RC606.pdf

Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido:03/01/2025 Aceptado:10/03/2025 Publicado: 25/04/2025 hps://doi.org/10.52973/rcfcv-e35606 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 7 Evaluaon of renal damage biomarkers in an experimental Pyelonephris model ınduced by uropathogenic Escherichia coli Evaluación de los biomarcadores de daño renal en un modelo experimental de Pielonefris inducida por Escherichia coli uropatógena Dilek Canlar-Akar 1* , Funda Kiral 2 ¹*Dokuz Eylül University, Faculty of Veterinary Medicine, Department of Biochemistry, İzmir, Türkiye ²Aydın Adnan Menderes Üniversitesi, Faculty of Veterinary Medicine, Department of Biochemistry, Aydın, Türkiye *Correspondence author: dilek.canlar@deu.edu.tr ABSTRACT This study aims to determine the levels of biomarkers in rats on diﬀerent days of the disease by creang a pyelonephris model using Uropathogenic Escherichia coli (UPEC). Forty rats were used in the study; 10 were designated as the control group and the remaining 30 rats were intrarenally administered UPEC to create a pyelonephris model. Blood and urine samples were collected on days 1, 4 and 7 of the experiment. Histopathologically, it was determined that pyelonephris occurred in all experimental groups. In serum samples, signiﬁcant changes were observed in the groups’ clusterin, L-FABP and clusterin/Cr levels. In urine samples: while no signiﬁcant changes were detected in Cr, clusterin, NGAL/Cr and clusterin/Cr levels, signiﬁcant alteraons were idenﬁed in NGAL, L-FABP, KIM-1, cystan C, KIM-1/Cr, cystan C/Cr and L-FABP/Cr levels. In the scope of the study, changes in the idenﬁed biomarkers in the serum and urine samples of rats with induced pyelonephris were parcularly evident. In evaluaons conducted on diﬀerent days of the disease, it was observed that urine NGAL, L-FABP, KIM-1 and cystan C levels increased up to the 4th day compared to the control group. These ﬁndings suggest that urine biomarkers, in parcular, may play a signiﬁcant role in diagnosing pyelonephris. Key words: Kidney; pyelonephris; uropathogenic Escherichia coli; biomarker; rat RESUMEN El objevo de este estudio es determinar los niveles de biomarcadores en diferentes días de la enfermedad en ratas, creando un modelo de pielonefris mediante el uso de Escherichia coli Uropatógena (UPEC). Se ulizaron cuarenta ratas en el estudio; 10 fueron designadas como grupo de control y a las 30 ratas restantes se les administró UPEC de forma intrarrenal para inducir el modelo de pielonefris. Se recolectaron muestras de sangre y orina los días 1, 4 y 7 del experimento. Desde el punto de vista histopatológico, se determinó que la pielonefris ocurrió en todos los grupos experimentales. En las muestras de suero, se observaron cambios signiﬁcavos en los niveles de clusterina, L-FABP y en la relación clusterina/Cr entre los grupos. En las muestras de orina, aunque no se detectaron cambios signiﬁcavos en creanina, clusterina, NGAL/Cr y la relación clusterina/Cr, se idenﬁcaron alteraciones signiﬁcavas en los niveles de NGAL, L-FABP, KIM-1, cistana C, KIM-1/Cr, cistana C/Cr y L-FABP/Cr. Dentro del alcance del estudio, los cambios en los biomarcadores idenﬁcados en las muestras de suero y orina de ratas con pielonefris inducida fueron parcularmente evidentes en las muestras de orina. En las evaluaciones realizadas en diferentes días de la enfermedad, se observó que los niveles de NGAL, L-FABP, KIM-1 y cistana C en orina aumentaron hasta el cuarto día en comparación con el grupo de control. Estos hallazgos sugieren que, en parcular, los biomarcadores en orina pueden desempeñar un papel signiﬁcavo en el diagnósco de la pielonefris. Palabras clave: Riñón; pielonefris; Escherichia coli uropatógena; biomarcador; rata

Renal damage biomarkers in an experimental Pyelonephris model / Canlar and Kiral UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Urinary tract infecons (UTIs) can occur in any part of the urinary system. The most common types include asymptomac bacteriuria, acute uncomplicated cyss and pyelonephris [1]. Uropathogenic Escherichia coli (UPEC) is the most common cause of these infecons [2 ,3,4]. Pyelonephris, which develops when bacteria travel from the bladder to the kidneys and can lead to serious complicaons, poses diagnosc challenges due to the lack of speciﬁc symptoms [5 , 6]. Therefore, the importance of biomarkers in the diagnosc process is increasingly recognized. Cystan C is a protein found in various body ﬂuids such as urine, plasma and saliva. Due to its stable structure, it has been proposed as an alternave to creanine (Cr) for measuring glomerular ﬁltraon rate. It is widely used in acute and chronic kidney diseases [7 , 8 , 9]. Clusterin (as a molecular chaperone) binds to misfolded proteins and directs them for lysosomal degradaon, regulang the protein-folding process [10 , 11]. Several studies have shown that clusterin has a higher diagnosc power in detecng kidney injury, especially proximal tubular damage than standard markers such as blood urea nitrogen and serum Cr [12 , 13]. Neutrophil gelanase-associated lipocalin (NGAL), a protein capable of detecng acute kidney injury (AKI) more quickly and accurately than serum Cr, also plays a role in combang bacterial infecons [14]. Numerous studies have demonstrated that NGAL is more sensive than serum Cr in idenfying AKI and suggested its potenal use as a beneﬁcial biomarker in clinical pracce [15 , 16]. Liver-type fay acid-binding protein (L-FABP) is an eﬀecve indicator for detecng oxidave stress and tubulointersal damage in the kidneys by facilitang the excreon of lipid peroxidaon products [17 , 18]. In clinical studies, the urinary level of this biomarker has been used to predict the progression of kidney disease [19 , 20]. Kidney injury molecule 1 (KIM-1), a protein highly expressed in kidney ssue, has emerged as an important biomarker for the early diagnosis of AKI and cardiovascular diseases and for monitoring the treatment process [21]. However, it has also been reported that KIM-1 levels ﬂuctuate signiﬁcantly in cats with AKI compared to healthy cats [22]. This study aims to determine the levels of KIM-1, NGAL, L-FABP, clusterin and cystan C, as well as changes in Cr, urea, total protein and albumin levels; it does so by tracking these markers on diﬀerent days. Speciﬁcally, it focuses on assessing speciﬁc biomarkers associated with acute and chronic kidney injury in an experimentally induced pyelonephris model. MATERIALS AND METHODS Forty non-pregnant adult female Sprague-Dawley rats (Raus norvegicus), 12 weeks old and weighing 250 ± 50 (OHAUS-Navigator) grams, were used in the study. The rats were kept under convenonal condions at 22 ± 1°C with a 12–hour light/dark cycle. They were divided into four groups, each with 10 animals. The Local Ethics Commiee approved the study for Animal Experiments of Aydın Adnan Menderes University (Protocol no: 2022/031). Preparaon of bacteria The UPEC was stored at –20°C (Bosch KDV4200NE, Germany) unl the day of the experiment. The night before the study, the bacteria were revived in an incubator (Memmert, Germany) set to 37°C. The revived bacteria were centrifuged (Nuve, Nf 800R, Turkey) at 7440 g for 3 min, washed three mes and resuspended in sterile physiological saline soluon (0.9% NaCl) [23]. Experimental study This study was conducted at Aydın Adnan Menderes University, Faculty of Veterinary Medicine, Experimental Animals Producon and Research Center. The rats were divided into four groups (1 control group and 3 experimental groups), with 10 animals in each group. Ten rats were assigned to the control group, while the remaining rats were anesthezed intraperitoneally with xylazine (Interchemie) -ketamine (Doğa İlaç). The abdominal area of the anesthezed rats was shaved, disinfected with alcohol (70%) and povidone-iodine (10%) and prepared for surgery. A midline incision was made in the upper abdominal region to expose the right kidney. To induce experimental pyelonephris, a freshly prepared 100 µl bacterial suspension (UPEC-ATCC 25922, 1 × 10⁹ CFU) was injected into the right kidney cortex and directed towards the medulla using a 1 mL syringe. Before returning the kidneys to the abdominal cavity, they were rinsed with sterile physiological saline. The abdomen was closed and the rats were returned to their cages [1 , 24 , 25]. In Group 1, blood samples were collected intracardially, one d aﬅer the injecon; in Group 2, four d aﬅer; and Group 3, seven d aﬅer. Blood samples were also collected from the control group at the end of the study. Rats were anesthezed intraperitoneally with a combinaon of ketamine (100 mg/kg) and xylazine (10 mg/kg) [26] and blood samples were collected according to established protocols [27]. Serum and urine clusterin, NGAL, L-FABP, KIM-1 and cystan C levels were measured using