with a combination of NIC and TAC (NIC + TAC group, n = 8). TAC (Prograf, Astellas Pharma, Ibaraki, Japan) and NIC (Sigma-Aldrich) doses were based on previous studies [9,10].

The antibodies used in this study for immunohistochemistry and immunoblotting were as follows: TGF-β1 (R&D Systems, Minneapolis, MN, USA), connective tissue growth factor (CTGF, #ab125943, Abcam, Cambridge, UK), TGF-β inducible gene-h3 (βig-h3, Proteintech, Chicago, IL, USA), ectodermal dysplasia-1 (ED-1, Serotec Inc., Oxford, UK), monocyte chemotactic protein-1 (MCP-1, Santa Cruz Biotechnology, Dallas, TX, USA), interleukin-1beta (IL-1β, #ab9722, Abcam), interleukin 18 (IL-18, #ab191152, Abcam), NOD-like receptor pyrin domain-containing protein 3 (NLRP3, #ab214185, Abcam), superoxide dismutase 1 (SOD1, #ab16831, Abcam), superoxide dismutase 2/manganese superoxide dismutase (SOD2/MnSOD, #ab13534, Abcam), NADPH oxidase-2 (NOX-2, #ab31092, Abcam), NADPH oxidase-4 (NOX-4, #ab109225, Abcam), B-cell lymphoma-2 (Bcl-2, #ab59348, Abcam), Bcl2-associated X (Bax, #ab32503, Abcam), cleaved caspase-3 (Millipore, Billerica, MA, USA), p62 (#ab56416, Abcam), light chain 3B (LC3B, #ab192890, Abcam), Parkin (#2132, Cell Signaling, Danvers, MA, USA), phosphate and tension homology deleted on chromsome ten-induced kinase 1 (PINK1, N4/15, #ab186303, Abcam), optic atrophy protein 1 (OPA1, #ab42364, Abcam), dynamin-related protein 1 (DLP1/Drp1, BD Transduction Laboratories, Lexington, KY, USA), C/EBP homologous protein (CHOP, L63F7, #2895, Cell Signaling), binding immunoglobulin protein (BiP, C50B12, Rabbit mAb, #3177, Cell Signaling), inositol-requiring protein-1α (IRE-1α, phospho S724, #ab48187, Abcam), activating transcription factor 6 (ATF-6, D4Z8V, Rabbit mAb, #65880, Cell Signaling), and β-actin (#ab8226, Abcam).

Before sacrifice, animals were housed individually in metabolic cages (ZH-B6, Anhui Zhenghua Biological Instrument Equipment, Anhui, China) for water intake measurement and 24-hour urine volume (UV) collection. On the following day, animals were anesthetized, and blood samples were withdrawn for further analysis. Serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C (Cys-C) were measured using a quantitative enzymetic colorimetric method (Roche Cobas 8000 Core ISE, Roche Diagnostics, Basel, Switzerland).

After the treatment was initiated, rats were pair-fed and their body weight (BW) was monitored daily. Systolic blood pressure (SBP) was recorded in conscious rats by the tail-cuff method with plethysmography using a tail manometer-tachometer system (XR900, Shanghai XinRuan Information Technology Co. Ltd., Shanghai, China). At least three readings were averaged for each rat. Urine protein was measured by the enzymatic colorimetric method (Modular DPP system, Roche, Hamburg, Germany). Whole-blood TAC level was measured using liquid chromatography mass spectrometry/mass-spectrometry.

Kidney specimens were preserved by in vivo perfusion. Rats were perfused using 0.01 mol/L phosphate-buffered saline (PBS) to flush blood via the abdominal aorta, and dissected kidneys were rapidly immersed in periodate-lysine-2% paraformaldehyde solution and embedded in wax. After dewaxing, 4-μm sections were processed and stained with periodic acid-Schiff (PAS) or Masson’s trichrome and hematoxylin. Glomerular injury (fractional mesangial area) and TIF were assessed in PAS or trichrome stained sections and quantified by a color image auto-analyzer (VHX-7000, Leica Microsystems, Wetzlar, Germany), as we previously described [11,12].

