Abstract

The plant-derived toxin aristolochic acid (AA) is the cause of herbal nephropathy and Balkan nephropathy. High-dose AA ingestion can cause acute kidney injury, while chronic low-dose AA ingestion can lead to progressive kidney disease. Ingested AA is taken up by renal tubular epithelial cells, leading to DNA damage and cell death. Procyclin D (CypD) is involved in mitochondria-dependent cell death, but whether this mechanism plays a role in acute or chronic AA-induced kidney injury is unclear. We addressed this question by exposing CypD-/- and wild-type (WT) mice to acute high-dose or chronic low-dose AA. 5 mg/kg AA gavage for 3 d in WT mice induced acute kidney injury as evidenced by loss of renal function, tubular cell injury and death, and neutrophil infiltration. All these parameters were significantly reduced in CypD-/- mice. chronic low-dose (2 mg/kg AA) administration in WT mice resulted in chronic renal disease with impaired renal function and renal fibrosis by 28 days. However, CypD-/- mice were not protected against AA-induced chronic kidney disease. In conclusion, CypD promotes AA-induced acute kidney injury, but CypD does not promote the transition from acute kidney injury to chronic kidney disease during sustained AA exposure.

Keywords

acute kidney injury; aristolochic acid; cell death; chronic kidney disease; cyclophilin D; inflammation; renal fibrosis;Cistanche tubulosa

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Introduction

Aristolochic acid (AA) is a nitro phenanthrene carboxylic acid found in the Aristolochiaceae family, which includes nearly 500 species of plants.AA consists mainly of a mixture of two metabolites, 8-methoxy-6-necrophilic-(3,4-d)-1,3-deoxy-5-carboxylic acid (AAI) and 6-necrophilic-(3,4-d)-1,3-deoxy-5-carboxylic acid (AAII) [1]. Investigation of endemic kidney disease in the Balkans identified AA as the nephrotoxin responsible for this environmentally related disease [2]. Aristolochia species growing in fields of cereal crops in the region contaminate baking flour, and long-term consumption of AA causes chronic kidney disease, kidney stones, and bladder cancer [1]. Aristolochia spp. is also used in the preparation of various herbal medicines in which AA has been identified as a causative toxin in herbal nephropathy [3]. In a study of 300 patients with herbal nephropathy, acute kidney injury and slower-progressing chronic kidney disease were associated with high or low levels of AA intake, respectively [4]. Identification of AA as a common toxin in these diseases leads to the term aristolochic acid nephropathy (AAN). There is no treatment for AAN, so it is crucial to understand the mechanisms by which AA induces acute and chronic kidney injury.

Renal tubular epithelial cells are very sensitive to AA toxicity because they express the organic anion transporter protein OAT1/3, which is able to efficiently take up AA into the cells [5]. Intracellularly, AA reacts with DNA bases to produce DNA adducts that can lead to a: T → T: a transformation, resulting in DNA damage and possibly cancer development [1]. In addition, AA induces the death of cultured tubular epithelial cells by inducing high levels of reactive oxygen species (ROS) [6]. Both mice and rats are sensitive to the toxic effects of AA and therefore the mechanism of AA-induced nephrotoxicity can be investigated in vivo. A single high dose of AA-induced acute renal failure with tubular necrosis in animals, whereas repeated low doses of AA-induced chronic kidney disease with tubular atrophy and fibrosis [7,8].

Procyclins are a group of widely expressed enzymes with peptidyl cis-trans isomerase (PPIase) activity that are involved in protein folding. Cyclophilin D (CypD), also known as Peptidylprolyl Isomerase F (PPIF), is a component of the mitochondrial membrane permeability transition pore (mPTP). After cell injury, mPTP is opened due to excess ROS or other stressors, leading to the release of cytochrome c into the cytoplasm and subsequent cell death [9]. CypD gene deletion mice are phenotypically normal, but CypD-/- mice are resistant to renal ischemia/reperfusion injury or cisplatin toxicity-induced tubular necrosis and acute renal failure [10-13]. In addition, CypD-/- mice are protected against the unilateral ureteral obstruction (UUO) model of renal fibrosis [14]. However, it is not clear whether CypD plays a role in AA-induced TEC injury. In the present study, we investigated the role of CypD in high-dose AA-induced acute kidney injury and chronic low-dose AA-induced renal fibrosis.

