In clinical practice, MTX is considered as a key drug in the treatment of both malignant tumors and RA. As the most frequently used disease-modifying drug in RA and a widely used drug in cancer, MTX occupies an important position in clinical usage of neoplastic and autoimmune diseases.
However, MTX treatment is often associated with various toxicities, such as myelosuppression, mucositis, and kidney injury, which unexpectedly result in the interruption or discontinuation of medical treatment. Since MTX is excreted through kidney, so the impairment of kidney function will lead to the increase of other MTX adverse effect. Even though many preventions of MTX-induced kidney injury were conducted in clinical practice, MTX-induced nephrotoxicity is still continuously observed.
Moreover, although it has been reported that the toxicity of MTX is
associated with the infusion duration 33,34, the mechanism of kidney injury caused by the difference of MTX administration duration has yet to be determined. In the present study, we successfully established the animal model reflecting the clinical MTX-induced kidney injury caused by longer duration of MTX administration, and found that MTX accumulation in renal tissue by long-MTX administration causes kidney injury through an increase in oxidative stress.
27
One of the interesting finding in this study is that, even though the
administrated dosage of MTX was same (25 mg/kg), long-MTX administration caused more severe kidney injury than short-MTX administration. Goldie et al.
reported the duration of MTX administration was important factor in
MTX-induced toxicity 33. Recent report also showed that shorter duration of MTX administration was more beneficial in view of reducing toxicity and enhancing central nervous system pharmacokinetics for lymphomas patients 34. In our
experiments, the dosage of MTX (25 mg/kg) were intraperitoneally injected to the rats by short-administration (25 mg/kg by 1 injection) or long-administration (5 mg/kg by 5 injections), respectively. While the same dosage of MTX was
administrated, longer duration of MTX administration caused more severe kidney injury than short duration of its administration in rat model (Figures 3-10). It is well-documented that neither dosage nor plasma concentration may be suitable index to predict MTX-induced kidney injury 28,32,57. Previous study showed that, in high dose of MTX, toxicity was not related to plasma MTX area under the curve 57. Meanwhile, MTX rapidly eliminated from the plasma at low dose, and plasma MTX concentration was also unrelated to toxic response, indicating MTX plasma levels may not be a reliable and not appropriate index for therapeutic drug
monitoring 57. As expected, no obvious correlation between serum MTX concentration and kidney tissue injury was observed. Interestingly, our further analysis focusing on MTX concentration in kidney tissue suggested that MTX accumulation in kidney by long-MTX administration caused kidney injury through increasing oxidative stress (Figure 19).
28
Figure 20. Chemical structure of MTX and MTX-PGs.
Moreover, recent studies revealed that MTX underwent polyglutamation and transformed into polyglutamated MTX (MTX-PGs) in the cell (Figure 20),
MTX-PGs were activated form of MTX 58 and MTX-PGs showed severe cytotoxicity 47,48. The MTX-PGs stayed at the cytoplasm for longer time until MTX-PGs changed back into MTX (Figure 21) 47,58. Because intracellular
MTX-PGs concentration increases and sustained over time while administration 59, MTX in kidney cells may be more likely to transfer into MTX-PGs, while low dosage of MTX was administrated with longer duration. Therefore, we determined the MTX-PG3 in renal tissue (data not shown). The result showed the increase of MTX-PG3 in Long-MTX group, suggesting that MTX-PG3 was accumulated in Long-MTX group.
29
Besides, previous articles showed MTX cause oxidative stress in renal tissue.
Long administration duration of MTX may also cause more severe kidney injury by oxidative stress by accumulation of intracellular MTX-PGs. Our results also showed the increased oxidative stress in MTX-PGs-accumulated kidney,
suggesting that higher intracellular MTX-PGs concentration may trigger higher oxidative stress and severe kidney injury.
Figure 21. The polyglutamation of MTX after entering the cell. MTX undergoes polyglutamation in the cell. Polyglutamated MTX (MTX-PGs) will retain in cell until hydrolyzed back to MTX 58.
30
Among all the MTX-PGs, MTX-PG3 was reported as the major MTX-PG in erythrocyte (49.4% of the total concentration of MTX-PG2-5) 58. Erythrocyte MTX-PG3-5 concentration was reported correlated to therapeutics effect 58. Even though previous report shows that the erythrocyte MTX-PGs have no correlation with adverse effect 58. Since only the nucleated cell will need to take up folic acid, and therefore be a candidate to take up MTX. However, circulating erythrocyte doesn’t have nuclear anymore. So, the erythrocyte no longer uptake MTX and erythrocyte MTX-PGs can’t represent real time internal MTX concentration 60. We still cannot exclude the possibility that kidney tissue MTX-PGs concentration correlates to MTX-induced kidney injury.