the ELISA, while total protein, urea, Cr and albumin levels were measured spectrophotometrically (Shimadzu UV-1601, China). Histopathological examinaon of kidneys At the end of the experiment, the right and leﬅ kidneys of the animals in the control and experimental groups were collected. The kidneys were bisected longitudinally using a scalpel and ﬁxed in 10% buﬀered formalin. The ﬁxed kidneys were promptly sent to a contracted laboratory. Tissues were processed in an automac ssue processor (Leica TP1020, USA), following a roune procedure that included dehydraon in graded alcohols, clearing in xylene (98,5%) and embedding in paraﬃn. Thin secons (5-6 µm) of paraﬃn- embedded ssues were prepared using a microtome (Leica RM2125 RTS, USA) and then stained with hematoxylin and eosin [28]. Using a light microscope (Carl Zeiss Axiolab 5, China), the prepared slides were evaluated and microphotographs (Olympus DP27, Japan) were taken for documentaon [28]. 2 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Stascal analyses The obtained data was analyzed using SPSS (Stascal Package for Social Sciences) for Windows version 21. The normality of the data distribuon was tested with the Shapiro–Wilk method. Diﬀerences between groups with normal distribuon were analyzed using one-way analysis of variance (ANOVA) and the signiﬁcance of these diﬀerences was tested using post hoc Tukey and Tamhane tests. For datasets not meeng the normality assumpon, group diﬀerences were examined via the Kruskal– Wallis test. The Bonferroni-adjusted Mann-Whitney U test was performed to determine the source of the diﬀerences. Stascal ﬁndings with a p-value below 0.05 were deemed signiﬁcant and all results are presented as mean ± standard error. RESULTS AND DISCUSSIONS The results of the serum samples are presented in TABLE I; the urine samples are presented in TABLE II. Serum Cr, urea, albumin and total protein levels signiﬁcantly increased in the study groups than the control group. In the urine, while Cr and urea levels decreased, albumin and total protein levels increased. Stascal analysis indicated that the changes in urea, albumin and total protein levels were signiﬁcant, whereas the change in Cr level was not. TABLE I. Mean serum biochemical values in rats with renal damage biomarkers in a UPEC-induced experimental Pyelonephris model Parameters n Control Day 1 Day 4 Day 7 P Creanine (mg/dl) 10 0,43±0,029 ab 0,44±0,0,035 ab 0,33±0,017 a 0,50±0,029 b 0,011* Urea (mg/dl) 10 0,48±0,05 a 1,19±0,15 b 1,00±0,06 b 1,12±0,07 b 0,0001*** Albumin (g/dl) 10 2,22±0,055 a 3,49±0,143 ab 3,91±0,040 b 3,77±0,067 b 0,0001*** Total Protein (g/)dl 10 4,43±0,22 a 5,82±0,33 b 7,06±0,20 c 7,48±0,42 c 0,000*** Cystan C (ng/mL) 10 10,87±0,57 a 10,08±0,48 a 10,57±0,44 a 11,59±0,62 a 0,269 NS Clusterin (ng/mL) 10 28,74±0,32 a 29,95±0,63 ab 32,76±2,17 b 33,72±1,61 b 0,003** NGAL (ng/mL) 10 10,11±0,55 a 10,65±0,37 a 10,53±0,74 a 10,35±0,44 a 0,906 NS L-FABP (ng/mL) 10 31,62±2,21 a 22,39±1,55 b 24,06±1,75 b 24,25±1,04 b 0,003** KIM-1 (ng/mL) 10 1,411±0,049 a 1,182±0,073 a 1,235±0,048 a 1,282±0,088 a 0,119 NS Cystan C/Cr (ng/mg Cr) 10 2366,03±302,02 a 3694,46±932,06 a 2867,51±455,21 a 2854,51 ±305,76 a 0,624 NS Clusterin/Cr (ng/mg Cr) 10 5724,94±745,64 a 10692,96±2431,47 ab 10079,12±615,64 b 8640,31±706,34 ab 0,007** NGAL/Cr (ng/mg Cr) 10 2206,30± 294,90 a 3874,71±730,22 a 3017,57±561,99 a 2549,01±357,10 a 0,197 NS L-FABP/Cr (ng/mg Cr) 10 7358,59±825,07 a 7616,21±1629,38 a 7793,52±749,75 a 6008,32±289,99 a 0,511 NS KIM-1/Cr (ng/mg Cr) 10 382,96±94,46 a 421,19±103,49 a 341,44± 56,03 a 306,33±34,02 a 0,853 NS Neutrophil gelanase-associated lipocalin: NGAL, Liver-type fay acid-binding protein: L-FABP, Kidney injury molecule 1: KIM-1, Creanine: Cr. All data are presented as means ± standard error. Diﬀerent leers abc in the same row indicate signiﬁcance (P < 0.05). *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05 and NS indicates no signiﬁcant diﬀerence between groups. TABLE II. Mean urine biochemical values in rats with renal damage biomarkers in a UPEC-induced experimental Pyelonephris model Parameteres n Control Day 1 Day 4 Day 7 p Creanine (mg/dl) 10 72,72±8,01 a 77,40±13,39 a 50,90±9,74 a 60,25±3,69 a 0,202 NS Urea (mg/dl) 10 8,74±0,76 a 5,79±0,53 b 6,29±0,47 b 7,42±0,70 ab 0,013* Albumin (g/dl) 10 0,103±0,012 a 0,152±0,030 ab 0,175±0,023 ab 0,177±0,010 b 0,007** Total Protein (g/dl) 10 0,29±0,03 a 0,26±0,05 a 0,50±0,07 ab 1,33±0,40 b 0,0001*** Cystan C (ng/mL) 10 7,57±0,68 a 12,74±0,75 bc 13,59±0,52 b 10,57±0,42 c 0,0001*** Clusterin (ng/mL) 10 99,46±18,79 a 80,13±17,47 a 97,36±7,87 a 72,44±4,91 a 0,233 NS NGAL (ng/mL) 10 8,76±0,52 ac 11,51±0,78 b 10,78±0,44 ab 7,33±0,54 c 0,0001*** L-FABP (ng/mL) 10 20,74±2,23 a 31,86±3,52 ab 29,26±2,10 b 21,94±1,48 a 0,005** KIM-1 (ng/mL) 10 0,805±0,048 a 1,224±0,077 b 1,221±0,049 b 0,990±0,053 a 0,0001*** Cystan C/Cr (ng/mg Cr) 10 9,69± 2,05 a 25,86± 5,38 ab 26,96± 5,62 b 20,73± 2,13 ab 0,013* Clusterin/Cr (ng/mg Cr) 10 112,66±22,88 a 147,03±29,70 a 159,41±44,71 a 131,85±5,79 a 0,385 NS NGAL/Cr (ng/mg Cr) 10 10,89± 2,17 a 23,00±4,58 a 25,59±5,87 a 15,10±1,60 a 0,077 NS L-FABP/Cr (ng/mg Cr) 10 25,23±5,89 a 62,95±13,56 b 58,31±15,38 b 43,63±5,51 b 0,041* KIM-1/Cr (ng/mg Cr) 10 0,94± 0,09 a 2,52±0,58 b 2,33±0,58 ab 1,93±0,19 b 0,017* Neutrophil gelanase-associated lipocalin: NGAL, Liver-type fay acid-binding protein: L-FABP, Kidney injury molecule 1: KIM-1, Creanine: Cr. All data are presented as means ± standard error. Diﬀerent leers abc in the same row indicate signiﬁcance (P < 0.05). *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05 and NS indicates no signiﬁcant diﬀerence between groups. 3 of 7

Renal damage biomarkers in an experimental Pyelonephris model / Canlar and Kiral UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Many studies evaluang humans and animals with kidney diseases classify cystan C as a helpful biomarker [29 , 30 , 31]. Studies invesgang pyelonephris have reported diﬀerent results regarding changes in serum cystan C levels. Some studies on this subject [32 , 33 , 34] have reported that serum cystan C levels in paents with acute pyelonephris were signiﬁcantly higher than those without. In contrast, Islekel et al. [35] stated no signiﬁcant change in serum and urinary cystan C levels in pyelonephris paents. In this study, while no signiﬁcant diﬀerence was detected among the groups in terms of serum cystan C and cystan C/Cr levels, a stascally signiﬁcant increase was observed in urinary cystan C and cystan C/Cr levels. These ﬁndings align with studies indicang that urinary cystan C levels strongly correlate with kidney funcon in kidney diseases. The results indicate that urinary cystan C levels may be a valuable biomarker for diagnosing pyelonephris. In the literature search, no study determined serum and urine levels of clusterin, except for a histopathological study examining the relaonship between clusterin and pyelonephris cases. In that histopathological study by Sansanwal et al. [36], weak immunohistochemical staining was observed in the kidneys of paents with pyelonephris. Weak staining indicates that clusterin is expressed at low levels in pyelonephris. In our study, serum clusterin and clusterin/Cr raos were signiﬁcantly elevated in the pyelonephris groups compared to the control group. The fact that the increase in serum clusterin/Cr rao was more pronounced than the increase in serum clusterin levels suggests that evaluang these two parameters together may be more useful in diagnosis. In a canine endotoxemia model designed to simulate kidney injury caused by Escherichia coli (E. coli)-derived lipopolysaccharides, it was reported that AKI occurred and urinary clusterin and urinary clusterin/Cr levels signiﬁcantly increased before any rise in serum Cr levels [37]. In this study, E. coli was used to induce pyelonephris and some similaries were observed between the current model and the canine model. However, unlike the model applied in dogs, no signiﬁcant increase was detected in urinary clusterin and clusterin/Cr levels. Addionally, the absence of a signiﬁcant increase in urinary clusterin and clusterin/Cr levels is inconsistent with previous studies on AKI [12 , 13]. Neutrophil gelanase-associated lipocalin is a widely used biomarker for diagnosing urinary system diseases such as chronic kidney diseases and urinary tract obstrucon.Studies evaluang NGAL as an indicator of kidney damage highlight the sensivity of urinary NGAL and NGAL/Cr raos in detecng AKI [37 , 38 , 39]. This study showed a notable increase in serum NGAL/Cr, urinary NGAL and urinary NGAL/Cr levels, especially on the ﬁrst day. While the increases in serum NGAL/Cr and urinary NGAL/Cr levels were not stascally signiﬁcant, the increase in urinary NGAL levels was substanal. These results suggest that NGAL measurement may be a diagnosc indicator for acute pyelonephris rather than a prognosc biomarker. Within the limited number of studies on the levels of L-FABP in pyelonephris, contradictory ﬁndings exist. In one study, it was emphasized that L-FABP levels do not help idenfy pyelonephris [40]. In contrast, other studies reported that urinary L-FABP and urinary L-FABP/Cr levels increase in children with pyelonephris [41] and signiﬁcant rise in urinary L-FABP in urinary tract infecons [42 , 43]. Among the parameters we evaluated, serum L-FABP levels signiﬁcantly decreased, whereas the L-FABP/Cr rao changes were not stascally signiﬁcant. The increase in urinary L-FABP and L-FABP/Cr rao in the pyelonephris groups is consistent with the literature, more studies are needed to examine the relaonship between pyelonephris and L-FABP. Studies in humans and animals have reported varying results regarding serum and urinary KIM-1 levels. Krzemien et al. [44] and Urbschat et al. [45] suggested that urinary KIM-1 is ineﬀecve in detecng pyelonephris. In contrast, Lee et al. [46] and Rius-Gordillo et al. [47] reported a signiﬁcant increase in urinary KIM-1/Cr levels during the acute phase of the disease. In this experimental pyelonephris model, serum KIM-1 levels decreased over me; however, this change was not stascally signiﬁcant. Skowron et al. [48] developed a pyelonephris model in rats using diﬀerent doses of UPEC and invesgated its dose- dependent eﬀects. Similarly, in the pyelonephris group induced with 1 × 10⁹ bacteria, urinary KIM-1 levels were reported to have increased signiﬁcantly within one week. In this study, consistent with the ﬁndings of Skowron et al., a signiﬁcant increase in urinary KIM-1 levels was observed in the pyelonephris group compared to the control group. Studies have shown that urinary KIM-1 is more speciﬁc than serum or plasma KIM-1 and may serve as a noninvasive biomarker for early diagnosis [49]. Similarly, the ﬁndings of this study support that urinary KIM-1 levels could be a valuable indicator for the diagnosis of pyelonephris. The histopathological ﬁndings are presented in detail in FIG. 1. In the histopathological evaluaon, pyelonephris was found in the kidneys of all animals in the experimental group, while no pathological ﬁndings were detected in the control group (FIGS. 1. A, B). In rats belonging to the ﬁrst- and fourth-d groups, dense neutrophilic leukocyte inﬁltraon, focal microabscess formaon, mild lymphocyc inﬁltraon in the renal intersum and thickening of the glomerular basement membrane were observed. In tubular epithelial cells, severe vacuolizaon, marked tubular lumen dilataon, hyaline cast formaon in some dilated tubules, tubular atrophy and intense neutrophilic leukocyte inﬁltraon in the collecng ducts were noted. Signs of urine stasis were detected in the corcal tubules. Squamous hyperplasia and leukocyte inﬁltraon were observed in the transional epithelium of the renal pelvis (FIGS. 1. C, D, E). In rats belonging to the seventh-d group, intense lymphoplasmacyc cells, less dense neutrophilic leukocyte inﬁltraon and widespread ﬁbrosis were observed in the intersum. Extensive necroc areas and severe glomerulosclerosis were noted in some rats. Glomeruli and tubules were uerly absent in parenchymal areas where ﬁbrosis was prevalent. In highly atrophied tubules and glomeruli, thickening of the basement membrane was observed. Squamous hyperplasia and inﬂammaon dominated by lymphocytes were observed in the transional epithelium of the renal pelvis (FIGS. 1. F, G, H). In this study, the changes in potenal biomarkers of kidney diseases and tradional diagnosc parameters in pyelonephris paents were invesgated, yielding the following ﬁndings. In serum samples, clusterin and clusterin/Cr levels signiﬁcantly increased, while L-FABP markedly decreased. In urine, signiﬁcant increases were detected in the levels of NGAL, L-FABP, KIM-1, cystan C, KIM-1/Cr, cystan C/Cr, and L-FABP/Cr. Among the roune biochemical parameters evaluated, Cr, urea, albumin, and total protein levels showed signiﬁcant increases in serum, whereas in urine, all parameters except Cr exhibited signiﬁcant changes. 