Hyperbaric oxygen therapy (HBOT) suppresses biomarkers of cell stress and kidney injury in diabetic mice

Abstract

The disease burden from diabetic kidney disease is large and growing. Effective therapies are lacking, despite an urgent need. Hyperbaric oxygen therapy (HBOT) activates Nrf2 and cellular antioxidant defenses; therefore, it may be generally useful for treating conditions that feature chronic oxidative tissue damage. Herein, we determined how periodic exposure to oxygen at elevated pressure affected type 2 diabetes mellitus-related changes in the kidneys of db/db mice. Two groups of db/db mice, designated 2.4 ATA and 1.5 ATA, were treated four times per week with 100 % oxygen at either 1.5 or 2.4 ATA (atmospheres absolute) followed by tests to assess kidney damage and function. The sham group of db/db mice and the Hets group of db/+ mice were handled but did not receive HBOT. Several markers of kidney damage were reduced significantly in the HBOT groups including urinary biomarkers neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (CyC) along with significantly lower levels of caspase-3 activity in kidney tissue extracts. Other stress biomarkers also showed trends to improvement in the HBOT groups, including urinary albumin levels. Expressions of the stress response genes NRF2, HMOX1, MT1, and HSPA1A were reduced in the HBOT groups at the end of the experiment, consistent with reduced kidney damage in treated mice. Urinary albumin/creatinine ratio (ACR), a measure of albuminuria, was significantly reduced in the db/db mice receiving HBOT. All of the db/db mouse groups had qualitatively similar changes in renal histopathology. Glycogenated nuclei, not previously reported in db/db mice, were observed in these three experimental groups but not in the control group of nondiabetic mice. Overall, our findings are consistent with therapeutic HBOT alleviating stress and damage in the diabetic kidney through cytoprotective responses. These findings support an emerging paradigm in which tissue oxygenation and cellular defenses effectively limit damage from chronic oxidative stress more effectively than chemical antioxidants.

Fig. 1

a Overview of the experimental protocol indicating the frequency and pressure of HBOT treatment. Also shown are the time points for blood (solid arrows) and urine (broken arrows) collection. b Average weekly weights over the duration of the 20-week HBOT regimen for the Hets, Sham, and 2.4 ATA groups. All groups are shown in Fig. S1. Weeks indicate animal age; for example week 5 is equivalent to week 1 of HBOT

Fig. 2

Urinary biomarkers of early onset kidney damage. a, c, e, g Results of NGAL, cystatin C, clusterin, and NAG at 4 and 10 weeks, respectively. b, d, f, h The same biomarkers at 20 weeks. Shown are the results for the Het, Sham, and 2.4 ATA groups. All groups are shown in Fig. S2. Dots are individual animals and group means are shown as bars. Asterisks indicate significant differences (p < 0.05). Groups in Figs. 2 and 5, and S2 contain different numbers of data points (mice) due to the removal of a small number of outliers (see statistical section of “Materials and methods”) and a small number of deaths

Fig. 3

Caspase 3 activity in extracts of kidney tissue taken from animals at the termination of the experiment. Asterisks indicate significant differences (p < 0.05). A protective effect is observed in the 2.4 ATA group relative to the Sham

Fig. 4

Antioxidant and cytoprotective gene expression levels in kidney tissue at 20 weeks post-HBOT. RNA quantification was performed by real-time PCR. Results are shown for Nrf2, HSPA1A, Mt1, and Hmox1, as indicated. Asterisks indicate significant differences (p < 0.05)

Fig. 5

a Total urinary albumin excreted over 22 h. An increase in excretion was observed in the db/db groups with a trend to reduced excretion in the HBOT groups. b The albumin to creatinine ratio (ACR) is shown for all groups. A significant reduction in the ACR was observed in the Sham versus 2.4 ATA groups (p < 0.05). c Creatinine clearance data for all groups is shown. Urinary creatinine was measured by an enzyme assay, and serum creatinine was quantified by HPLC, as described in the “Materials and methods.” All db/db groups showed reduced creatinine clearance relative to Hets

Fig. 6

Histological appearance of db/db kidney sections stained with periodic acid-Schiff reagent (PAS) and counterstained with hematoxylin. All three of the db/db mouse groups contained the lesions noted in the images. a Segmental expansion of mesangium of a glomerulus (arrow). b Tubules with highly vacuolated epithelial cells (arrows) and PAS-positive material in lumen (asterisks). c Numerous variably enlarged clear to PAS-positive nuclei (so-called glycogenated nuclei) in tubular epithelial cells (arrows). d Accumulations of PAS-positive material in tubular epithelial cells. All scale bars represent 10 μm