**5. Discussion**

Although AKI is a common finding in the patients undergoing alloHSCT, most of the classifications assessing the degree of renal impairment concentrate on the functional indices. However, damage markers seem a promising and objective alternative in the assessment of kidney injury. The fact that the number of potential AKI markers is increasing proves that the problem is emerging. Thus, our aim was to confront the known indices of tubular injury (KIM-1) and glomerular function (cystatin C, eGFR) with a new marker of cellular stress and damage. Clusterin has not been analyzed in a specific population of patients undergoing HSCT so far.

Every patient in this study group, irrespective of the indication for HSCT, demonstrated the features of cellular damage seen even before the procedure of transplantation. The interpretation of this unexpected result was quite challenging, because up-to-date studies on the populations undergoing HSCT have never compared their records to those of age-matched controls with normal renal function and no history of kidney injury. The pre-transplantation therapies, including chemotherapeutics and nephrotoxic drugs, seem of paramount importance as an explanation for this phenomenon. Indeed, all patients transplanted due to oncological reasons have undergone series of chemotherapies, whereas many of the non-oncological children were treated with potentially nephrotoxic antibiotics prior to alloHSCT. All these interventions were related to their primary diagnoses and could trigger the pre-transplantation subclinical kidney damage. Irrespective of the underlying cause, such observation would sugges<sup>t</sup> that subclinical background is a common finding and, thus, the key player in the HSCT-related AKI.

The post-transplantation aggravation of cellular damage was rather predictable, taking into account the nephrotoxic and cytotoxic potentials of conditioning regimens, anti-GvHD prophylaxis and treatment of infections. The essential finding was that these signs of injury concerned both serum and urine, depicting not only renal, but also systemic cell damage after HSCT.

Among all analyzed parameters, the serum and urinary clusterin values have shown the most spectacular changes in children before HSCT compared to controls and before HSCT compared to 24 h after HSCT. The serum and urinary values before transplantation were at least two times higher than in controls and rose over 2.5-fold after transplantation. In the meantime, the other serum markers increased by no more than 60%, whereas the urinary values became 14% higher (KIM-1) or remained unchanged (cystatin C). Only in the case of clusterin the post-HSCT response was more spectacular than the di fference between control group and pre-HSCT values.

Clusterin is a 75–80 kDa heat shock protein secreted by both epithelial and secretory cells in response to stress. Its protective and anti-apoptotic roles against renal ischemia-reperfusion injury have been demonstrated in murine kidneys [21]. Interestingly, clusterin was detected in both renal tubular epithelial and mesangial cells. Its decreased expression aggravated postischemic renal injury, as well as proteinuria in the course of glomerulopathy. Clusterin knockout mice su ffered from the progression of renal inflammation and fibrosis after ischemia–reperfusion injury [22]. Investigation concerning humans is restricted to single reports on the urinary clusterin in patients with diabetes mellitus and promising results of a diagnostic multi-biomarker kit including urine clusterin in the scope of indices of chronic kidney injury [23,24].

Taking into account the experimental data, the above mentioned reports and our results, we could put forward the hypothesis about the protective role of clusterin in the kidney injury due to HSCT. Such presumption would provide the explanation for increasing levels in both serum and urine as a response to systemic and kidney stress conditions. Moreover, clusterin has turned out the most accurate marker predicting drug-induced AKI in adults, better than cystatin C or KIM-1 [25]. Interestingly, all three markers became higher compared to non-AKI controls already 1–3 days before the onset of AKI. This may sugges<sup>t</sup> the reason for the early pre-transplant elevation of all indices compared to healthy controls. It also shows the added value of all tested markers in diagnosing nephrotoxicity. Moreover, yet, testing big cohorts may give unequivocal results in the pediatric population, questioning for example the accuracy of KIM-1 as a predictor of AKI in children [26].