4 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Histopathological ﬁndings in an experimental Pyelonephris model. A) Control group medulla (Bar: 200 µm). B) Control group cortex (Bar: 200 µm). C) 1 d group. Widespread neutrophil inﬁltraon (star) and tubular dilataon (arrow) (Bar: 200 µm) D) 4 d group. Neut- rophil inﬁltraon in tubules (Bar: 50 µm) E) 4 d group. Squamous hyperplasia (arrow) in pelvic epithelium with leukocyte inﬁltraon (star) (Bar: 200 µm). F) 7 d Group. Widespread ﬁbrosis, glomeruli and tubules are lost (Bar: 500 µm) G) 7 d group. Widespread ﬁbrosis, glomeruli and tubules are lost (Bar: 200 µm) H) 7 d group. Lymphocyte inﬁltraon (arrowheads) in the intersum of the cortex and tubules (arrows) and atrophy (Bar: 100 µm). Hematoxylin and eosin stain. CONCLUSION AND RECOMMENDATIONS In conclusion, the obtained ﬁndings suggest that urinary biomarkers may potenally be superior in the diagnosis of pyelonephris. Addionally, clusterin, invesgated for the ﬁrst me in the serum and urine samples of pyelonephris paents, showed signiﬁcant increases in serum clusterin and clusterin/ Cr levels, indicang its diagnosc value. However, the lack of signiﬁcant changes in urinary clusterin levels highlights the need for further research into its role. These ﬁndings support the potenal of urinary biomarkers for clinical use in pyelonephris diagnosis, while more comprehensive studies are required to clarify their diagnosc and prognosc value in the future. ACKNOWLEDGEMENT This study was supported by the Scienﬁc and Technological Research Council of Türkiye (TÜBİTAK) under Grant Number 122O941. The authors thank TÜBİTAK for their support. Conﬂict of Interest Statement The authors declare no conﬂict of interest. BIBLIOGRAPHIC REFERENCES [1] Gupta K, Donnola SB, Sadeghi Z, Lu L, Erokwu BO, Kavran M, Hijaz A, Flask CA. Intrarenal Injecon of Escherichia coli in a Rat Model of Pyelonephris. J. Vis. Exp. [Internet]. 2017; 2017(125):e54649. doi: hps://doi.org/ph5v [2] Ramakrishnan K, Scheid DC. Diagnosis and management of acute pyelonephris in adults. Am. Fam. Physician. [Internet]. 2005 [cited Feb.26 2025]; 71(5):933-942. Available in: hps://goo.su/lcGqP [3] Piccoli GB, Consiglio V, Deagosni MC, Serra M, Biolca M, Ragni F, Biglino A, De Pascale A, Frascisco MF, Veltri A, Porpiglia F. The clinical and imaging presentaon of acute “non complicated” pyelonephris: a new proﬁle for an ancient disease. BMC Nephrol. [Internet]. 2011; 12:68. doi: hps://doi.org/dx9nzz [4] Sobel JD, Kaye D. Urinary Tract Infecons. In: Benne JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Benne’s Principles and Pracce of Infecous Diseases. 8th ed. Philadelphia: Elsevier Saunders; 2015. p. 886- 913. [5] Whalan EJ. A Toxicologist’s Guide to Clinical Pathology in Animals: Hematology, Clinical Chemistry; Urinalysis. 1st ed. Switzerland: Springer Internaonal Publishing; 2015. [6] Cooper KL, Badalato GM, Rutman MP. Infecons of the Urinary Tract. In: Parn AW, Dmochowski RR, Kavoussi LR, Peters CA, editors. Campbell-Walsh-Wein Urology. 12th ed. Philadelphia, PA: Elsevier; 2021. p.1129-1201. [7] Löerg H, Grubb AO. Quantaon of gamma-trace in human biological ﬂuids: indicaons for producon in the central nervous system. Scand. J. Clin. Lab. Invest. [Internet]. 1979 ;39(7):619-626. doi: hps://doi.org/ cqx7rk [8] Adingwupu OM, Barbosa ER, Palevsky PM, Vassalo JA, Levey AS, Inker LA. Cystan C as a GFR Esmaon Marker in Acute and Chronic Illness: A Systemac Review. Kidney Med. [Internet]. 2023; 5(12):100727. doi: hps://doi. org/ph6t [9] Munavalli V, Dhumale AJ, Kothiwale V A. Serum Cystan C concentraon levels as a marker of acute Kidney injury in crically ill Paents - A cross seconal study. APIK J. 5 of 7

Renal damage biomarkers in an experimental Pyelonephris model / Canlar and Kiral UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Int. Med. [Internet]. 2016; 4(3):9-14. doi: hps://doi. org/ph6v [10] Satapathy S, Wilson MR. The Dual Roles of Clusterin in Extracellular and Intracellular Proteostasis. Trends Biochem. Sci. [Internet]. 2021; 46(8):652-660. doi: hps://doi.org/gqj3rt [11] Itakura E, Chiba M, Murata T, Matsuura A. Heparan sulfate is a clearance receptor for aberrant extracellular proteins. J. Cell Biol. [Internet]. 2020; 219(3):e201911126. doi: hps://doi.org/ghswdt [12] Dieterle F, Perentes E, Cordier A, Roth DR, Verdes P, Grenet O, Pantano S, Moulin P, Wahl D, Mahl A, End P, Staedtler F, Legay F, Carl K, Laurie D, Chibout SD, Vonderscher J, Maurer G. Urinary clusterin, cystan C, beta2-microglobulin and total protein as markers to detect drug-induced kidney injury. Nat. Biotechnol. [Internet]. 