Acute heat stress upregulates Akr1b3 through Nrf-2 to increase endogenous fructose leading to kidney injury

Abstract

In recent years, the prevalence of extremely high-temperature climates has led to an increase in cases of acute heat stress (HS), which has been identified as a contributing factor to various kidney diseases. Fructose, the end product of the polyol pathway, has been linked to kidney conditions such as kidney stones, chronic kidney disease, and acute kidney injury. However, the relationship between acute HS and kidney injury caused by endogenous fructose remains unclear. The study found that acute HS triggers the production of reactive oxygen species, which in turn activate the Nrf-2 and Akr1b3 leading to an increase in endogenous fructose levels in kidney cells. It was further demonstrated that the elevated levels of endogenous fructose play a crucial role in causing damage to kidney cells. Moreover, inhibiting Nrf-2 effectively mitigated kidney damage induced by acute HS by reducing endogenous fructose levels. These findings underscore the detrimental impact of excessive fructose resulting from acute stress on kidney function, offering a novel perspective for future research on the prevention and treatment of acute HS-induced kidney injury.

Keywords: Akr1b3; Nrf-2; fructose; heat stress; kidney.

Figure 1

Acute heat stress induces kidney injury and increases ROS.A, mice kidneys were stained with hematoxylin-eosin at different time points after HS (“↑”: hemorrhage in tissue; “Δ”: renal tubular injury; “O”: glomerular edema, and there were six mice in each group). B–C, ROS fluorescence intensity at different time points after HS (Error bars indicate SD, ∗∗p < 0.01, ∗∗∗p < 0.001; there were six mice in each group, and the images were selected from the same position on the kidney). HS, heat stress; ROS, reactive oxygen species.

Figure 2

Transcriptome data analysis.A, a heat map of heat stress–related protein expression changes. B, the number of differentially expressed genes in the HS group compared with the control group, 134 genes downexpression and 58 genes upexpression (p value<0.05, |log₂fold change|≥1, there were five mice in each group). C, the top 20 enrichment of GO: BP pathway analysis between the control group and HS group. D, heat map of expression changes of enriched genes in response to water deprivation. E, schematic representation of the polyol pathway. F, Akr1b3 mRNA expression of different time points post-HS (error bars indicate SD, ∗∗p < 0.01, ∗∗∗p < 0.001, and there were three mice in each group). G, fructose content of different time points after HS (error bars indicate SD, ∗∗∗p < 0.001, and there were three mice in each group). H–J, the protein expression levels of AKR1B3 and SORD were analyzed by Western blotting (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01). Akr1b3, aldo-keto reductase family 1 member B3; HS, heat stress; SORD, sorbitol dehydrogenase.

Figure 3

Acute heat stress upregulates Nrf-2 expression.A, Nrf-2 mRNA expression change in the kidney between the control group and 0h, 4h, 8h, 16h, and 24h after acute heat stress (error bars indicate SD, ∗∗∗p < 0.001). B–C, NRF-2 protein expression change in the kidney between the control group and 0h, 4h, 8h, 16h, and 24h after acute heat stress (the β-actin gel blots reused with the fourth row of Figure 2H for the same analysis, and error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01). D–E, NRF-2 protein expression change in TCMK-1 cells between the control group and heat stress for 0.5 h, 1h, 1.5 h, 2h, and 2.5 h (error bars indicate SD, ∗∗p < 0.01). F–G, immunofluorescence of NRF-2 and AKR1B3 in the control group and HS group (acute heat stress 2.5 h) (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01, and mean fluorescence intensity related to the amounts of cells). HS, heat stress.

Figure 4

Nrf-2 is a key factor in the regulation of renal endogenous fructose production in response to acute heat stress.A, after Nrf-2 overexpression the mRNA expression changes in TCMK-1 cells between different groups (error bars indicate SD, ∗∗∗p < 0.001). B–C, after Nrf-2 overexpression the protein expression change in TCMK-1 cells between different groups (error bars indicate SD, ∗p < 0.05, ∗∗∗p < 0.001). D–E, after Nrf-2 overexpression, the AKR1B3 protein expression changes in TCMK-1 cells between different groups (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01). F, after four siRNA sequences transfected into TCMK-1 cells, the Nrf-2 mRNA expression change (error bars indicate SD, ∗∗∗p < 0.001). J–K, AKR1B3 protein expression change between the control, siRNA1, and siRNA1+HS group (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01). L, the fructose content of TCMK-1 cells in different groups (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01). AKR1B3, aldo-keto reductase family 1 member B3.

Figure 5

Inhibition of Nrf-2 restores endogenous fructose production induced by acute heat stress and cell viability after fructose treatment.A, molecular structure of ML385. B, flow chart of experimental treatment of TCMK-1 cells supplemented with ML385. C–E, immunofluorescence detection of NRF-2 and AKR1B3 in different treatment groups (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and mean fluorescence intensity related to the amounts of cells). F, changes of fructose content in TCMK-1 cells in different treatment groups (error bars indicate SD, ∗∗p < 0.01, and there were six mice in each group). G–H, Nrf-2 and Akr1b3 mRNA levels were tested in different treatment groups (error bars indicate SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and there were three mice in each group). I, kidney injury of mice in different treatment groups,“↑”: hemorrhage in tissue (there were three mice in each group). Akr1b3, aldo-keto reductase family 1 member B3.

Figure 6

Mechanisms of acute heat stress–regulated kidney endogenous fructose. Acute heat stress can induce renal injury by generating endogenous fructose through the actions of NRF-2 and AKR1B3. ROS, reactive oxygen species; AKR1B3, aldo-keto reductase family 1 member B3.