1. Volume 56, Issue 1, Pages 104-117 (July 1999)
Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack 
Richard A. Zager, M.D., Benjamin M. Sacks, Kristin M. Burkhart, Ali C. Williams 
Kidney International 
Volume 56, Issue 1, Pages (July 1999)
DOI: /j x
Copyright © 1999 International Society of Nephrology Terms and Conditions

2. Figure 1
Representative two-dimensional thin layer chromatography patterns, obtained during the course of these experiments. (A) Phospholipid standards, demonstrating separation of PC, PE, PS, PI, SM, CL, LPC, and LPE (abbreviations are in the Appendix). (B) Sample from control PTS, obtained after 30 minutes of oxygenated incubation. (C) Sample obtained after 30 minutes of 0.05 units/ml Naja PLA2 treatment. (D) Sample obtained after combined Ca2+ ionophore + Naja PLA2 treatment. Note that (C) and (D) demonstrate substantial amounts of LPC and LPE compared with control tubules. The circles drawn around each of the samples represent the areas that were scraped and analyzed for phospholipid phosphate.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

3. Figure 2
Results obtained from hypoxia ± low-dose (0.025 units/ml) Naja PLA2 experiments. (A) Hypoxia induced no loss of total plasma membrane phospholipid content (PC + PE + PS + PI + SM) compared with control (C) tubules (values given as nmol phosphate). Low-dose Naja PLA2 caused significant phospholipid loss in both control and hypoxic tubules (significantly greater in the latter). Despite this, PLA2 significantly attenuated the extent of hypoxic cell death (discussed in text). (B) Hypoxia did not alter the PS/PC ratio compared with control tubules. Naja PLA2 significantly increased the PS/PC ratio (because of outer membrane PC attack). Slightly higher, rather than lower, PS/PC ratios were observed in the hypoxic tubules when challenged with PLA2, implying an absence of plasma membrane “flip flop.”
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

4. Figure 3
Results obtained from hypoxia ± high-dose (0.05 units/ml) Naja PLA2 experiments. The results obtained in these experiments mirrored those produced with low-dose Naja PLA2. (For details, see Figure 2 legend and text.)
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

5. Figure 4
Results obtained from hypoxia ± pancreatic (panc.) PLA2 experiments. (A) As with the results presented in Figure 2 and Figure 3, hypoxia once again failed to cause any decrease in total plasma membrane phospholipid phosphate (Figure 2 legend). As expected, pancreatic PLA2 caused significant phospholipid losses in both control and, to a greater degree, in hypoxic PTS. (B) Again, as with Figure 2 and Figure 3, hypoxia did not alter the PS/PC ratio. Pancreatic PLA2 caused a significant decrease in the PS/PC ratio in control PTS (consistent for its ability to penetrate the plasma membrane and its greater activity against PS). This response was accentuated in hypoxic tubules, consistent with increased susceptibility to PLA2 attack.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

6. Figure 5
Results obtained from the iron (Fe) + PLA2 experiments (Naja, 0.05 units/ml; pancreatic, pancr., PLA2, 10 units/ml). (A) Iron caused a 12 to 15% loss of total plasma membrane phospholipid [P < vs. coincubated control (C) tubules]. The addition of either Naja or pancreatic PLA2 significantly reduced total plasma membrane phospholipids, particularly in the tubules exposed to concomitant iron treatment (that is, the iron predisposed to PLA2 attack). (B) The iron-mediated phospholipid reductions were associated with a significant decrease in the PS/PC ratio (P < 0.01), consistent with preferential inner leaflet damage. However, no evidence of membrane “flip flop” was apparent because the iron-exposed tubules developed a rising, rather than falling, PS/PC ratio under conditions of Naja PLA2 attack. Pancreatic PLA2 dramatically reduced PS/PC ratios in control and particularly in iron-exposed PTS.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

7. Figure 6
Results obtained with Ca ionophore (CaI) + Naja PLA2. (A) CaI caused a significant (6%) reduction in total phospholipids, compared with control (C) tubules. The CaI-treated tubules were also highly vulnerable to Naja PLA2, as evidenced by more than 50% losses of total phospholipid mass. (B) CaI did not independently alter the PS/PC ratio (suggesting that the phospholipid losses affected both inner and outer leaflet phospholipid constituents). CaI treatment did not appear to induce membrane “flip flop” because in CaI-loaded tubules, Naja increased rather than decreased the PS/PC ratio.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

8. Figure 7
Free fatty acid concentrations (nmol/μmol total phospholipid phosphate) in control, hypoxic, Ca ionophore (CaI), and iron (Fe)-challenged PTS. As shown in (A), hypoxia caused approximate eightfold FFA increments. CaI approximately doubled FFA levels (**P < 0.04; *P < 1×10-4), whereas Fe induced only an approximate 25% increase (P = NS). (B) Depiction of the mean phospholipid losses with each of these challenges observed over the course of this investigation. If one contrasts these losses versus the FFA increments, a striking inverse correlation is apparent.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

9. Figure 8
Effect of arachidonic acid on Naja PLA2-mediated phosphatidylcholine (PC) hydrolysis. PLA2 caused an approximate 60% reduction in PC and striking LPC increments. When this PLA2/PC reaction was run in the presence of 50 or 100 μ M arachidonic acid (C20:4), dose-dependent inhibition resulted (PC preservation/LPC decrements; P < 0.03, P < 0.01, respectively).
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions