Mechanism of activation of TLR4/NF-κB/NLRP3 signaling pathway induced by heat stress disrupting the filtration barrier in broiler

Abstract

Background: High-temperature environment can cause acute kidney injury affecting renal filtration function. To study the mechanism of renal injury caused by heat stress through activates TLR4/NF-κB/NLRP3 signaling pathway by disrupting the filtration barrier in broiler chickens. The temperature of broilers in the TN group was maintained at 23 ± 1 °C, and the HS group temperature was maintained at 35 ± 1℃ from the age of 21 days, and the high temperature was 10 h per day, and one broiler from each replicate group at the age of 35 and 42 days was selected for blood sampling, respectively.

Results: The ELISA results demonstrated that in comparison to the TN group, serum CORT content of broilers in the HS group was all remarkably elevated (P < 0.01); the levels of IL-6 and TNF-α in the serum were remarkably elevated (P < 0.05 or P < 0.01); serum CAT and SOD activities were all remarkably reduced (P < 0.05 or P < 0.01), and serum LDH activity and MDA content were all remarkably decreased (P < 0.05); serum BUN and CRE levels were remarkably elevated (P < 0.01). Pathological sections and transmission electron microscopy demonstrated that the structure of the renal filtration barrier in the HS group damaged gradually with the prolongation of heat stress in comparison to the TN group, but the damage was reduced at 42 days of age; the levels of TLR4, MyD88, NF-κB, NF-κB-p65, NLRP3, caspase-1 and IL-1β mRNAs were all up-regulated (P < 0.05 or P < 0.01) in renal tissues of the HS group, indicating that heat stress caused damage to the morphological structure and function of the renal filtration barrier and that TLR4/NF-κB/NLRP3 pathway was also affected by heat stress, leading to increased activity (P < 0.05 or P < 0.01).

Conclusions: It demonstrated that heat stress caused detrimental effects on both the morphological structure and function of the renal filtration barrier, and the initiation of the TLR4/NF-κB/NLRP3 signaling pathway exacerbated the inflammatory damage, leading to increased thermal damage to renal tissues and glomerular filtration barriers; however, with the prolongation of heat stress, broilers gradually developed heat tolerance, and the damage to the renal tissues and filtration barriers triggered by heat stress was mitigated.

Keywords: Antioxidant capacity; Broiler; Heat shock protein; Heat stress; Renal filtration barrier; TLR4/NF-κB/NLRP3.

Fig. 1

Mechanism of inflammatory damage in broiler kidney tissue due to heat stress. Note: Heat stress stimulates the organism to induce extracellular or extracellular HSP70 overexpression, HSP70 binds to TLR4 and activates TLR4, which activates downstream MyD88 to indirectly activate NF-κB or TLR4 activation can directly activate NF-κB, resulting in the release of inflammatory cytokines such as (TNF-α, IL-6) from NF-κB. The activation of NF-κB causes NLRP3 overexpression; NLRP3 activates downstream caspase-1 and promotes the release of IL-1β and IL-18, leading to an increased renal inflammatory response and an impaired filtration barrier

Fig. 2

The impact of heat stress on serum IL-6 (A), TNF-α (B) and CORT (C) concentrations in broilers of different days of age. TN denotes the normal group; HS denotes the heat stress group; data were expressed as mean ± SD (n = 5). P < 0.01 indicates a highly significant difference, P < 0.05 indicates a significant difference and P > 0.05 indicates NS (∗P < 0.05, ∗∗P < 0.01)

Fig. 3

The impact of heat stress on antioxidant-related indices CAT (A), MDA (B), SOD (C) and LDH (D) in broilers of different ages. TN indicates the normal group; HS indicates the heat stress group; data were presented as mean ± SD (n = 5). P < 0.01 indicates a highly significant difference, P < 0.05 indicates a significant difference (∗P < 0.05, ∗∗P < 0.01)

Fig. 4

The impact of heat stress on the production of BUN (A) and CRE (B), indicators of renal function, in broilers of different days of age.TN denotes the normal group; HS denotes the heat stress group; data are presented as mean ± SD (n = 5). P < 0.01 indicates a highly significant difference, P < 0.05 indicates a significant difference (∗P < 0.05, ∗∗P < 0.01)

Fig. 5

The impact of heat stress on histopathological changes of kidney in broilers of different ages (HE staining; scale bar: 20X, 100 μm). A1, 35-day-old TN group; B1, 35-day-old HS group; A2, 42-day-old TN group; B2, 42-day-old HS group. Note: blue arrows: renal tubules demonstrated granular and vacuolar degeneration, partial exfoliation of epithelial cells, rupture and necrosis; red arrows: basement membranes were bare; yellow arrows: part of the inner cells of the vascular glomeruli were infiltrated, atrophied and fragmented; black arrows: interstitial congestion and/or hemorrhage

Fig. 6

The impact of heat stress on the ultrastructure of glomerular filtration barrier in broiler chickens of different days of age (Transmission electron microscopy observation; Scale bar: 2000X, 5 μm). A1, 35-day-old TN group; B1, 35-day-old HS group; A2, 42-day-old TN group; B2, 42-day-old HS group Note: red arrows: thickening of the basilar membrane; dark blue arrows: disappearance of breaks in the peduncle and shortening of its length; yellow arrows: widening of the subendothelial pellucid zone; green clippings: narrowing of the lacunar membrane; purple arrows: gradual narrowing of the subendothelial pellucid zone from wide to narrow

Fig. 7

The impact of heat stress on the production of HSP60 (A), HSP70 (B), HSP90 (C) and VEGF (D) in kidney tissues of broilers of different ages. Data were shown as mean ± SD (n = 5). P < 0.01 indicates a highly significant difference, P < 0.05 indicates a significant difference and P > 0.05 indicates NS (∗P < 0.05, ∗∗P < 0.01)

Fig. 8

The impact of heat stress on mRNA activation of TLR4 (A), MyD88 (B), NF-κB (C), NF-κB-p65 (D), NLPR3 (E), caspase-1 (F) and IL-1β (G) in renal tissues of broilers of different days of age, with the data presented as mean ± SD (n = 5). P < 0.01 indicates a highly significant difference, P < 0.05 indicates a significant difference (∗P < 0.05, ∗∗P < 0.01)