Following dewaxing, sections were incubated with 0.5% Triton X 100/PBS solution for 30 minutes and washed with PBS three times. Non-specific binding sites were blocked with normal horse serum diluted 1:10 in 0.3% bovine serum albumin for 30 to 60 minutes, and then incubated for 2 hours at 4°C in mouse antiserum against ED-1 diluted in 1:1,000 in humid environment. After rinsing in tris-buffered saline (TBS), sections were incubated in peroxidase-conjugated rabbit anti-mouse immunoglobulin G (IgG, Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 30 minutes. For coloration, sections were incubated with a mixture of 0.05% 3,3′-diaminobenzidine containing 0.01% H₂O₂ at room temperature until a brown color was visible, washed with TBS, counterstained with hematoxylin and examined under light microscopy. Analysis of ED-1-positive cells was examined automatically by counting the percentage of positive areas in each section of twenty nonoverlapping fields using a color image auto-analyzer (VHX-7000, Leica Microsystems).

Kidney tissues were post-fixed with 1% osmium tetroxide-N-methylmorpholine N-oxide (OSO4) and embedded in Epon 812 following fixation in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Ultrathin sections were cut and stained with uranyl acetate/lead citrate, and photographed with a JEM-1400 Flash transmission electron microscope (JEM-1400 Flash HC, JEOL Ltd., Tokyo, Japan).

Immunoblot analysis was performed as described previously [9]. Briefly, renal tissue lysates were incubated with specific antibodies, and immunoblotting images analyses were performed using an image analyzer (Odyssey CL Imaging System, LI-COR Biosciences, Lincoln, NE, USA). Optical densities were obtained using the VH group as 100% reference and normalized with β-actin.

Apoptosis in tissue sections was identified using the ApopTag in situ apoptosis detection kit (Sigma-Aldrich). The number of TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells was counted on 20 randomly selected non-overlapping fields in each section at ×200 magnification (VHX-7000, Leica Microsystems).

The levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) were evaluated using a competitive enzyme-linked immunosorbent assay (ELISA) to determine the levels of DNA adduct 8-OHdG in serum and urine (Japan Institute for the Control of Aging, Shizuoka, Japan).

Serum and urine concentrations of cotinine, a stable NIC metabolite, were evaluated using commercial ELISA kits (Calbiotech, San Diego, CA, USA) according to the manufacturer’s instructions.

The dates are expressed as mean ± standard error. Multiple comparisons among groups were fulfilled by one-way analysis of variance (ANOVA) with the Bonferroni post hoc test (SPSS software version 21.0, IBM, Armonk, NY, USA). Statistical significance was accepted as p < 0.05.

Table 1 outlines the effects of NIC on basic parameters in the experimental groups. Both NIC and TAC increase UV (polyuria), which was further increased by NIC and TAC treatment. Neither NIC nor TAC prevented BW loss. Urine protein excretion, Scr, BUN, and Cys-C were markedly higher in both the NIC and TAC groups compared with the VH group; these levels further increased with combination of the two drugs, implying that NIC accelerates TAC-induced renal dysfunction. There were no significant between-group SBP differences. Four-week TAC treatment increased blood TAC concentration to 11.0 ± 0.9 ng/mL, while NIC did not influence blood TAC concentration (12.8 ± 1.5 ng/mL, p > 0.05 vs. TAC). In addition, treating normal rats with NIC resulted in serum and urine cotinine concentration increases by 56.5 ± 1.7 ng/mL and 856 ± 192.6 μg/day, respectively. These serum and urine cotinine concentrations mimics those observed in humans who are active smokers [13].

