Circulating nucleic acids as possible damage-associated molecular patterns in different stages of renal failure

Figures & data

FIGURE 1. (a,b) Spectrophotometric scan analysis of plasma circulating nucleic acids and corresponding standards. For the isolation of RNA from plasma samples, the commercial RNA isolation reagent set TRI Reagent BD (T3809) was used. Purified RNAs and related oligonucleotides were employed for spectrophotometric scan analysis. For spectrophotometric scan analysis the solutions of rRNA, DNA polyA, polyC, polyU, polyI:C, polyA:U, and CpG were used. The UV spectra obtained in our study are in a close agreement with recent revised UV extinction coefficients of RNA and related nucleotides.¹⁹

TABLE 1.  Main clinical characteristics of investigated groups and circulating RNA level

Stage/type of renal disease	N	Urea (mmol/L)	Creatinine (μmol/L)	Circulating RNA (mg/L)
Control	14	3.56 ± 1.39	76.96 ± 8.22	28.2 ± 10.33
Chronic renal failure (stage II–III)	10	24.35 ± 10.70	315.68 ± 45.56	50.95 ± 10.11
		p < 0.001	p < 0.001	p < 0.001
		p < 0.001**	p < 0.001**
Chronic renal failure (stage IV–V)	9	34.2 ± 8.36	572 ± 107.51	54.37 ± 7.25
		p < 0.001	p < 0.001	p < 0.001
		p < 0.001**	p < 0.001**	p < 0.05**
Renal hemodialysis	13	21.83 + 8.05	664.43 + 151.09	62.4 ± 12.78
		p < 0.001	p < 0.001	p < 0.001
		p < 0.001**	p < 0.001**	p < 0.001**
Renal transplantation	6	13.13 ± 5.20	186.96 ± 60.22	43.25 ± 11.23
		p < 0.001	p < 0.001	p < 0.001

Notes: Patients with the different stages of chronic kidney disease were recruited from the Clinic for Nephrology and Hemodialysis Medical Faculty, University of Nis. Blood laboratory markers of kidney damage were present in establishing a diagnosis of stages of chronic kidney disease. They were selected according to the National Kidney Foundation (NKF) criteria.

*p < 0.001, statistical significance compared to the control.

**p < 0.001, statistical significance compared to the patients who underwent renal transplantation.

FIGURE 2. (a–e) NF-κB, p38, MDA-5, IRF-3, and IRF-7 level after incubation of residential macrophages with isolated nucleic acids. Residential macrophages, cultured in 24-well plates, were allocated into different groups: the cells exposed to media supplemented with physiological saline solution (intact macrophages); the cells exposed to media supplemented with RNAs purified from plasma of control healthy subjects; the cells exposed to media supplemented with RNAs purified from different groups of patients. The number of samples of each group corresponded to the number of circulating nucleic acid samples, except that untreated group of cells exposed to media and physiological saline solution consisted of 10 samples. The residential macrophages were cultured with isolated nucleic acids for 4 hours, afterwards the cells were transferred into five 96-well plates for determination of NF-κB, p38, MDA-5, IRF-3, and IRF-7. The MFI (logarithmic scale) was determined, and the results presented here are the values of MFI following subtraction of values for unstained matched control cells, analyzed on Victor Perkin Elmer–Wallac multiplate reader. *p < 0.001, statistical significance compared to the cells exposed to media supplemented with RNAs purified from plasma of control healthy subjects. **p < 0.001, statistical significance compared to the cells exposed to media supplemented with RNAs purified from plasma of patients who underwent renal transplantation. ***p < 0.05, statistical significance compared to the cells exposed to media supplemented with physiological saline solution (intact macrophages).

Cavaluzzi M, Borer P. Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 2004;32:e1–e13.

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