**5. MiRNAs as Biomarkers of Septic Acute Kidney Injury**

Research is currently focused on miRNAs as new potential biomarkers and/or therapeutic tools for many conditions including AKI. MiRNAs are small molecules (18–31 nucleotides) of noncoding RNAs, representing a large part of genetic information not translated from the DNA matrix into final protein production. The evidence of their abundance, developmentally regulated fashion and often subcellular localization points to their important biological role in many biochemical and pathophysiological processes and pathways on the cellular and molecular level [21]. Influence on post-transcriptional gene regulation, cell metabolism, cytokine production, cell differentiation and programmed cell death are only a small percentage of miRNAs' effects and their target genes. In the AKI condition, some act protectively and can become potential therapeutic targets but others can increase the toxic activity and renal damage. Anti-inflammatory and/or anti-apoptotic activity in AKI has been described for the following miRNAs: *miR-10a*, *miR-21*, *miR-26a*, *miR-122*, *miR-126*, *miR-146a*, *miR-199a*, *miR-296* and *miR-494* [22]. Some miRNAs involved in the pathophysiological inflammatory process of sepsis based on endotoxin (LPS) activation of TLR4 in the signaling pathway of NF-κB activation, pro-inflammatory cytokine production (IL-6, IL-1β, TNFα) and subsequent neutrophil activation, damage of endothelial permeability and tissue injury are: *miR-146 a*/*b*, *miR-223*, *miR-155*, *miR-203*, *miR-15a*, *miR-16*, *miR-126*, *miR-199a* and *miR-9*. Each regulates positively or negatively a different part of the biochemical cascade to final cytokine production and tissue damage according to their target genes [23,24]. In the development of septic AKI, severe metabolic alterations of tubular epithelial cells may play a crucial role via *miR-21-3p* influence on the *AKT*/*CDK2-FOXO1* pathway, with induction of cycle cell arrest and apoptosis [25]. According to one human study (Ge et al., 2017), many other signaling pathways are involved in septic AKI development, including oxidative stress and mitochondrial dysfunction pathways (*HIF-1*, *PI3K-Akt*, *mTOR* and *TGF*β). In septic, critically ill patients, significantly overexpressed *miR-4321* was observed, with the predicted oxidative-stress-associated target genes: *AKT1*, *MTOR*, *NOX5*, *IL17RA* and *IL26* [26]. The mitochondrion is assumed to be a key organelle in the development of septic acute kidney injury, and has major pathophysiological significance in ROS production and apoptosis [27]. In one hybrid human and experimental study including 50 patients with sepsis, an effect was found of *miR-106a* on caspase-3 activity, *BCl-2* expression and proinflammatory cytokine production after LPS stimulation [28]. The authors found an association between *miR-106a* and an aggravation of LPS-induced inflammation, and apoptosis in sepsis-induced AKI. A target gene for *miR-106a* was established as thrombospondin *THBS2*, which takes part in a number of processes such as regulation of cell motility, death and cytoskeleton formation [28].

#### **6. Medication-Induced AKI in Septic Patients**

Drug-induced nephrotoxicity varies from a relatively mild form of acute tubulointerstitial nephritis (ATIN), several types of glomerulonephritis, crystal nephropathy and osmotic nephrosis to acute tubular necrosis represented mostly by severe renal impairment with the need for RRT. The last-mentioned may be associated with development of chronic nephropathy and the need for chronic hemodialysis treatment. The incidence of medication-associated nephrotoxicity accounts for approximately 18–27% of all AKI patients in US hospitals—the main causative drugs are NSAIDs, aminoglycosides, amphotericin B and calcineurin inhibitors [29].

Potentially nephrotoxic medications, which are considered essential and commonly used in sepsis and critically ill patients, are antimicrobial agents (antibiotics, antiviral and antifungal treatment), human albumin in septic shock or proton pump inhibitors to prevent stress ulcers [30] (Table 1). Iodine contrast agents are used for radiocontrast imaging examinations in septic, critically ill patients when there is need for sepsis source finding or surgical interventions [31].

The nephrotoxicity of some antimicrobial drugs is a common problem. Of these, the most nephrotoxic are: vancomycin, aminoglycosides and polymyxins, which cause acute tubular necrosis and apoptosis depending on dose, among other factors. Many antimicrobials and other drugs frequently used in critically ill patients can also cause ATIN, accounting for 60–70% of all ATIN cases [32].


**Table 1.** Nephrotoxicity of commonly used medications in critically ill patients [30–36].

NSAIDs—nonsteroidal anti-inflammatory drugs, ROS—reactive oxygen species.

#### **7. Vancomycin-Induced Nephrotoxicity Pathophysiology and Biomarkers**

Vancomycin is a glycopeptidic antimicrobial agent with substantial bactericidal effect on Gram-positive bacterial infections and is frequently used in the treatment of Methicillin-resistant *Staphylococcus aureus* (MRSA). It also acts against *Streptococcus* sp., *Enterococcus* sp., *Actinomyces* sp., *Clostridium* sp. and *Eubacterium* sp. Its pharmacokinetics and pharmacodynamics are time-dependent, but according to recommendations for vancomycin treatment, the best parameter for evaluating its efficacy is the ratio of the 24 h area under the curve (AUC) to the minimum inhibitory concentration of AUC/MIC ≥400 mg.h/L with an MIC of pathogen <2 mg/L [37,38]. Vancomycin is not metabolized in the human body and is eliminated renally by glomerular filtration. Its binding to plasma proteins is less than 50%, the elimination half-life ranges from 6 to 12 h and volume of distribution is 0.4–1.0 L/kg [37]. The recently revised consensus guidelines of the American Society of Health-System Pharmacists for vancomycin therapy and monitoring for serious MRSA infections (2020) recommends, in adults and pediatric patients, a daily AUC/MIC ratio between 400 to 600 mg.h/L. A higher loading dose (20–25 mg/kg based on actual body weight) should be considered in critically ill patients treated by any type of RRT or in need of continuous vancomycin infusion. In obese patients with serious infections, the maximum loading dose is 3000 mg intravenously [39]. However, vancomycin nephrotoxicity with risk of AKI development is usually associated with higher vancomycin exposure, as measured by AUC0–24. Additionally, a significantly increased risk for nephrotoxicity has been observed in patients with AUC0–24 of 563 mg.h/L [40]. However, higher trough vancomycin serum concentrations >16.5 mg/L are also at greatest risk for new onset of AKI in critically ill patients [41]. In these cases, it is very difficult, in clinical practice, to maximize antibiotic efficacy, and, at the same time, to minimize its nephrotoxicity. The pathophysiology of vancomycin nephrotoxicity has been intensively investigated mostly in experimental research. Sakamoto et al. (2017) uncovered the possible nephrotoxic effect based on peroxidation of the mitochondrial membrane cardiolipin by vancomycin-induced production of intracellular ROS and activation of apoptosis in proximal tubular cells [42]. Apoptotic cell death induced by vancomycin may be associated with activation of specific caspases: caspase 9 and caspase 3/7 and extensive ROS production [34]. In the prospective multicenter Sapphire study (NCT01209169 ClinicalTrials.gov) including 723 critically ill adult patients, the pharmacokinetics

of two urinary biomarkers TIMP-2 and IGFBP7 were evaluated in patients receiving vancomycin, piperacilin-tazobactam, or their combination. The concentration of biomarkers and the risk of death or need for dialysis treatment within 9 months, were the highest in the combination group. However, the AKI progression to an aggravated stage 2/3 was comparable in vancomycin monotherapy and in the combination treatment [43]. Selected urinary AKI biomarkers—clusterin, cystatin C, NGAL/lipocalin-2, osteopontin and KIM-1—were investigated in animals receiving vancomycin. A higher vancomycin exposure presented by an AUC0–24 and maximum serum concentration significantly correlated with increase in urinary AKI biomarkers but did not correlate with histopathological score [44]. Serum creatinine, urinary NGAL and KIM-1 were measured in 87 patients without chronic kidney disease (12.6% developed AKI) before and during the vancomycin therapy. According to the results, both urinary biomarkers NGAL and KIM-1 successfully discriminated patients with and without vancomycin-induced AKI earlier than serum creatinine [45]. However, in one animal study (Pais et al., 2019) KIM-1 and clusterin were more sensitive to vancomycin-induced AKI than NGAL [46].

One possible explanation for vancomycin-induced AKI and tubular cell apoptosis is based on DNA methylation by activation of Methyl-CpG binding domain protein 2 (*MBD2*). Experimental inhibition of *MBD2* can downregulate *miR-301-5p* with subsequent restoration of anti-apoptosis gene expression e.g., hepatoma-derived growth factor (*HDGF*) and microphthalmia-associated transcription factor (*MITF*) and can increase MDM-4 expression for reduction of p53 [47].