2010; 28(5):463-469. doi: hps://doi.org/ b6p92f [13] Udupa V, Prakash V. Gentamicin induced acute renal damage and its evaluaon using urinary biomarkers in rats. Toxicol. Rep. [Internet]. 2019;6:91-99. doi: hps:// doi.org/ph6x [14] Crescenzi E, Leonardi A, Paciﬁco F. NGAL as a Potenal Target in Tumor Microenvironment. Int. J. Mol. Sci. [Internet]. 2021; 22(22):12333. doi: hps://doi.org/ gpw8wz [15] Defauw P, Schoeman JP, Leisewitz AL, Goddard A, Duchateau L, Aresu L, Meyer E, Daminet S. Evaluaon of acute kidney injury in dogs with complicated or uncomplicated Babesia rossi infecon. Ticks Tick Borne Dis. [Internet]. 2020; 11(3):101406. doi: hps://doi.org/ ph62 [16] Monari E, Troìa R, Magna L, Gruarin M, Grise C, Fernandez M, Balboni A, Giun M, Dondi F. Urine neutrophil gelanase-associated lipocalin to diagnose and characterize acute kidney injury in dogs. J. Vet. Intern. Med. [Internet]. 2020; 34(1):176-185. doi: hps://doi. org/ph63 [17] Li B, Hao J, Zeng J, Sauter ER. SnapShot: FABP Funcons. Cell. [Internet]. 2020; 182(4):1066-1066.e1. doi: hps:// doi.org/gp8k68 [18] Xu Y, Xie Y, Shao X, Ni Z, Mou S. L-FABP: A novel biomarker of kidney disease. Clin. Chim. Acta. [Internet]. 2015; 445:85-90. doi: hps://doi.org/f7c9kd [19] Yokoyama T, Kamijo-Ikemori A, Sugaya T, Hoshino S, Yasuda T, Kimura K. Urinary excreon of liver type fay acid binding protein accurately reﬂects the degree of tubulointersal damage. Am. J. Pathol. [Internet]. 2009; 174(6):2096-2106. doi: hps://doi.org/bkrntq [20] Kamijo-Ikemori A, Kimura K. Clinical ulity of tubular markers in kidney disease: a narrave review. J. Lab. Precis. Med. [Internet]. 2022;7:27. doi: hps://doi.org/ ph64 [21] Medić B, Rovčanin B, Basta Jovanović G, Radojević- Škodrić S, Prostran M. Kidney Injury Molecule-1 and Cardiovascular Diseases: From Basic Science to Clinical Pracce. Biomed. Res. Int. [Internet]. 2015; 2015:854070. doi: hps://doi.org/f84jnb [22] Bland SK, Clark ME, Côté O, Bienzle D. A speciﬁc immunoassay for detecon of feline kidney injury molecule 1. J. Feline Med. Surg. [Internet]. 2019; 21(12):1069-1079. doi: hps://doi.org/gfns9x [23] Jiang Y, Xiong Z, Huang J, Yan F, Yao G, Lai B. Eﬀecve E. coli inacvaon of core-shell ZnO@ZIF-8 photocatalysis under visible light synergize with peroxymonosulfate: Eﬃciency and mechanism. Chin. Chem. Le. [Internet]. 2022; 33(1):415–423. doi:hps://doi.org/ph65 [24] Kasap B, Soylu A, Ertoy Baydar D, Kiray M, Tuğyan K, Kavukçu S. Protecve eﬀects of bilirubin in an experimental rat model of pyelonephris. Urology. [Internet]. 2012; 80(6):1389.e17-1389.e22. doi:hps:// doi.org/f25j [25] Sabetkish N, Sabetkish S, Mohseni MJ, Kajbafzadeh AM. Prevenon of Renal Scarring in Acute Pyelonephris by Probioc Therapy: an Experimental Study. Probiocs Anmicrob. Proteins. [Internet]. 2019; 11(1):158-164. doi:hps://doi.org/ph66 [26] Suckow MA, Danneman P, Brayton C. The Laboratory Mouse. Boca Raton, London, Newyork, Washington DC: CRC Press LLC; 2001. Available in: hps://goo.su/Hgcq [27] Waynforth HB, Flecknell PA. Experimental and Surgical Technique In the Rat. 2nd ed. Amsterdam, Boston, Heıdelberg, London, Newyork, Oxford, Paris, San Diego, Sanfrancisco, Singapore, Sydney, Tokyo: Academic Press; 1995. Available in: hps://goo.su/vXYDhK8 [28] Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. London; Boston: Buerworths. 1985. [29] Poświatowska-Kaszczyszyn I. Usefulness of serum cystan C measurement for assessing renal funcon in cats. Bull Vet. Inst. Pulawy. [Internet]. 2012; 56(2):235– 239. doi:hps://doi.org/ph67 [30] Miyagawa Y, Takemura N, Hirose H. Evaluaon of the measurement of serum cystan C by an enzyme-linked immunosorbent assay for humans as a marker of the glomerular ﬁltraon rate in dogs. J. Vet. Med. Sci. [Internet]. 2009; 71(9):1169-1176. doi: hps://doi.org/ ﬅ65qf [31] Yan Y, Liu Y, Guo Y, Li B, Li Y, Wang X. Early diagnosc model of pyonephrosis with calculi based on radiomic features combined with clinical variables. Biomed. Eng. [Internet]. 2024; 23(1):97. doi: hps://doi.org/ph69 [32] Zhang L, Li Y, Wang Z, Jiang J, Jin S, Zhu L, Lu Q, Chen J. The relaonship between pathogenic bacteria distribuon and the expression of serum YKL-40 and IL-8 in paents with acute pyelonephris. Chin. J. Microecol. [Internet]. 2022; 34(10):1186-1190. doi:hps://doi.org/ph7b [33] Yim HE, Yim H, Bae ES, Woo SU, Yoo KH. Predicve value of urinary and serum biomarkers in young children with febrile urinary tract infecons. Pediatr. Nephrol. [Internet]. 