Nicotine Exacerbates Tacrolimus-induced Renal Injury By Programmed Cell Death
Mar 23, 2022
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Yu Ji Jiang, Sheng Cui, Kang Luo, Jun Ding
INTRODUCTION
Despite the discovery of new immunosuppressants, tacrolimus (TAC) remains the cornerstone of immunosuppressive regimens in solid organ transplantation. However, long-term use of TAC increases the risk of adverse effects (e.g., nephrotoxicity, neurotoxicity, infections, malignancies, diabetes, and gastrointestinal complaints) [1]. Among these, acute kidney injury or chronic TAC nephrotoxicity has been reported in 46% of lung transplant recipients [2] and 22.4% of kidney transplant recipients [3]. While acute kidney injury is considered reversible after TAC dose reduction or a complete withdrawal, chronic nephrotoxicity, which leads to allograft loss, is irreversible. Chronic TAC nephrotoxicity is characterized by glomerulopathy, hyalinosis of afferent arterioles, and striped tubulointerstitial fibrosis (TIF) [4]. Although the exact mechanism of this clinical dilemma remains unknown, we recently demonstrated that oxidative stress-originated inflammation, transforming growth factor β1 (TGF-β1), and programmed cell death may be important players [5]. Cigarette smoking is a critical public health challenge and societal financial burden which reduces the quality of life. Epidemiological reports show that there are currently more than one billion cigarette smokers worldwide and six million deaths annually owing to tobacco use. These rates are both increasing, particularly in developing or undeveloped countries [6]. It is well recognized that cigarette smoking is a risk factor for various cancer types, cardiovascular events (myocardial infarction and stroke), and obstructive lung diseases. In the kidney, smoking increases renal dysfunction severity in patients with diabetes, hypertension, polycystic kidney disease, and post-kidney transplant [7]. Moreover, smoking may also cause de novo kidney damage even in a healthy population without pre-existing chronic kidney disease (CKD) [8]. This study sought to evaluate: (1) whether nicotine (NIC), a primary toxic component of cigarette smoking, aggravates TAC-induced renal injury; and, if so, (2) which mechanism accounts for NIC(nicotine)-accelerated nephrotoxicity.
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RESULTS
Effect of NIC(nicotine) on basic parameters
Table 1 outlines the effects of NIC(nicotine) on basic parameters in the experimental groups. Both NIC(nicotine) and TACincrease UV(polyuria), which was further increased by NIC(nicotine) and TAC treatment. Neither NIC(nicotine) nor TAC prevented BW loss. Urine protein excretion, Scr, BUN, and Cys-C were markedly higher in both the NIC(nicotine) and TAC groups compared with the VH group; these levels further increased with the combination of the two drugs, implying that NIC(nicotine) accelerates TAC-induced renal dysfunction. There were no significant between-group SBP differences. Four-week TAC treatment increased blood TAC concentration to 11.0±o.g ng/mL, while NIC(nicotine) did not influence blood TAC concentration(12.8±15 ng/mL,p>o.o5 vs. TAC).In addition, treating normal rats with NIC(nicotine) result-ed in serum and urine cotinine concentration increases by 56.5±17 ng/mL and 8;6±192.6 μg/day， respectively. These serum and urine cotinine concentrations mimic those observed in humans who are active smokers [13].
Table 1. Effect of NIC(nicotine) on functional parameters
Effect of NIC(nicotine) on histopathology in chronic TAC nephrotoxicity
Chronic TAC nephrotoxicity is characterized by unique histological features including 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. IA, IC, and D), this may link to increment urine protein excretion. On quantitative analyses, both NIC(nicotine) and TAC increased the fractional mesangial area, with a further increase observed in the NIC(nicotine) and TAC group. The major changes in chronic TAC nephrotoxicity were confined to tubulointerstitial areas, as delineated by tubulointerstitial fibrous deposition, tubular atrophy, and striped fibrosis (Fig.1B,1E, and F). Figures clearly show that the TIF score in the NIC(nicotine)+TACgroup was higher than in either the NIC(nicotine) group or the TACgroup.
Figure 1. Representative photomicrographs of periodic acid-Schiff(PAS) (A), Masson trichrome (B),transmission electron mi-croscopy(C-F), and quantitative analysis of glomerular injury and tubulointerstitial fibrosis(TIF);(C)glomerular basement membrane thickening (arrows);(D)effacement of podocyte foot processes (arrows);(E)swelling of the tubulointerstitium and extensive collagen deposition (scatters);(F) atrophied tubules (arrows).VH, vehicle; NIC(nicotine), nicotine; TAC, tacrolimus. p<o.o1 vs. VH, Pp< o.05 Vs.NIC(nicotine),$p<0.05 vs.TAC or NIC(nicotine).
Effect of NIC(nicotine) on profibrotic cytokine expression in chronic TAC nephrotoxicity
The present study sought to compare the expression of the profibrotic cytokine TGF-β1 and CTGF, and the extracellular matrix (ECM) component of βig-h; between the experimental groups. Consistent with the TIF findings, NIC(nicotine) augmented TAC-induced overexpression of pro-fibrotic TGF-β1and CTGF and ECMβig-h3(Fig.2)
Figure 2. Representative photomicrographs of immunoblotting analysis for(A)transforming growth factor β1 (TGF-B1),(B TGF-β-induced gene-h(βig-h3), and (C) connective tissue growth factor(CTGF).Values for protein expression are represented usingthe vehicle (VH)group as 1oo% reference and normalized to β-actin. NIC(nicotine), nicotine;TAC,tacrolimus. 3p<o.o1 vs.VH, 5p <0.05 Vs. NIC, "p<o.05 Vs.TACor NIC(nicotine).
Effect of NIC(nicotine) on inflammation in chronic TAC nephrotoxicity
To define the effect ofNIC 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 the expression of pyroptosis-related cytokines (ⅡL-1, IL-18, and NLRP3)and MCP-1, resulting in ED-1-positive cell infiltration, and further increases were found by a combined treatment of NIC(nicotine) and TAC.
Figure 3. Representative photomicrographs ofimmunohistochemistry for ectodermal dysplasia-1 (ED-1) (A) and immunoblot-ting analysis for proinflammatory cytokines (B). VH, vehicle; NIC(nicotine), nicotine; TAC, tacrolimus; MCP-1, monocyte chemotactic protein-1;IL, interleukin; NLRP3, NOD-like receptor pyrin domain-containing protein3. p<o.o1 vs.VH,pp<o.os vs. NIC(nicotine),cp <O.05 vs.TAC or NIC(nicotine).
Effect of NIC(nicotine) on oxidative stress in chronic TAC nephrotoxicity
As shown in Fig.4, chronic(nicotine) 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 of8-OHdG, an oxidative stress marker, were 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.
Figure 4.Representative photomicrographs of immunoblotting analysis of a series of oxidative stress-related proteins (A) and serum (B) and urinary (C) 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations. VH, vehicle; NIC(nicotine), nicotine; TAC, tacrolim-us; SOD, superoxide dismutase; NOX,NADPH oxidase.Bp<o.01 vs.VH, p<o.o5vs. NIC,$p<o.05 vs. TAC or NIC(nicotine).
Effect of NIC(nicotine) on programmed cell death in chronic TAC nephrotoxicity
Either type I(apoptosis)or type II (autophagy) programmed cell death is involved in the pathogenesis of chronic TAC-induced renal injury [1s]. Using TUNEL assay, we observed that most TUNEL-positive cells or apoptotic bodies were localized to tubular epithelial cells and interstitial vascular endothelial cells, where tubular atrophy and typical TIF progressed(Fig.5A). Quantitative analysis showed that both NIC(nicotine) 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, the addition of NIC(nicotine) 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).
Figure 5. Representative photomicrographs of TdT-mediated dUTP-biotin nick end labeling(TUNEL) assay (A) and immuno-blotting analysis for apoptosis-controlling genes (B). Nicotine treatment increases TUNEL-positive cells (arrows) in the tacro-limus-treated rat kidneys.VH, vehicle; NIC(nicotine),nicotine; TAC,tacrolimus; Bcl, B-celllymphoma; Bax, Bcl2-associated X. p<o.o1 vs.VH,Pp<o.o5 vs. NIC, 'p<0.05vs.TACor NIC.
Effect of NIC(nicotine) on mitochondrial dysfunction in chronic TAC nephrotoxicity
By transmission electron microscopy, we clearly observed that both NIC(nicotine) 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 organelle fission)(Fig.6). Quantitative analysis results showed that NIC(nicotine) 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 Drpi) induced by either Nicor TAC was exacerbated by coadministration of NICand TAC (Fig. 6).
Figure 6. Representative transmission electron micrographs of autophagy and mitochondrial morphology(A-F), immunoblot-ting analysis(G, H), and quantitation of mitochondrial number and size in each treatment group. (A)Autophagy formation in the kidneys of tacrolimus (TAC) and/or nicotine (NIC) treated rats (arrow);(B) an autophagic compartment containing mitochondria the kidneys of 'TAC and/or NIC(nicotine) treated rats (mitophagy, arrow); (C) normal mitochondria;(D) reduced mitochondrial number in the kidneys of TAC and/or NIC(nicotine) treated rats;(E) mitochondrial fusion seen in the kidneys of TAC and/or NIC treated rats (circle);(F) mitochondrion divided into two or more daughter organelles in the kidneys ofTAC and/or NIC treated rats(fission, circle). VH, vehicle;LC3B, light chain 3B;PINK1, phosphate and tension homolog deleted on chromosome ten-induced kinase 1;OPA, optic atrophy protein; Drp, dynamin-related protein.p<o.o vs.VH, pp<o.o5 vs. NIC,$p<o.o5vs. Tacori NIC(nicotine).
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Effect of NIC(nicotine) on endoplasmic reticulum stress in chronic TAC nephrotoxicity
As shown in Fig.7, Nicor TAC induced degranulation of ribosomes, disconnected and dilated cisternae, and peroxisome vacuolization in the rough endoplasmic reticulum(ER), whereas smooth ER structure remained almost normal (Fig.7B). Immunoblotting analysis revealed that either NIC(nicotine) or TAC increased the 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).
Figure 7. Representative photomicrographs of transmission electron microscopy(A, B) and immunoblotting analysis of endo-plasmic reticulum(ER) stress-related genes (C). (A) Normal rough ER (arrows);(B) ribosomes degranulation,disconnected and dilated cisternae, and vesicle dilatation of rough ER in nicotine(NIC) and/or tacrolimus(TAC)-treated rat kidney. VH, vehicle;CHOP, C/EBP homologous protein;Bip, binding immunoglobulin protein; IRE-1c,inositol-requiring protein-1c; ATF, acti-vating transcription factor. p< o.01 vs.VH, p<o.05 vs. NIC(nicotine), cp<o.05 vs. TAC or NIC(nicotine).
DISCUSSION
Cigarette smoking is an important modifiable risk factor for the progression of CKD in both healthy populations and patients with underlying diseases(e.g, diabetes, hypertension). Clinical trials show that cigarette smoking increases urinary albumin excretion and adversely affects kidney function [16]; in addition, smoking cessation may improve proteinuria in patients with CKD and type 2 diabetes and slow the progression to end-stage renal disease[1-,18]. Moreover, either recipient or donor smoking contributes to rejection episodes and chronic allograft nephropathy, leading to allograft loss in kidney transplantation [19,2o]. These deleterious effects of smoking(NIC(nicotine))are mirrored by studies in rodent models of s/6 nephrectomy[2al and diabetic nephropathy [o]. In the present study, we found that NIC(nicotine) aggravated TAC-induced fractional mesangial area and TIF. These pathological changes led to more pronounced renal function impairment and boosting urinary protein excretion. Based on these results and other reports, we suggest that cigarette smoking may accelerate renal allograft loss in transplant recipients who receive TAC-based immunosuppressants.
Inflammation plays a pivotal role in the evolution of chronicTACnephrotoxicitybecauseit precedes ongoing renal scarring. Increased proinflammatory mediators in response to injurious stimuli may recruit inflammatory cells, which in turn overexpress pro-inflammatory and profibrotic cytokines such as chemoattractants, adhesion molecules, and TGF-β1. We have recently demonstrated that NLRP3-dependent and independent inflammasome are involved in the pathogenesis of chronic TAC nephrotoxicity [s]. Our results show that administering NIC(nicotine) to TAC-treated rats amplifies the expression of pyroptosis-related genes (NLRP3, IL-1β, and I-18), proinflammatory mediators MCP-1, and profibrotic cytokines TGF-β and CTGF, leading to excessive ED-1-positive cells influx and βig-h upregulation; these responses reflect the severity of glomerular and tubular injuries. Our findings are consistent with those of Arany et al.[22]and Agarwal et al.[23],which showed the effects of NIC on renal inflammation and fibrosis.