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Results

Cyclophilin D Deletion Protects against Aristolochic Acid-Induced Acute Kidney Injury

Plasma creatinine levels in healthy wild-type (WT) C57BL6/J mice ranged from 8 to 16 μ mol/L. The administration of 5 mg/kg AA to WT mice resulted in an acute decrease in renal function on day 3, defined as a 3-fold increase in plasma creatinine levels (range 30 to 53 μ mol/L). Compared with WT control mice, tubular epithelial cells of WT mice were significantly and histologically damaged on day 3 after AA administration. The apical brush border was lost, cells were swollen, nuclei were lost, cells were shed into the tubular lumen, and casts were formed in the tubular lumen. Tubular necrosis was visible under high magnification. We also assessed cell death by staining for cleaved caspase 3. WT control mice lacked cleaved caspase 3 stainings, but on day 3 after AA administration, a large number of tubular epithelial cells showed cleaved caspase 3 stainings. Consistent with these histological features of cell injury and cell death, mRNA levels of the tubular injury marker Kim1 were significantly elevated, whereas mRNA levels of the protective protein α-Klotho were significantly reduced.

The kidney structure and function were normal in CypD gene deletion (CypD-/-) mice. A single administration of 5 mg/kg AA significantly protected CypD-/- mice from acute kidney injury. Although plasma creatinine levels were mildly elevated in 5/10 CypD-/- mice, mean plasma creatinine levels were significantly lower than in the WT AA group and not significantly different from those of CypD-/- not treated with AA. A similar picture was evident in the analysis of renal tubular injury, with the CypD-/- day 3 AA group showing significant reductions in histological tubular injury, cleaved caspase-3-stained cell numbers, and Kim1 mRNA levels, as well as a significant protection against reduced α-Klotho mRNA levels.

Cyclophilin D Deletion Protects against Acute Aristolochic Acid-Induced Leukocyte Infiltration

Neutrophils were largely absent in the kidneys of the WT and CypD-/- control groups. However, a large number of Ly6G+ neutrophil infiltrates were evident in the area of tubular injury in WT mice on day 3 after AA administration. neutrophil infiltration was significantly reduced in the CypD-/- a group. In contrast to neutrophils, there were large numbers of resident F4/80+ macrophages in WT and CypD-/- control kidneys. Although macrophages were increased in the area of tubular injury, they did not reach statistical significance in either the WT AA group or the CypD-/- AA group. t cells were largely absent in WT and CypD-/- control kidneys. A small but significant T-cell infiltrate was seen in the kidneys of both WT and CypD-A mice at day 3 AA, although there were no differences between WT and CypD-/- mice.

Cyclophilin D Deletion Does Not Protect against Chronic Aristolochic Acid-Induced Kidney Disease

WT mice given 2 mg/kg AA every 2 days for 28 days resulted in chronic kidney disease with a 2.6-fold increase in plasma creatinine levels. Histological examination revealed prominent tubular atrophy and dilatation, partial tubular casting formation, and essentially normal glomeruli. day 28 AA of WT did not show significant tubular necrosis, but there was significant lysis of a large number of caspase 3-stained cells (probably apoptotic cells), although this was reduced by 66% compared with day 3 of WT AA (P < 0.001). Kim1 mRNA levels increased and a-Klotho mRNA levels decreased in the WT AA group on day 28; these changes were greater than in the WT AA group on day 3 (;p < 0.001). CypD-/- mice were not protected against chronic AA-induced kidney disease. They exhibited plasma creatinine levels, histological damage, Kiml and a-Klotho mRNA levels, and a number of cleaved caspase 3-stained cells comparable to those of day 28 AA WT mice.

Cyclophilin D Deletion Does Not Protect against Chronic Aristolochic Acid-Induced Renal Fibrosis

Interstitial fibrosis is a hallmark of chronic kidney disease and is characterized by collagen deposition, aggregation of α-SMA+ myofibroblasts, macrophage infiltration, and loss of peritubular capillaries [15]. Compared to WT and CypD-/- control kidneys, where type IV collagen was localized to the glomerular and tubular basement membranes (as well as the vessel wall), diffuse type IV collagen deposition was increased in the interstitial region of 28-day AA WT mice. A 2-fold increase in interstitial type IV collagen deposition was associated with a significant increase in renal type I collagen mRNA levels. a 3.5-fold increase in A - SMA mRNA levels was seen with a significant accumulation of myofibroblasts. In WT mice, there was also significant macrophage infiltration at day 28 AA, as evidenced by elevated levels of CD68 mRNA and significantly elevated expression of CD206 - an alternatively activated macrophage marker associated with the promotion of renal fibrosis [15]. In addition, AA WT mice showed a significant absence of CD31+ peritubular capillaries on day 28 compared to WT control mice.