2014; 29(11):2181-2189. doi:hps://doi.org/ f6jg2x [34] Lezhenko HO, Zakharchenko NA. The role of nitric oxide synthase and cystan C in the mechanisms of anmicrobial protecon in children with urinary tract infecons considering the eological factor. Zaporozhye Med. J. [Internet]. 2022; 24(4):459-463. doi: hps://doi. org/ph7c [35] Islekel H, Soylu A, Altun Z, Yis U, Turkmen M, Kavukcu S. Serum and urine cystan C levels in children with post- pyelonephric renal scarring: a pilot study. Int. Urol. 6 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Nephrol. [Internet]. 2007; 39(4):1241-1250. doi:hps:// doi.org/bc38t6 [36] Sansanwal P, Li L, Sarwal MM. Inhibion of intracellular clusterin aenuates cell death in nephropathic cysnosis. J. Am. Soc. Nephrol. [Internet]. 2015; 26(3):612-625. doi: hps://doi.org/f64bqz [37] Steblaj B, Kuer APN, Srn M, Daminet S, Major A, Zini E. Endotoxic kidney injury in Beagle dogs assessed by serum creanine and symmetric dimethylarginine, and urinary neutrophil gelanase-associated lipocalin and clusterin. Res. Vet. Sci. [Internet]. 2023;162:104966. doi: hps://doi.org/gs3q9x [38] Zhou X, Ma B, Lin Z, Qu Z, Huo Y, Wang J, Li B. Evaluaon of the usefulness of novel biomarkers for drug-induced acute kidney injury in beagle dogs. Toxicol. Appl. Pharmacol. [Internet]. 2014; 280(1):30-35. doi: hps:// doi.org/f6h7z6 [39] Palm CA, Segev G, Cowgill LD, LeRoy BE, Kowalkowski KL, Kanakubo K, Westropp JL. Urinary Neutrophil Gelanase- associated Lipocalin as a Marker for Idenﬁcaon of Acute Kidney Injury and Recovery in Dogs with Gentamicin- induced Nephrotoxicity. J. Vet. Intern. Med. [Internet]. 2016; 30(1):200-205. doi: hps://doi.org/ph7d [40] Mizutani M, Hasegawa S, Matsushige T, Ohta N, Kiaka S, Hoshide M, Kusuda T, Takahashi K, Ichihara K, Ohga S. Disncve inﬂammatory proﬁle between acute focal bacterial nephris and acute pyelonephris in children. Cytokine. [Internet]. 2017; 99:24-29. doi: hps://doi. org/ph7f [41] Raﬁei A, Abedi Arzefuni F, Mohammadjafari H, Yazdani- Chara J. The Urinary Level of Liver-type Fay Acid Binding Protein in Children with Febrile UTI. Iran J. Kidney Dis. [Internet]. 2020 [cited Feb.26 2025]; 14(3):191-197. Available in: hps://goo.su/P6qzNQ6 [42] Ni AN, Shumatova TA, Sergeeva EV, Prikhodchenko NG, Zernova ES, Bykova OG. Endogenous Proteins in Infants and Toddlers with Urinary Tract Infecons. Doctor Ru Magazine. [Internet]. 2021; 20(10):44–47. doi:hps:// doi.org/ph7g [43] Sergeeva EV, Nee A, Shumatova TA, Bykova OG, Prikhodchenko NG, Zernova ES. The role of increased intesnal permeability markers in developing urinary tract infecon in children of the ﬁrst three years of life. Russ. J. Infect. Immun. [Internet]. 2022; 12(2):339-346. hps://doi.org/ph7h [44] Krzemień G, Turczyn A, Pańczyk-Tomaszewska M, Kotuła I, Demkow U, Szmigielska A. Prognosc value of serum and urine kidney injury molecule-1 in infants with urinary tract infecon. Cent. Eur. J. Immunol. [Internet]. 2019; 44(3):262-268. doi:hps://doi.org/ph7j [45] Urbschat A, Obermüller N, Paulus P, Reissig M, Hadji P, Hofmann R, Geiger H, Gauer S. Upper and lower urinary tract infecons can be detected early but not be discriminated by urinary NGAL in adults. Int. Urol. Nephrol. [Internet]. 2014; 46(12):2243-2249. doi:hps:// doi.org/f6sgv7 [46] Lee HE, Lee SH, Baek M, Choi H, Park K. Urinary Measurement of Neutrophil Gelanase Associated Lipocalin and Kidney Injury Molecule-1 Helps Diagnose Acute Pyelonephris in a Preclinical Model. J. Biomark. [Internet]. 2013; 2013:413853. doi: hps://doi.org/ gb6935 [47] Rius-Gordillo N, Ferré N, González JD, Ibars Z, Parada- Ricart E, Escribano J, DEXCAR Study Group. Role of dexamethasone in controlling the proinﬂammatory cytokine cascade in the ﬁrst episode of paediatric acute pyelonephris. Acta Paediatr. [Internet]. 2024; 113(3):564-572. doi: hps://doi.org/ph7k [48] Skowron B, Baranowska A, Dobrek L, Ciesielczyk K, Kaszuba-Zwoinska J, Wiecek G, Malska-Wozniak A, Strus M, Gil K. Urinary neutrophil gelanase-associated lipocalin, kidney injury molecule-1, uromodulin, and cystan C concentraons in an experimental rat model of ascending acute kidney injury induced by pyelonephris. J. Physiol. Pharmacol. [Internet]. 2018; 69(4):625-637 doi:hps://doi.org/ph7m [49] Anandkumar DG, Dheerendra PC, Vellakampadi D, Ramanathan G. Kidney injury molecule-1; is it a predicve marker for renal diseases? J. Nephropharmacol. [Internet]. 2023; 12(2):e10572. doi: hps://doi.org/ph7n 7 of 7