Attempts have been made to establish animal models showing MTX-induced kidney injury, as of this moment, the suitable model to study the effect of
administration duration on MTX-induced kidney injury is not yet available. In this study, we successfully established the animal model with renal failure caused by longer duration of MTX administration. We observed the significant decrease in body weight, water intake, and urine volume in this model (Figures 4, 5). As
expected, urea nitrogen and Cr in urine were significantly decreased in Long-MTX group (Figure 6), while BUN and Cr in serum were increased in Long-MTX group (Figure 7). Additionally, in Long-MTX group, both N-gal and Kim-1, kidney injury markers, were also markedly increased (Figures 9, 10). Moreover, 4-HNE and MDA were significantly increased in Long-MTX group (Figures 18, 19), indicating that, at least in significant parts, this model showed the clinical
phenotype of MTX-induced kidney injury. Interestingly, our model showed that
31
both N-gal and Kim-1 were significantly increased in kidney tissue by longer duration of MTX administration (Figures 9, 10). Recent studies have shown that both N-gal and Kim-1 are recognized as sensitive and early diagnostic markers for kidney injury 51,52. Thus, N-gal and Kim-1 levels in urine or serum may have potential to be a useful biomarker for kidney injury caused by long-MTX administration. In clinical case report, it has been reported that serum Cr is
increased during MTX induced kidney injury 32. However, in this model, urine Cr was obviously decreased in Long-MTX group without the significant increase of serum Cr. It may due to the deceased Cr synthesis in Long-MTX group. Because tetrahydrofolate (THF) is necessary for Cr synthesis 61, and MTX shows its
therapeutic effect by inhibiting the synthesis of THF 60, MTX administration may lead to a decrease in Cr synthesis. It should be noted that folic acid was used with MTX in clinical usage, but not in animal experiment 24,32, indicating the difference between animal model and clinical case in Cr synthesis. Further investigation is needed to determine the more detailed molecular mechanism and verify the usefulness of this model for studying MTX-induced kidney injury.
In conclusion, we provided first hand evidence that longer duration of MTX administration caused more severe kidney injury in rat model. MTX accumulation in renal tissue by long-MTX administration caused kidney injury through an increase in oxidative stress. These findings may bring new insights into
understanding of MTX-induced kidney injury, and may lead to clinical application for appropriate treatment with MTX.
32
Summary and Perspective
Through this study, the following new information on MTX induced kidney injury were obtained.
1） Longer administration duration of MTX amplifies MTX induced adverse effect including kidney injury.
2） Longer administration duration leads to less MTX excretion through urine and higher MTX and MTX-PGs concentration in kidney tissue.
3） MTX accumulation in kidney leads to the increase of oxidative stress and causes kidney injury.
I observed very good correlation between administration duration of MTX and MTX-induced adverse effect. Given with same dosage, Long-MTX group showed serious adverse effect including kidney injury. Then, I observed that MTX
excretion in urine decreased in Long-MTX group. It led to the increase of kidney MTX concentration. According to previous article, MTX may undergo
polyglutamate after entering the cell, and MTX transformed into MTX-PGs.
MTX-PGs may be detained in the cell until hydrolyzed back to MTX. Because MTX-PGs have cytotoxicity, thus MTX-PGs may be a key role in MTX-induced kidney damage. Indeed, our preliminary result showed the increase of MTX-PG3 in Long-MTX group. So, I concluded longer administration duration leads to more polyglutamation and results in MTX retention. One step further, I determined the marker of oxidative stress and observed the increase of oxidative stress in
Long-MTX group. Thus, we concluded MTX retention in kidney led to increase of
33
oxidative stress.
Moreover, apart from MTX, other antimetabolites such as 5-FU, also showed longer administration duration amplifies adverse effect in clinical. Similar to MTX, 5-FU is also activated in the cell. Thus, the effect of administration duration of other antimetabolites can also be studied in the future.
This study can give new knowledge that not only bring new insights into understanding of MTX-induced kidney injury, but also help for clinical MTX application.
34
4. Materials and Methods