Relationship between cytokeratins CK8/18&19 and KIM-1 level in urine with apoptosis and necrosis of nephrotheliocytes in rats with toxic nephropathy

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Abstract


The aim of the article. The aim of this work was to elucidate the role of apoptosis and necrosis in kidney tissue in the development of acute renal damage in poisoning rats with uranyl acetate. The research objectives included modeling acute poisoning in rats, collecting urine and kidney tissue with identifying markers of programmed cell death, tissue polypeptide antigen (TPA, fragments of cytokeratin CK8/18 & 19), and KIM-1 level in urine. An analysis of the relationship between an early increase in urinary excretion of the TPA and apoptosis level, a kidney injury molecule KIM-1, and necrosis of the tubular epithelial cells during rat poisoning with nephrotoxin uranyl acetate dihydrate.

Materials and methods. Uranyl acetate dihydrate (CAS # 6159-44-0) was administered to 18-week old female Sprague-Dawley rats weighing 175–199 g by intragastrically at a dose of 30 mg / 100 g body weight once through an atraumatic probe. Rats were divided into 2 groups: group 1 — intact animals (12 individuals), group 2 — animals with induced AKI (36 individuals). Daily urine was collected before, on the 1st, 3rd, and 7th day after poisoning in metabolic cages. The concentration of creatinine, KIM-1, tissue polypeptide antigen was measured in urine. In the kidney tissue samples, the fraction of dead cells and nephrothelial cells with apoptotic signs of nuclear changes by fluorescence microscopy with AMD — Hoechst 33342 staining was determined. Data processing was performed using GraphPad Prism 6.0.

Results. Acute kidney injury in rats with uranyl acetate dihydrate leads to a rapid increase in urinary excretion of cytokeratin fragments CK8/18 & 19 due to subtotal damage to nephrothelial cells due to activation of apoptosis, and then an increase in KIM-1 as a marker of necrotic cell death. Fluorescence microscopy of nuclear chromatin stained renal tubule cells showed a significant increase in the proportion of cells with apoptotic bodies, chromatin condensation, and a change in the shape of the nuclei.

Conclusion. Examination of the curves of risk function showed that only creatinine in blood (p = 0.0002) and urine KIM-1 (p = 0.0005) had a significant level of association with rat mortality and necrosis of the nephrothelial cells. A comparative analysis of the relationship between apoptosis biomarker levels — TPA (cytokeratin fragments CK8/18 & 19) and urinary nephrotoxicity marker KIM-1 with the proportion of kidney cells dying by the mechanism of necrosis and apoptosis revealed positive correlations of Spearman in pairs of “cytokeratin CK8/18 & 19 — apoptosis” (r = 0.73, 95% CI 0.45–0.88, p < 0.0001), “KIM-1 — necrosis” (r = 0.98, 95% CI 0.96–0.99, p < 0.0001). The revealed relationship indicated the possibility of determining urinary tissue polypeptide antigen TPA as a marker of the early stage of acute kidney damage as a surrogate marker of tubular cell apoptosis, and KIM-1 as a marker for necrosis of nephrothelial cells.


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About the authors

Konstantin V. Sivak

Smorodintsev Research Institute of Influenza

Author for correspondence.
Email: kvsivak@gmail.com
ORCID iD: 0000-0003-4064-5033

Russian Federation, Saint Petersburg

PhD in Biology, Head of the Department of Preclinical Trials

Ruslan G. Guseynov

Saint Petersburg Clinical Hospital of St. Luke

Email: rusfa@yandex.ru
ORCID iD: 0000-0001-9935-0243

Russian Federation, Saint Petersburg

MD, Head of the Urology Department No 2

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Supplementary files

Supplementary Files Action
1.
Fig. 1. Micrograph of urine sediment of a rat poisoned with uranyl acetate dihydrate, magnification ×200 (a); macroscopic picture of acute cortical necrosis, magnification ×2 (b)

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2.
Fig. 2. Dynamics of the level of tissue polypeptide antigen in urine in rats: a — box-plot with 5–95 percentile, ** p < 0.01 differences vs. background; b — Spearman’s rank correlations between the normalize level of TPA in urine and the proportion of proximal tubule cells with morphological signs of apoptosis after 24 h

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3.
Fig. 3. Micrograph of apoptotic bodies in renal tubular epithelium of proximal tubules (arrows, magnification ×400)

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4.
Fig. 4. Dynamics of the level of KIM-1 in urine in rats: a — box-plot with 5-95 percentile, *** p < 0.01 differences vs. background; b — Spearman’s rank correlations between the normalize level of KIM-1 in urine and the proportion of proximal tubule cells with morphological signs of necrosis after 24 h

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5.
Fig. 5. Risk function curves for serum creatinine (a) and KIM-1 in rat urine (b)

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Copyright (c) 2020 Sivak K.V., Guseynov R.G.

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