J Pathol Transl Med.  2024 Sep;58(5):219-228. 10.4132/jptm.2024.07.12.

Paricalcitol prevents MAPK pathway activation and inflammation in adriamycin-induced kidney injury in rats

• Deluque AL
• Almeida LFd
• Oliveira BM
• Souza CS
• Maciel ALD
• Francescato HDC
• Giovanini C
• Costa RS
• Coimbra TM

• KMID: 2559067
• DOI: http://doi.org/10.4132/jptm.2024.07.12

Abstract

Background
Activation of the mitogen-activated protein kinase (MAPK) pathway induces uncontrolled cell proliferation in response to inflammatory stimuli. Adriamycin (ADR)-induced nephropathy (ADRN) in rats triggers MAPK activation and pro-inflammatory mechanisms by increasing cytokine secretion, similar to chronic kidney disease (CKD). Activation of the vitamin D receptor (VDR) plays a crucial role in suppressing the expression of inflammatory markers in the kidney and may contribute to reducing cellular proliferation. This study evaluated the effect of pre-treatment with paricalcitol on ADRN in renal inflammation mechanisms.
Methods
Male Sprague-Dawley rats were implanted with an osmotic minipump containing activated vitamin D (paricalcitol, Zemplar, 6 ng/day) or vehicle (NaCl 0.9%). Two days after implantation, ADR (Fauldoxo, 3.5 mg/kg) or vehicle (NaCl 0.9%) was injected. The rats were divided into four experimental groups: control, n = 6; paricalcitol, n = 6; ADR, n = 7 and, ADR + paricalcitol, n = 7.
Results
VDR activation was demonstrated by increased CYP24A1 in renal tissue. Paricalcitol prevented macrophage infiltration in the glomeruli, cortex, and outer medulla, prevented secretion of tumor necrosis factor-α, and interleukin-1β, increased arginase I and decreased arginase II tissue expressions, effects associated with attenuation of MAPK pathways, increased zonula occludens-1, and reduced cell proliferation associated with proliferating cell nuclear antigen expression. Paricalcitol treatment decreased the stromal cell-derived factor 1α/chemokine C-X-C receptor type 4/β-catenin pathway.
Conclusions
Paricalcitol plays a renoprotective role by modulating renal inflammation and cell proliferation. These results highlight potential targets for treating CKD.

Keyword

Vitamin D; Inflammation; Macrophages; Cellular proliferation; Renal insufficiency

Figure

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  Fig. 1. Western blot analysis of CYP24A1 in kidney. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as loading control. Data from the control (dots), paricalcitol (squares), adriamycin (ADR; upward facing triangles) and ADR + paricalcitol (downward facing triangles) groups. n = 4–6 for each group. One-way ANOVA and Newman-Keuls multiple comparisons; data are expressed as mean ± standard error of the mean. *p < .05.

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  Fig. 2. Analysis of macrophages in kidney tissue. (A) Immunolocalization of CD68 in renal compartments. Arrows indicate positive expression of CD68 in the glomerulus. Arrowheads indicate positive CD68 expression in the tubulointerstitial compartments. Percentage of CD68-positivity in the glomerulus (B), in the cortex (C), and in the outer medulla (D). (E) Densitometric analysis of CD68. Cytokines and macrophage profile in kidney tissue. Enzyme-linked immunosorbent assay for interleukin-1β (IL-1β) (F) and tumor necrosis factor-α (TNF-α) (G). Densitometric analysis of arginase I (H) and arginase II (I) in the kidney. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as loading control. Data from the control (dots), paricalcitol (squares), adriamycin (ADR; upward facing triangles) and ADR + paricalcitol (downward facing triangles) groups. n = 4–6 for each group. One-way ANOVA and Newman-Keuls multiple comparisons; data are expressed as mean±standard error of the mean. *p < .05, **p < .01, ***p < .001.

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  Fig. 3. Activation of mitogen-activated protein kinase pathways. Analysis of p-p38 (A), p-JNK (B), p-ERK1/2 (C), zonula occludens-1 (ZO-1) (D), and proliferating cell nuclear antigen (PCNA) (E). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as loading control. Data from the control (dots), paricalcitol (squares), adriamycin (ADR; upward facing triangles) and ADR + paricalcitol (downward facing triangles) groups. n = 4–6 for each group. For (A–D), one-way ANOVA and Newman-Keuls multiple comparisons; data are expressed as mean±standard error of the mean. *p < .05, **p < .01, ***p < .001.

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  Fig. 4. Analysis of pro-inflammatory pathways in renal tissue. Analysis of nuclear factor-κB (NF-κB) (A), nuclear factor κB alpha inhibitor (IκBα) (B), nuclear factor κB beta inhibitor (IκBβ) (C), stromal cell-derived factor 1α (SDF-1α) (D), C-X-C chemokine receptor type 4 (CXCR4) (E), and β-catenin (F). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as loding control. Data from the control (dots), paricalcitol (squares), adriamycin (ADR; upward facing triangles) and ADR + paricalcitol (downward facing triangles) groups. n = 4–6 for each group for (A, B, C, D, F). One-way ANOVA and Newman-Keuls multiple comparisons; data are expressed as mean ± standard error of the mean. (E) Kruskal-Wallis and Dunn’s post-test; data are expressed as median and percentiles (25%–75%). *p < .05, **p < .01, ***p < .001.

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Reference

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