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Discussion

Tubular necrosis is a feature of acute kidney injury when toxic chemicals, such as drugs (e.g., cisplatin, vancomycin, gentamicin, and mucin) and Phytonephrotoxins (e.g., aristolochic acid and Cynarin) are preferentially taken up by renal tubular epithelial cells [16]. A common feature of this toxin-induced tubular cell death is mitochondrial damage. For example, in addition to causing DNA damage, acute kidney injury induced by high doses of cisplatin or aa is directly associated with severe mitochondrial damage in renal tubular epithelial cells [17,18].

CypD plays an important role in toxin-induced mitochondria-dependent cell death through the opening of mPTP [9]. The present study demonstrates for the first time that CypD is required in high-dose aa-induced tubular epithelial cell death and acute kidney injury. This conclusion is based on CypD-/- mice exhibiting significantly better renal function, reduced tissue tubular injury and tubular cell death (as shown by cleaved caspase 3 staining), reduced expression of the tubular injury marker Kim-1, and a protective effect against loss of a-Klotho expression. These findings are consistent with previous in vitro studies that AA induces tubular epithelial cell death via induction of mitochondria-derived reactive oxygen species [19] and that CypD is required for reactive oxygen species-induced cell death in cultured tubular epithelial cells [14]. Furthermore, in the acute AA model, the accumulation of neutrophils was significantly reduced in CypD-/- mice. Little is known about the function of CypD in neutrophils; however, the reduction in neutrophil infiltration may only be an indirect effect of reduced tubular cell injury and cell death, in which a reduced release of risk-related molecular patterns leads to reduced neutrophil recruitment.10 Although neutrophils promote a "second wave" of tubular cell death in models of renal ischemia/reperfusion injury "[20-22], the role of neutrophils themselves in acute AA-induced acute kidney injury has not been determined. There was also a slight but significant infiltration of T cells on day 3 of AA, but there were no differences between WT and CypD-/- mice. There was no significant difference in the number of renal macrophages on day 3 of the AA model compared to controls.

CypD-/- mice are protected against high-dose AA-induced acute kidney injury, which is consistent with studies that CypD-/- mice are protected against tubular epithelial cell death and loss of renal function after cisplatin or renal ischemia/reperfusion injury (IRI) [10-12], and that treatment with procyclidine inhibitors protects against IRI-induced acute kidney injury [23].

Having determined that CypD-/- mice are protected from high-dose AA-induced acute kidney injury, we hypothesized that these mice would also be protected from long-term exposure to low-dose AA-induced tubular cell death and progressive kidney disease. However, this was not the case, and CypD-/- mice exhibited levels of tubular cell injury (based on Kim-1 and α-Klotho), renal failure, and renal fibrosis after chronic low-dose AA administration that did not differ from WT mice.

There may be several reasons for the lack of protection in CypD-/- mice during chronic low-dose AA exposure. Histological analysis showed tubular necrosis in the acute high-dose AA model, but not in the chronic low-dose AA model at 28 days, when damage was evident in the form of atrophy and apoptotic cell death (as shown by lysis caspase 3 staining). This suggests that high-dose AA induces tubular necrosis but not low-dose AA. This may be due to differences in reactive oxygen species induction induced by low and high-dose AA, or that surviving tubular cells are resistant to AA-induced necrosis. Further detailed studies are needed to isolate these possible mechanisms or to identify another potential explanation, such as tubular cell damage caused by accumulated DNA damage in a chronic AA exposure model. However, it is clear that CypD gene deletion does not protect tubules from repeated damage by low doses of AA. Indeed, similar findings were found in these acute and chronic AA exposure models [24] using JUN amino-terminal kinase (JNK) inhibitors. both JNK and CypD are involved in ROS-induced, mitochondria-dependent tubule cell death [14,24]. jNK inhibitor treatment inhibits tubule cell death in acute high-dose AA models, but in chronic low-dose AA models [24] had no effect on tubular cell death or the development of renal fibrosis.