In our study group, urinary KIM-1 behaved similarly to urinary clusterin, although the elevation after HSCT did not reach 50%. However, the rising trend persisted until the third week and then a significant decrease was noticed. Yet, the values remained higher than before transplantation. Such scheme would talk into the tubular damage aggravating since the HSCT procedure. The serum KIM-1 concentrations kept the systematic growth throughout the whole observation period, except for the plateau phase between 24 h and one week after HSCT. These results have suggested the ongoing tubular damage in the course of HSCT procedure, triggered most probably by nephrotoxic drugs. Increased serum KIM-1 could point at its release by the cells damaged in the course of conditioning therapy, anti-GvHD prophylaxis or current infection treatment, as well as the accumulation of a molecule that cannot be filtered freely through glomeruli due to its molecular mass of 90–110 kDa. The possible long-term consequence has been discovered in experimental studies, when chronic KIM-1 elevation in mice promoted fibrosis, thus linking AKI to CKD [27].

However, both abovementioned markers seemed similar in the mode of early reaction to kidney injury. The usefulness of clusterin and KIM-1 could be strengthened by the fact that their elevated serum and urine values were noticed even before transplantation, both in oncological and non-oncological patients, whereas pre-transplantation eGFR changes concerned only oncological children. In detail, their eGFR values were significantly higher compared to controls and most of the patients demonstrated hyperfiltration. This finding was confirmed by other authors, who put the main stress on hypermetabolic states and previous chemotherapy as causative factors of hyperfiltration [28]. The eGFR discrepancies could also be the consequence of many transplant-related covariates like catabolism, inflammation or weight loss, directly influencing serum creatinine [29].

Whether this hyperfiltration could be the early sign of progression into chronic kidney injury, remains unexplained, because the longer time of observation would be needed. However, the elevation of both damage parameters and eGFR could be the proof for the pre-transplantation kidney injury in oncological patients. This finding justifies the current attempts to prevent renal injury or at least to minimize the impact of potential nephrotoxins on the kidney. The reduction of nephrotoxic exposure is one of the e ffective tools already used in oncological patients [30]. The animal models sugges<sup>t</sup> the possibility to prevent AKI with the use of phosphodiesterase-5 inhibitors prior to potentially nephrotoxic treatment [31,32]. Independent of the chosen strategy, the threat of renal injury should urge careful follow-up of the patient in order to avoid additional insults triggering irreversible damage to the kidney.

Cystatin Cis an established marker of glomerular function and a good predictor of AKI in children, so it seemed a good candidate to verify the abovementioned discrepant eGFR results. However, it did not confirm di fferences between oncological and non-oncological patients [33]. Out of the three examined markers, cystatin C was the only one with low molecular weight (13 kDa) freely filtered through glomeruli. Therefore, the fact that the elevation of cystatin C in serum has outrun the increase of the urinary value was the direct proof of the serum origin of cystatin C found in the urine. Having said that, the increased values of urinary clusterin and KIM-1, both of higher molecular weight than cystatin C, must have been of tubular cell origin. Therefore, their elevation was probably proportionate to the degree of cell damage and, in case of clusterin, to the intensity of protective mechanisms against kidney injury.

Summarizing, in our study clusterin has outperformed the targeted glomerular (cystatin C) and tubular (KIM-1) indices of kidney function. Therefore, it seems a promising early marker of the sublethal kidney injury, covering both tubular and glomerular spectrum of renal damage. Whether clusterin may become a duplex renal functional and damage marker, is ye<sup>t</sup> to be established in the studies performed on a larger group of patients.

We also must acknowledge the limitations of our study. First, the clinical data were collected according to the hematological protocols, not taking into account all nephrological aspects. Thus, the full information about urine output is missing. This is the first report on the clusterin serum and urinary values in children, so it is impossible to confront them with the age-related reference values. We are also aware of the low number of patients and the heterogeneity of examined groups, which limits the power of conclusions and urges the continuation of the study throughout a longer period on a larger group of patients.
