Damage of the kidneys, as an essential organ eliminating xenobiotics, is a possible adverse effect of multiple medicinal substances. Many drug classes are characterised with potential nephrotoxicity. These include anti-infectives (aminoglycoside antibiotics, amphotericin B, sulphonamides), anti-retrovirus agents, non-opioid analgesics, immunosuppressants, contrast agents, agents containing heavy metals, statins, proton-pump inhibitors, anticonvulsants, biphosphonians, angiotensin-converting-enzyme inhibitors, AT1-receptor antagonists for angiotensin II, loop and thiazide diuretics, antiplatelet thienopyridines and other [1, 2]. Herbal preparations can also impair renal functions (“Chinese herbs” containing aristolochic acid) [3, 4]. Furthermore, a significant class of drugs demonstrating nephrotoxicity are antineoplastic chemothera- peutics, including cyclophosphamide (CP) and ifosfamide (IF) [5, 6].

Those compounds are prodrugs and undergo metabolic biotransformation by CYP3A4, CYP2B6 and CYP2C9 isoenzymes leading to the subsequent production of the active nitrogen mustard, which exerts cytotoxic activity by DNA crosslinking. The process of hepatic and extrahepatic biotransformation of both CP and IF also causes the formation of other by-products, determining toxicities of those drugs, such as acrolein and chloroacetaldehyde (CAA) [7, 8]. Acrolein (2-propenal) is a highly reactive unsaturated aldehyde disturbing the cell redox balance leading to increased generation of reactive oxygen species synthesis and glutathione depletion. Moreover, it reacts with cysteine, lysine and histidine residues of many proteins, causing the addition of the aldehydic carbonyl group to their structure. The cellular carbonylated proteins are involved in many cellular dysfunctions, including energy production deficiency and loss of cytoskeletal integrity [9]. CAA, like acrolein, is also a factor initiating severe oxidative stress and reacts with protein and non-protein sulfhydryl groups of many compounds, thereby impairs proliferation and apoptosis regulatory mechanisms. CAA also inhibits lysosomal cysteine proteases (e.g., cathepsin B), resulting in disturbed digestion of endocytosed proteins. Moreover, CAA-induced depletion of reduced glutathione diminishes the capacity of the cells to scavenge free radicals, thereby increasing their susceptibility to oxidative stress [10].

Acrolein, excreted with urine, is a compound considered to damage mainly the urinary bladder, whereas CAA impairs kidneys, mostly affecting proximal tubules. Due to the fact that both CP and IF share the same metabolic pathways during their biotransformation processes, both compounds are considered to be uro- and nephrotoxic drugs [11, 12]. However, there are some quantitative differences in the metabolism of CP and IF – during IF biotransformation, the higher formation of CAA takes place, hence this drug is seen as more nephrotoxic compared to CP [8]. To reduce the risk of these adverse effects, during CP or IF therapies, forced diuresis is applied and compounds with uro- and nephroprotective effects are administered, such as mesna (sodium 2-mercaptoethane- sulfonate). It is a compound reducing the excessive oxidative stress and the resulting cellular oxidative damage caused by acrolein and CAA and rebuilding the glutathione resources in the cells [13]. A compound characterised, similarly to mesna, with the presence of sulfhydryl groups, essential from the point of view of glutathione regeneration and antioxidating effective, is N-acetylcysteine (NAC), currently applied as a secretolytic agent in obstructive conditions of the respiratory tract and as an antitoxin in paracetamol poisoning [14]. The registered indications for administration of this drug do not, however, include its application as a protective agent in IF therapies [15].

Thus, the objective of our experiment was the assessment of the nature of kidney dysfunction in rats treated with IF as well as verification of the expected alleviating, uro- and potential nephroprotective effect of NAC. Our assessment was based on the microscopic evaluation of kidneys and urinary bladders as well as laboratory analysis of classical parameters evaluating the renal functions and new protein markers of kidney functions: cystatin C (CysC), kidney injury molecule-1 (KIM-1) and nephrin (NPH). These compounds are modern indicators of kidney injury, and their increased occurrence in urine precedes obvious functional and structural kidney dysfunction.

The analysis of the results obtained in our experiment shows that the administration of IF to rats at the total dose of 250 mg/kg b.w., divided into 5 daily doses, resulted in the development of urinary bladder inflammation, without significant effect on the structure of the kidneys visible in histopathological analysis, but with functional disorders of those organs. Both the 24-hour diuresis value and urine pH did not differ significantly in the analysed groups, even though literature data mention the possibility of renal tubular acidosis and nephrogenic diabetes insipidus during IF therapies. At the same time, literature reports indicate that IF is a potential etiological factor of damage to proximal tubules and development of tubulopathy, which clinically manifests with a wide spectrum of abnormalities and may lead to Fanconi syndrome [5, 23]. The pathogenesis of IF-induced tubulopathy, as mentioned in the Introduction, is related to the emerging IF metabolite – chloroacetaldehyde, mostly synthesised with hepatic CYP3A4, CYP2B6 and CYP2C9 isoenzymes [7, 8], but also by renal isoenzymes CYP3A4, CYP3A5 and CYP2B6 [24]. As mentioned in the Introduction, CAA is a compound disturbing the mitochondrial function, causing energy deficiency, increased tubules cell sensitivity to the oxidative stress and damaging their structure [10, 25]. Furthermore, CAA impairs intracellular calcium homeostasis in proximal tubules due to the inhibition of the Na⁺/Ca²⁺ exchanger and affecting the cyclic adenosine monophosphate and protein kinase A signalling. It results in the progressive tubular cell dysfunction and their final necrosis [26].

The results obtained by us regarding CysC, KIM-1 and NPH confirm the hypothesis of the development of functional tubulopathy, without any pathomorphological changes visible at the level of classical light microscopy. The results of classical parameters, determined routinely to estimate the kidney functions, were ambiguous. The creatinine and electrolyte concentration was comparable in all groups. Elevated urea and BUN concentration was demonstrated in group 2 in relation to the control 1 group. Taking into account the serum creatinine level within the norm in all study groups, this growth could reflect increased protein catabolism. However, in individuals from group 2, the parallel increase in total protein concentration in blood was demonstrated. This finding, therefore, negated the hypothesis of protein hypercatabolism. The tendency of increase in the value of the BUN/creatinine quotient in groups 2 and 3 in comparison with group 1 was noticeable (nevertheless, with no statistical significance). This parameter is an azotemia discriminator and its elevated value suggests a hypercatabolic state or prerenal disturbances, such as hypovolemia and decreased renal perfusion [27]. Reduced kidney perfusion could explain the increase of CysC in serum in rats treated with IF. Nevertheless, the value of daily diuresis in all groups was comparable - no oliguria was found in group 2 or 3. Furthermore, as mentioned above, the creatinine level in all groups did not differ significantly, which excludes disturbances in glomerular filtration. We also did not perform any imaging of the kidneys that would allow to verify renal perfusion. Therefore, the results obtained in the serum do not allow for a clear interpretation.