Although NIC(nicotine)-based exacerbation of chronic TAC nephrotoxicity in this model may be multifactorial, it is likely related to the impact of oxidative stress. It is well known that chronic TAC treatment is closely associated with afferent arteriolopathy-related hypoxia, which subsequently links to oxidative stress, which in turn activates profibrotic TGF-β1 overexpression, leading to fibrosis. This construct is supported by the observation that antioxidant therapies(e.g, coenzyme Q10)attenuate TGF-β1 expression and TIF via preservation of mitochondrial integrity in chronic TAC nephrotoxicity 24]. Recently, overwhelming evidence from in vitro studies demonstrates an interrelationship between chronic NIC exposure and oxidative stress in a variety of renal injuries [o,25,26. Herein, we found that NIC increases serum and urinary 8-OHdG levels and augmented oxidant protein expression, while it suppresses antioxidant protein expression, thereby boosting TAC-induced oxidative stress as well as fibrosis. Indeed, NIC aggravation of TAC-treated kidneys in this study may be attributed to oxidative stress.
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It is also possible that both apoptosis(type I pro-gramed cell death) and autophagy (type II programmed cell death) are involved in NIC(nicotine)-accelerated chronic TAC nephrotoxicity. As previously illustrated, NIC directly induces podocyte and renal proximal tubule cell apoptosis [27,28]; it also indirectly induces apoptotic cell death via oxidative stress[2g] or activation of TGF-β1[22].NIC(nicotine) may also induce autophagy in pancreatic stellate cells Bo], neonatal mouse cardiac myocytes 31], and vascular smooth muscle cells 32]. Thus, oxidative stress and programmed cell death are interrelated. Using TUNEL assay and electron microscopy, we clearly observed that NIC significantly increased the number of TUNEL-positive cells in tubular epithelial cells, interstitial vascular endothelial cells, and autophagic compartments in TAC-treated rat kidneys. These morphological changes were accompanied by dysregulation of apoptosis- or autophagy-related genes in rat kidneys, leading to cell death. This cumulative evidence suggests that NIC(nicotine) itself not only induces oxidative stress and programmed cell death but also promotes the adverse effect of TAC on the kidney, resulting in renal dysfunction and architectural damage, as previously reported in models of streptozotocin (STZ)-induced diabetic nephropathy [o] and CKD (s/6 nephrectomy) B3].
In addition, oxidative stress-originated intracellular organelles such as mitochondrial dysfunction and ER stress play a critical role in chronic TAC nephrotoxicity integrity, trigger ER stress, and dysregulate mitochondrially and ER stress-controlling genes and the effects are worsened by combined treatment with the two. Thus, deterioration of mitochondrial fitness and ER stress may account for the effect of NIC(nicotine) on TAC-induced renal injury
Our study revealed that NIC(nicotine) at a dose of15mg/kg aggravates TAC-induced renal injury through oxidative stress, inflammation, and programmed cell death, consistent with previous studies [27,37,38]. However, others have observed opposite findings. Sadis et al.Bg] showed that NICat a dose ofo.s mg/kg protects the kidney from ischemia/reperfusion injury through the cholinergic anti-inflammatory pathway. Another study by Agarwal et al.[23] reported that long-term oral treatment with NIC(nicotine) (28 weeks)confers renoprotective effects in a rat model of spontaneous proteinuria （Munich-Wistar-Frömter rats). The reasons for this discrepancy in the role of NIC on the kidney are unknown but may depend on NIC dose, drug treatment duration, or rodent model. Further studies are required to resolve these issues. This study clearly demonstrates that NIC exacerbates TAC-induced renal injury in a rat model of chronic TAC nephrotoxicity. Aggravation of oxidative stress and programmed cell death may be one mechanism underlying the deleterious effects of NIC. Our findings provide greater insight into the impacts of smoking among transplant recipients.
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