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The specific role of CypD in renal fibrosis has been described in three previous studies. In the unilateral ureteral obstruction (UUO) model, systemic administration of procyclidine inhibitors or the use of CypD-/- mice prevented tubular cell death, myofibroblast accumulation, collagen deposition, and peritubular capillary loss [14,23]. However, cultured WT and CypD-/- renal fibroblasts did not differ in PDG-induced cell proliferation and TGF-β1-induced activation and collagen production [14], suggesting that CypD does not directly affect collagen-producing myofibroblasts in the development of renal fibrosis. Thus, in the UUO model, the protective effect of CypD-/- on renal fibrosis in mice was attributed to a reduction in tubular epithelial cell death and loss of peritubular capillaries [14]. In contrast, another study found that CypD-/- mice exhibited more severe glomerulosclerosis in a streptozotocin-induced type 1 diabetic nephropathy model, whereas treatment with a procyclidine inhibitor failed to alter the development of glomerulosclerosis in a DB/DB model of type 2 diabetic nephropathy [25]. The current study found that CypD deficiency had no effect on the development of renal fibrosis in a chronic low-dose AA exposure model, nor on tubular epithelial cell death or peritubular capillary loss. These comparative results in three different disease models suggest that the role of CypD in the development of renal fibrosis is highly dependent on the nature of the underlying renal injury and may not be of general importance for renal fibrosis.

Conclusions

The present study confirms the involvement of CypD in acute tubular necrosis and acute kidney injury induced by high-dose AA exposure. In contrast, CypD does not contribute to the toxic effects of chronic low-dose AA exposure-induced chronic kidney disease.

REFERENCES

1. Jadot, I.I.; Declèves, A.-E.; Nortier, J.; Caron, N. An Integrated View of Aristolochic Acid Nephropathy: Update of the Literature. Int. J. Mol. Sci. 2017, 18, 297.

2. Debelle, F.D.; Vanherweghem, J.L.; Nortier, J.L. Aristolochic acid nephropathy: A worldwide problem. Kidney Int. 2008, 74, 158–169.

3. Yang, B.; Xie, Y.; Guo, M.; Rosner, M.H.; Yang, H.; Ronco, C. Nephrotoxicity and Chinese Herbal Medicine. Clin. J. Am. Soc. Nephrol. 2018, 13, 1605–1611.

4. Yang, L.; Su, T.; Li, X.-M.; Wang, X.; Cai, S.-Q.; Meng, L.-Q.; Zou, W.-Z.; Wang, H.-Y. Aristolochic acid nephropathy: Variation in presentation and prognosis. Nephrol. Dial. Transplant. 2011, 27, 292–298.

5. Bakhiya, N.; Arlt, V.M.; Bahn, A.; Burckhardt, G.; Phillips, D.; Glatt, H. Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy. Toxicology 2009, 264, 74–79.

6. Yu, F.Y.; Wu, T.S.; Chen, T.W.; Liu, B.H. Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells. Toxicol. In Vitro 2011, 25, 810–816.

7. Zhou, L.; Fu, P.; Huang, X.R.; Liu, F.; Chung, A.C.K.; Lai, K.N.; Lan, H.Y. Mechanism of chronic aristolochic acid nephropathy: Role of Smad3. Am. J. Physiol. Physiol. 2010, 298, F1006–F1017.

8. Zhou, L.; Fu, P.; Huang, X.R.; Liu, F.; Lai, K.N.; Lan, H.Y. Activation of p53 Promotes Renal Injury in Acute Aristolochic Acid Nephropathy. J. Am. Soc. Nephrol. 2009, 21, 31–41.

9. Elrod, J.W.; Molkentin, J.D. Physiologic Functions of Cyclophilin D and the Mitochondrial Permeability Transition Pore. Circ. J. 2013, 77, 1111–1122.

10. Devalaraja-Narashimha, K.; Diener, A.M.; Padanilam, B.J. Cyclophilin D gene ablation protects mice from ischemic renal injury. Am. J. Physiol. Physiol. 2009, 297, F749–F759.