The hypothesis that IF-induced kidney dysfunction has the nature of tubulopathy is confirmed, in particular, by the results of the protein markers of renal functions analysed by us. Rats treated with IF were characterised with higher concentrations in urine and daily excretion of CysC, KIM-1 and NPH in the urine. Cystatin C is a new alternative for creatinine in terms of glomerular filtration markers [28]. This protein is an inhibitor of endogenic proteases responsible for the catabolism of multiple peptides, produced by all nuclear cells and released at a constant rate to blood. CysC, as a low-weight cellular protein, undergoes glomerular filtration and, subsequently, endocytosis in a mechanism dependent on the megalin receptor as well as irreversible degradation in the proximal tubules. Therefore, CysC concentration in the blood depends on the filtration rate of this protein, whereas in physiological terms, only low amounts of this protein are found in urine [29, 30]. The blood concentration of the classical glomerular filtration marker - creatinine - did not differ significantly in all investigated groups, which suggested a normal filtrating function. Thus, it can be assumed that increased CysC excretion in urine shown in our experiment could prove the impairment of tubular catabolism of this compound, constituting a premise supporting the hypothesis regarding the development of post-ifosfamide tubulopathy in the study rats. This hypothesis is also confirmed by the analysis of the results of the next evaluated compound – KIM-1. It is a protein physiologically present in urine in trace amounts (thus, its concentration value is expressed as [pg/mL]). KIM-1 is an ectodomain of a large transmembrane protein, which is a phosphatidylserine receptor of proximal tubules, mediating phagocytosis of apoptotic and necrotic bodies. Various toxic or ischemic factors, damaging renal tubules and interstitial tissue and leading to the development of acute kidney injury (AKI), cause an increased expression of the receptor, accompanied by a parallel increase in the concentration of KIM-1 in the urine. In the course of AKI, the surviving proximal tubules phagocytose debris of exfoliated cells, using the above-mentioned KIM-related receptor. The clearance protects the obstruction of the tubular lumen and infiltration by inflammatory cells, thus decreases the tubular hydrostatic pressure and reduces the risk of acute renal failure [31, 32]. Urine KIM-1 is, therefore, a good indicator of kidney damage and the protein itself, along with CysC, is one of the elements in the panel of laboratory markers, which should be currently determined to assess kidney function [33, 34]. In the described experiment, urine KIM-1 concentration and excretion of that protein in urine was 4 times higher after administration of IF, which indicates damage to renal tubules, not visible at the level of classic optical microscopy. Moreover, IF treatment also led to structural damage to the filtration barrier, which is proved by the increased presence of another protein – NPH – in the urine of IF-treated animals. Nephrin is a structural protein of gap junctions in the foot processes of podocytes [35, 36]. These visceral epithelial cells are highly-specialized units lining the outer surface of the glomerular capillaries and that neighbor cells of the Bowman’s capsule [37]. Podocytes exert essential functions, which all depend on their unique structure: controlling and maintaining the selectivity of glomerular membrane permeability, co-formation of glomerular capillary structures, participation in remodeling of the glomerular basement membrane and endocytosis of filtered proteins in the glomerulus [37, 38].

The nephrine-dependent glomerular disturbances are a pivotal element of the early stage of glomerular proteinuria pathogenesis. The immuno-inflammatory processes involving the glomerular basement membrane alter the structure and consequently NPH function (and co-occurring protein - podocin). It contributes to the dilatation and unsealing of the slit diaphragm, leading to its disappearance, impairing the filtration barrier and inducing proteinuria [39]. In the adult patient population, NPH was mainly evaluated as an early-stage biomarker of diabetic nephropathy, nephrosis (including lupus nephritis) and preeclampsia, and it was demonstrated that the urinary amount of nephrine correlated with microalbuminuria [38]. Nephrine-related disturbances are also a pathogenetic element of the inherited podocytopathies. It is now recognized that the common feature in minimal change disease (MCD), membranous glomerulopathy, crescentic glomerulonephritis, collapsing glomerulopathy, focal segmental glomerulosclerosis (FSGS) diagnosed in pediatric patients is through podocyte damage and dysfunction, manifesting by early increased urinary nephrine excretion [38]. Moreover, nephrine NPHS1 gene mutations are also the most common cause of rare and severe congenital Finnish-type nephrotic syndrome, characterized by the enlarged placenta, premature birth and massive proteinuria that begins in utero [40].

Thus, it can be concluded that the relationship between NPH abnormalities and the podocyte dysfunction is well documented and currently indisputable and the increased amount of this protein in the urine should be considered an early prognostic biomarker of developing proteinuria.

In our experiment, the revealed amount of NPH in urine allows, therefore, to identify the early phase of glomerular injury, which extends the description of post-ifosfamide tubulopathy revealed in our experiment with glomerulopathy developing in parallel. Therefore, we can state that the post-ifosfamide form of kidney dysfunction induced in our experiment combined the features of both tubulopathy and glomerulopathy.

