Severity of AKI after various renal ischemia times.

<p>The most sensitive parameter was (P) the tubular dilation score, which increased already after 10-min ischemia (C, H and M) compared to control-operated (ctrl-op; B, G and L) (* p<0.05 vs. non-operated (non-op) and p<0.001 vs. control-operated (ctrl-op); ** p<0.001 vs. non-op and ctrl-op). (Q) Tubular necrosis and (R) casts were present mostly after 20- (D, I and N) and 30-min (E, J and O) ischemia († p<0.05 vs. non-op, p<0.01 vs. ctrl-op, 10- and 30-min ishemia; ‡ p<0.0001 non-op, ctrl-op and 10-min ischemia, p<0.01 vs. 20-min ischemia; ¶ p<0.05 vs. non-op, p<0.01 vs. ctrl-op and 10-min ischemia, p<0.0001 vs. 30-min ischemia; *¶ p<0.0001 vs. non-op, ctrl-op, 10- and 20-min ischemia). There was no significant histologic change between non-op (A, F and K) and ctrl-op. (S) Renal function measured by blood urea nitrogen retention worsened after 20-min ischemia (# p<0.0001 vs. non-op, ctrl-op (ctrl-op) and 10-min ischemia). (non-op: n = 4; ctrl-op: n = 6; 10-min: n = 7; 20 min: n = 7; 30 min: n = 16).</p

Urinary NGAL was sensitive enough to detect 10-min ischemia.

<p>(A) Urinary NGAL normalized to urinary creatinine correlated significantly with 24-hour urinary NGAL (r = 0.9082, p<0.0001). (B) Plasma NGAL levels and (C) NGAL renal mRNA expression increased both after 20-min ischemia and control operation (# p<0.01 vs. non-op, 20- and 30-min ischemia; *# p<0.01 vs. non-op and ctrl-op; † p<0.0001 vs. non-op, 20- and 30-min ischemia, ‡ p<0.0001 vs. non-op, ctrl-op and 10-min ischemia). (D) Urinary NGAL increased significantly after 10-min ischemia, but also after control operation (* p<0.0001 vs. non-op, p<0.05 vs. 10-min ischemia, p<0.0001 vs. 20-min ischemia; *& p<0.0001 vs. non-op and p<0.05 vs. ctrl-op; & p<0.0001 vs. non-op and ctrl-op). (E) Urinary NGAL further normalized to (divided by) plasma NGAL increased significantly after ischemia-reperfusion injury, but not after control operation (¶ p<0.001 vs. non-op and ctrl-op). (F) The ratio between urinary NGAL and calculated filtrered NGAL was significantly higher only after 10-min ischemia and not after ctrl-op vs. non-op ($ p<0.001 vs. ctrl-op and non-op). (non-op: urine and plasma n = 23, kidney RNA n = 10; ctrl-op: n = 6; 10 min: n = 6; 20 min: n = 5; 30 min: plasma and kidney RNA n = 17).</p

The ability of plasma and urinary NGAL and BUN levels to discriminate between 10-min renal ischemia and control operation compared to non-operated (non-op.) group.

<p>The ability of plasma and urinary NGAL and BUN levels to discriminate between 10-min renal ischemia and control operation compared to non-operated (non-op.) group.</p

The intensity and extent of the NGAL immunostaining increased progressively with renal ischemia time.

<p>In the non-op kidneys (A, F and K) only the cortex (A) was NGAL positive, where the intracellular punctate staining pattern intensified after ischemia-reperfusion injury (R) compared to non-op (P) and control-op (Q). After control operation (ctrl-op; B, G and L) slight staining of the outer (G) and inner stripe (L) could be also observed. However, NGAL staining score increased further after 10- (C, H and M), 20- (D, I and N) and 30-min (E, J and O) renal ischemia in the outer medulla (U and V). Without the primary antibody no nonspecific staining was visible (S). * p<0.0001 vs. non-op; † p<0.05 vs. non-op and to 20-min, and p<0.001 vs. 30-min ischemia; ‡ p<0.001 vs. non-op, and p<0.05 vs. 30-min ischemia; ¶ p<0.0001 vs. non-op, and p<0.05 vs. ctrl-op; § p<0.0001 vs. non-op, p<0.001 compared ctrl-op, and p<0.05 vs. 10-min ischemia; †* p<0.01 vs. 20-min, and p<0.001 vs. 30-min ischemia; ¶* p<0.0001 vs. non-op, and p<0.01 vs. ctrl-op. (non-op: n = 4; ctrl-op: n = 7; 10 min: n = 9; 20 min: n = 8; 30 min: n = 17).</p

The ability of plasma and urinary NGAL and BUN levels to discriminate between the severity of renal ischemia-reperfusion injury compared to control operation.

<p>The ability of plasma and urinary NGAL and BUN levels to discriminate between the severity of renal ischemia-reperfusion injury compared to control operation.</p

Inflammation after surgery induced NGAL production, but NGAL was not derived from neutrophils.

<p>Systemic inflammation markers (A) IL-6 and the (B) p40 subunit of IL-12/-23 peaked at 3 and 6 hours after both 30-min renal I/R injury and control. (C) In the Mcl-1^ΔMyelo bone marrow chimeric mice neutrophil deficiency was verified by FACS. There was no difference in (D) BUN, (E) pNGAL and (F, G) urinary NGAL levels normalized to urinary creatinine and to plasma NGAL in Mcl-1^ΔMyelo compared to wild type (WT) bone marrow chimeras. *, **, ***: p<0.05, 0.01, 0.001 vs. the groups indicated. (IL-6 and p40 kinetics n = 5/group/time-point; WT-control-op n = 7; WT-IR20 n = 17; Mcl-1^ΔMyelo-control-op n = 6; Mcl-1^ΔMyelo-IR20 n = 8).</p

Bodyweight.

<p>The control group treated with saline (saline) is represented with filled circles and solid line, the group treated with novobiocin (NB) is represented with filled squares and solid line, the groups treated with LPS are represented with triangles and dashed line (LPS(p)), solid line (LPS(L)) and dotted line (LPS(p+L)), the groups treated with LPS + NB are represented with diamonds and solid line (NB + LPS(L)) and dotted line (LPS/ND + LPS(L)).</p

Western blot analysis of Hsp70 (A), and Hsp90 (B) protein expression.

<p>Results are normalized to actin protein expression, and presented as fold increase relative to the saline-treated group with its mean set to 1. **, ***: p<0.01, 0.001 vs. the saline-treated controls, respectively, or between the groups indicated (n = 6/group).</p

Survival.

<p>The control group treated with saline (saline) is represented with filled circles and solid line, the group treated with novobiocin (NB) is represented with filled squares and solid line, the groups treated with LPS are represented with triangles and dashed line (LPS(p)), solid line (LPS(L)) and dotted line (LPS(p+L)), the groups treated with LPS + NB are represented with diamonds and solid line (NB + LPS(L)) and dotted line (LPS/ND + LPS(L)).</p

Plasma urea levels at the time of organ harvest i.e. at 24 hours after the second treatment.

<p>LPS: bacterial lipopolysaccharide endotoxin, p: preconditioning, L: lethal, NB: novobiocin. *, **, ***: p<0.05, 0.01, 0.001 vs. the saline-treated controls, respectively, or between the groups indicated.</p