11. Jang, H.-S.; Noh, M.; Jung, E.-M.; Kim, W.-Y.; Southekal, S.; Guda, C.; Foster, K.W.; Oupicky, D.; Ferrer, F.A.; Padanilam, B.J. Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury. Kidney Int. 2020, 97, 327–339.

12. Linkermann, A.; Bräsen, J.H.; Darding, M.; Jin, M.K.; Sanz, A.B.; Heller, J.-O.; De Zen, F.; Weinlich, R.; Ortiz, A.; Walczak, H.; et al. Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 2013, 110, 12024–12029.

13. Park, J.S.; Pasupulati, R.; Feldkamp, T.; Roeser, N.F.; Weinberg, J.M. Cyclophilin D and the mitochondrial permeability transition in kidney proximal tubules after hypoxic and ischemic injury. Am. J. Physiol. Physiol. 2011, 301, F134–F150.

14. Hou, W.; Leong, K.G.; Ozols, E.; Tesch, G.H.; Nikolic-Paterson, D.J.; Ma, F.Y. Cyclophilin D promotes tubular cell damage and the development of interstitial fibrosis in the obstructed kidney. Clin. Exp. Pharmacol. Physiol. 2017, 45, 250–260.

15. Tang, P.M.K.; Nikolic-Paterson, D.J.; Lan, H.-Y. Macrophages: Versatile players in renal inflflammation and fifibrosis. Nat. Rev. Nephrol. 2019, 15, 144–158.

16. Petejova, N.; Martínek, A.; Zadrazil, J.; Teplan, V. Acute toxic kidney injury. Ren. Fail. 2019, 41, 576–594.

17. Jiang, Z.; Bao, Q.; Sun, L.; Huang, X.; Wang, T.; Zhang, S.; Li, H.; Zhang, L. Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity. Toxicol. Appl. Pharmacol. 2013, 266, 198–203.

18. Yang, Y.; Liu, H.; Liu, F.; Dong, Z. Mitochondrial dysregulation and protection in cisplatin nephrotoxicity. Arch. Toxicol. 2014, 88, 1249–1256.

19. Huang, X.; Wu, J.; Liu, X.; Wu, H.; Fan, J.; Yang, X. The protective role of Nrf2 against aristolochic acid-induced renal tubular epithelial cell injury. Toxicol. Mech. Methods 2020, 30, 580–589.

20. Klausner, J.M.; Paterson, I.S.; Goldman, G.; Kobzik, L.; Rodzen, C.; Lawrence, R.; Valeri, C.R.; Shepro, D.; Hechtman, H.B. Postischemic renal injury is mediated by neutrophils and leukotrienes. Am. J. Physiol. Physiol. 1989, 256, F794–F802.

21. Ryan, J.; Kanellis, J.; Blease, K.; Ma, F.Y.; Nikolic-Paterson, D.J. Spleen Tyrosine Kinase Signaling Promotes Myeloid Cell Recruitment and Kidney Damage after Renal Ischemia/Reperfusion Injury. Am. J. Pathol. 2016, 186, 2032–2042.

22. Singbartl, K.; Ley, K. Protection from ischemia-reperfusion induced severe acute renal failure by blocking E-selectin. Crit. Care Med. 2000, 28, 2507–2514.

23. Leong, K.G.; Ozols, E.; Kanellis, J.; Badal, S.S.; Liles, J.T.; Nikolic-Paterson, D.J.; Ma, F.Y. Cyclophilin Inhibition Protects Against Experimental Acute Kidney Injury and Renal Interstitial Fibrosis. Int. J. Mol. Sci. 2020, 22, 271.

24. Yang, F.; Ozols, E.; Ma, F.Y.; Leong, K.G.; Tesch, G.H.; Jiang, X.; Nikolic-Paterson, D.J. c-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury. Front. Physiol. 2021, 12.

25. Lindblom, R.S.; Higgins, G.C.; Nguyen, T.-V.; Arnstein, M.; Henstridge, D.C.; Granata, C.; Snelson, M.; Thallas-Bonke, V.; Cooper, M.E.; Forbes, J.M.; et al. Delineating a role for the mitochondrial permeability transition pore in diabetic kidney disease by targeting cyclophilin D. Clin. Sci. 2020, 134, 239–259.

Khai Gene Leong, Elyce Ozols, John Kanellis, Frank Y. Ma, and David J. Nikolic-Paterson