Fig 1.
Representative pictomicrograph of rat kidney cortical tissue sections of normoxia control and in different durations of hypobaric hypoxia exposure.
(A-D), Haematoxylin & Eosin Staining: Renal cortical sections showing characteristic histological signs of injury with afferent arteriolopathy, mild to moderate tubular degeneration, tubular dilation (thinning of tubular epithelium) and leukocyte infiltration of tubulointerstitium in all hypoxia exposure group as compared to normoxia control group of rats. (E-H) and (M-P), Masson’s Trichome and Picrosirius Red staining: representative pictures showing an increase interstitial collagen deposition in 3rd day and 7 day hypobaric hypoxia exposure group of rats. (I-L), Periodic Acid Schiff staining (PAS): representative pictures showing enhanced mesangial proliferation and glomerular basement membrane thickening observed in 3 and 7 day hypobaric hypoxia exposure group of rats (n = 5). G = Glomerulus, AA = Afferent Arteriole, T = tubular epithelial cells. Scale bar refers to 100μm at 400x.
Fig 2.
Effect of hypobaric hypoxia on kidney function.
Biochemical and hemodynamic parameters estimated in control (normoxia) and experimental groups (different intervals of hypobaric hypoxia exposure) (n = 5). (A) Plasma Creatinine. (B) Blood urea nitrogen (BUN). (C) Uric acid. (D) Urea. (E) Urinary Protein concentration. (F) Systolic blood pressure. Data represented here is Mean ± S. E. M. Values are significant if P< 0.05. *stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. control.
Fig 3.
Effect of hypobaric hypoxia on kidney injury biomarkers.
(A) Cystatin C in plasma. (B)Netrin-1 in urine. (C) Kim-1 in urine. (D) IL-18 in urine. Data represented here is Mean ± S. E. M. Values are significant if P<0.05. * stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. control.
Fig 4.
Effect of hypobaric hypoxia on inflammatory cell adhesion molecules.
(A)ICAM -1 (in kidney). (B) VCAM-1 (in kidney). Data represented here is Mean ± S. E. M. Values are significant if P<. 05. * stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. Control.
Fig 5.
Immunohistochemical analysis of tubulointerstitial injury and other cellular changes under hypobaric hypoxia.
(A) Tubular apoptosis by tunnel method. (B) Renal injury by Kim-1. (C) Hypoxia response by HIF-1alpha. (D-E) Fibrosis: Collagen-1 and Fibronectin. (F) Macrophage infiltration CD14. Data represented here is Mean ± S. E. M. Values are significant if P<. 05. * stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. control. Scale bar refers to 100μm at 400x and 200μm at 200x.
Fig 6.
Representative image of master gel showing differentially expressed spots in 2DEfrom rat kidney tissues.
Firstly, the proteins were resolved according to their Isoelectric point (pI) in the (3–10 pH) and then separated according to their MW on 4–15%gradient SDS-PAGE gradient gel followed by silver staining. Numbers marked on protein spots were differentially expressed in 7 day hypoxia exposed group.
Fig 7.
A. Heat map showing differently expressed spots obtained after 2DE. Spots that showed up regulation in hypobaric hypoxia condition shown in orange. While spots that showed down regulation shown in blue. Fig 7 (B-F). Magnified comparison maps of spot 31316, 30660 31162, 30340 and 30428 in the 2DE patterns of control and 7 day hypoxia. Spot 31316, 30660 and 30428 had decreased expression in the 7 day hypobaric hypoxia exposure group while 31162 and 30340 had increased expression in 7 day hypoxia group.
Table 1.
List of differently expressed spots with proteomics spot ID and their respective fold changes.
Table 2.
List of differently expressed spots during hypobaric hypoxia exposure, identified by MALDI-TOF/TOF.
Table 3.
List of differently expressed spots during hypobaric hypoxia exposure, identified by LC MS.
Fig 8.
Network analysis and gene ontology annotations of the proteins identified by MALDI-TOF MS.
A) Network analysis of identified proteins with their predicted interactions using STRING db 10.0 software. B) Gene ontology classifications (Pie chart analysis) of proteins were obtained by using PANTHER 10.0 software and proteins were distributed according to their Biological Process, Molecular Function, Cellular Component and Pathway Involved.
Table 4.
List of pathways obtained by KEGG pathway enrichment analysis of some of these identified proteins.
Fig 9.
Validation of proteomic data by Immunoblottting and immunofluorescence.
(A) Western blot analysis of SOD1 and cytoplasmic 1 actin of control and 7 day hypoxia group. Kidney tissue and their respective optical densities (ROD) and densitometry analysis of results from western Blot indicating significant change between two groups compared by Student ‘s t-test (B) Immunofluorescence analysis of SOD1 in control and 7 day hypoxia exposure group. Results have been represented in term of %. Values are significant if P<0.05. * stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. control.
Fig 10.
Biochemical assays showing redox parameters and ATP levels in 7 day hypobaric hypoxia.
A) ATP levels (B) ROS levels (C) SOD activity levels (D) GST activity levels. Results have been represented in term of %. Values are significant if P<0.05. * stands for level of significance when P<0.05, **when P<0.05, *** when P<0.05 vs. control.
Table 5.
Effect of hypobaric hypoxia on pH, blood gas variables and blood electrolytes in 7 days hypobaric hypoxia exposed rats.
Fig 11.
A hypothetical schematic diagram showing molecular events occurs during hypobaric hypoxia in kidney.
Increased oxidative stress, inflammation and decreased ATP levels together contribute towards progression of renal injury in hypobaric hypoxia.