Employment of additional NAC treatment in group 3 was connected with the alleviation of histopathological disturbances in the urinary bladders as well as reduction of functional disturbances in kidneys found in animals treated exclusively with IF, as mentioned above. The urea and BUN concentrations in blood were significantly lower than in group 2 and reached values similar to those recorded in the control group. Similarly, concentrations and daily excretion in urine of all evaluated protein markers reached significantly lower values in comparison to rats in group 2. Thus, it can be concluded that NAC, administered in parallel to the applied IF, had a uroprotective and nephroprotective effect. The pathogenesis of the said effects is complex, with key antioxidative effects featured by NAC, which, due to the presence of –SH groups, is a direct “sweeper” of oxygen and nitrogen free radicals. This compound also regenerates endogenic glutathione resources and, thus, decreases the cell prooxidative status. NAC also inhibits multiple intracellular pathways (c-Jun N-terminal kinase, p38 MAP kinase, SAPK/INK, c-Fos pathways, as well as nuclear factor κB) regulating the expression of genes coding the proinflammatory and antiapoptotic factors [41, 42]. Therefore, intervening in the above-mentioned acrolein/CAA initiated mechanisms, NAC reduces IF toxicity.

The concept of NAC as a nephroprotector has already been mentioned in the literature [39, 40]. It was not our intention to demonstrate for the first time that NAC exerts a nephroprotective effect, but to broaden the knowledge of the pathophysiological description of kidney dysfunction in the course of IF-induced cystitis and to contribute to the discussion of the NAC role in kidney protection. Our experiment assessing the positive effect of NAC was, to our best knowledge, the first based on the analysis of new protein markers reflecting the kidney functioning, and not only on the analysis of a panel of classical laboratory parameters used in the routine kidney evaluation. In our opinion, it is the novelty of our study. Based on the results of the evaluated novel markers of kidney function, we confirmed that IF caused renal tubulopathy (expressed by increased urinary KIM-1 and CysC excretion), which has previously been mentioned [5, 23, 43], but we also showed that administration of IF lead to damage of the structural elements of the glomerulus (manifested by increased NPH excretion with urine). NAC treatment alleviated the urinary excretion of the aforementioned proteins, which confirmed the nephroprotective effect of the compound. Our results are consistent with the work of Chen et al. [44], who proved in their experiment that i.p. administration of NAC in the dose of 1200 mg/kg b.w. alleviated post-IF renal dysfunction, reducing hypercreatininemia and excretion of beta-2 microglobuline in the urine. Moreover, they demonstrated that the application of NAC resulted in increased activity of glutathione S-transferase and an increase in glutathione concentration in kidney homogenates. Our conclusions regarding the nephroprotective effect of NAC are, therefore, consistent with the results quoted above. However, it must be emphasized again that our results were based on the evaluation of the novel biomarkers of kidney function, and Chen et al. [44] concluded their findings primarily from the assessment of a classic laboratory panel of low-molecular renal parameters.

The increasing number of reports from experimental studies on the nephroprotective effect of NAC is also reflected in clinical data. Hanly et al. [43] in their review paper briefly and synthetically characterized the rationale for using NAC in patients undergoing chemotherapy with IF. They also described cases of paediatric oncology patients suffering from renal dysfunction during IF treatment, which was mitigated with parallel NAC administration, and it was again convincing support for the nephroprotective use of NAC. They concluded that taking into consideration the fact that excessive oxidative stress is an essential element of the pathogenesis of renal damage during IF treatment, NAC as an antioxidant and glutathione regenerating compound appears to be potentially useful nephroprotector in a clinical setting. Therefore, it should be expected that consistent premises from both experimental studies (including our one) and clinical reports will result in the official approval of the use of NAC as a nephroprotective agent in the near future.

1. Intraperitoneal IF administration of the total dose of 250 mg/kg b.w. divided into 5 doses in rats caused the development of inflammatory changes in the urinary bladder and functional disturbances in the kidneys of tubulopathy and early glomerulopathy character, as was proven by the increased excretion of CysC, KIM-1 and NPH in the urine.

2. In parallel to IF treatment, p.o. administration of the total dose of 1000 mg/kg b.w. of NAC divided into 5 doses alleviated the inflammatory changes in urinary bladders and resulted in reduced renal dysfunction, manifested as a reduction in excretion of the above-mentioned proteins in urine.

3. Thus, NAC had both an uro- and nephroprotective effect. Due to its effect, NAC can be considered an alternative for mesna, currently applied as a routine uroprotective agent.