Chronic TAC nephrotoxicity is characterized by unique histological features includes striped TIF and glomerulopathy. By histological staining and electron microscopy, we found that TAC-caused glomerular injury is manifested by glomerular basement thickening and effacement of podocyte foot processes (Fig. 1A, 1C, and 1D), this may link to increment urine protein excretion. On quantitative analyses, both NIC and TAC increased the fractional mesangial area, with further increase observed in the NIC and TAC group. The major changes in chronic TAC nephrotoxicity were confined to tubulointerstitium areas, as delineated by tubulointerstitial fibrous deposition, tubular atrophy, and striped fibrosis (Fig. 1B, 1E, and 1F). Figures clearly show that the TIF score in the NIC + TAC group was higher than in either the NIC group or the TAC group.

The present study sought to compare the expression of the profibrotic cytokine TGF-β1 and CTGF, and the extracellular matrix (ECM) component of βig-h3 between the experimental groups. Consistent with the TIF findings, NIC augmented TAC-induced overexpression of pro-fibrotic TGF-β1 and CTGF and ECM βig-h3 (Fig. 2).

To define the effect of NIC on inflammation in TAC-induced renal injury, we studied the expression of proinflammatory mediators and pyroptosis-related genes by immunoblotting and immunohistochemistry. Fig. 3 showed that TAC treatment upregulated expression of pyroptosis-related cytokines (IL-1β, IL-18, and NLRP3) and MCP-1, resulted in ED-1-positive cell infiltration, and further increases were found by a combined treatment of NIC and TAC.

As shown in Fig. 4, chronic TAC treatment is closely associated with an imbalance between oxidant and antioxidant enzymes [12,14]. Immunoblotting analysis showed that NIC upregulated TAC-induced NOX-2 and NOX-4 expression but suppressed SOD1 and SOD2 expressions. In addition, serum and urinary levels of 8-OHdG, an oxidative stress marker, was higher in the NIC and TAC groups compared with the VH group and highest in the NIC + TAC group. These findings imply a synergistic effect of NIC and TAC on oxidative stress in this model.

Either type I (apoptosis) or type II (autophagy) programmed cell death is involved in the pathogenesis of chronic TAC-induced renal injury [15]. Using TUNEL assay, we observed that most TUNEL-positive cells or apoptotic bodies were localized to tubular epithelia cells and interstitial vascular endothelial cells, where tubular atrophy and typical TIF progressed (Fig. 5A). Quantitative analysis showed that both NIC and TAC significantly increased TUNEL-positive cells compared with the VH groups; this was more pronounced in the NIC + TAC group. At a molecular level, addition of NIC to TAC-treated rats resulted in a significant dysregulation of Bcl-2/Bax and cleaved caspase-3 expression toward cell death (Fig. 5B). In addition, electron microscopy displayed that TAC treatment induced an abundant formation of autophagic compartments, such as initial autophagic vacuoles (AVi), degradative autophagic vacuoles (AVd), and mitophagy (a form of selective autophagy) in the NIC, TAC, and NIC + TAC groups (Fig. 6). Quantitative immunoblotting revealed that overexpression of p62, LC3B, PINK1, and Parkin proteins seen in TAC-treated rat kidneys was further increased with NIC (Fig. 6G and 6H).

By transmission electron microscopy, we clearly observed that both NIC and TAC destroyed mitochondrial architectures, as illustrated by reduced mitochondrial size and number, disorganized cristae, vacuolization, fusion, mitophagy formation, and mitochondria divided into two or three daughter organelles (fission) (Fig. 6). Quantitative analysis results showed that NIC treatment further decreased the number and size of mitochondria compared with NIC or TAC treatment alone. Immunoblotting analysis revealed that dysregulation of mitochondrial-related proteins (OPA1 and Drp1) induced by either NIC or TAC was exacerbated by coadministration of NIC and TAC (Fig. 6).

As shown in Fig. 7, NIC or TAC induced degranulation of ribosomes, disconnected and dilated cisternae, and peroxisome vacuolization in rough endoplasmic reticulum (ER), whereas smooth ER structure remained almost normal (Fig. 7B). Immunoblotting analysis revealed that either NIC or TAC increased expression of ER stress-related genes including CHOP, Bip, IRE-1α, and ATF-6, but their expression was further increased by the combination of the two (Fig. 7C).