Modulation of aquaporin-2/vasopressin2 receptor kidney expression and tubular injury after endotoxin (lipopolysaccharide) challenge

Abstract

Objective: Sepsis-induced organ dysfunctions remain prevalent and account for >50% of intensive care unit admissions for acute renal failure with a mortality rate nearing 75%. In addition to the fact that the mechanisms underlying the pathophysiology of sepsis-related acute renal failure are unclear, the impact on septic-induced acute renal failure of either norepinephrine, a gold-standard vasopressor, and arginine vasopressin, a candidate alternative, are not well understood.

Design: Randomized and controlled in vivo study.

Setting: Research laboratory and animal facilities.

Subjects: Adult rats treated with endotoxin (lipopolysaccharide) and/or vasopressors.

Interventions: Rats were intraperitoneally injected with lipopolysaccharide (12 mg/kg) or saline and then infused with either saline, 0.375 microg/microL arginine vasopressin, or 32.5 microg/microL norepinephrine for 18 hrs. These vasopressor rates yielded respective targeted blood levels observed in human septic shock.

Measurements and main results: Renal function, including glomerular filtration rate and fraction, renal blood flow, aquaporin-2, and arginine vasopressin-2 (V2 receptor) networking, water and salt handling, and urinary protein excretion, were evaluated. After lipopolysaccharide challenge arginine vasopressin infusion: 1) impaired creatinine clearance without affecting renal blood flow, glomerular filtration rate, and fraction but reduced free-water clearance, both of which being partially restored by the V2 receptor antagonist SR-121463B; 2) decreased the recognized ability of arginine vasopressin alone to recruit aquaporin-2 to the apical membrane increase its mRNA expression and urinary release; 3) increased urinary protein content but decreased specific kidney injury molecule-1, and Clara cell protein-16 release (p < 0.05 vs. lipopolysaccharide alone). Conversely, norepinephrine infusion did not add to lipopolysaccharide-induced alteration of urine biochemistry, except for improved creatinine clearance and increased microalbuminuria.

Conclusion: In this endotoxic model, dose-targeted arginine vasopressin infusion increased lipopolysaccharide-induced renal dysfunction without affecting renal blood flow and glomerular function, but with particular disruption of aquaporin-2/V2 receptor networking, consecutive decreased salt and water handling ability. This is in clear contrast with norepinephrine infusion and suggests specific arginine vasopressin-induced "tubular epithelial dysfunction."

Figure 1

Study design; LPS, lipopolysaccharide; NE, norepinephrine; AVP, arginine vasopressin.

Figure 2

Modulation of renal function after endotoxin (lipopolysaccharide, LPS) challenge in the presence or absence of vasopressors (arginine vasopressin [AVP] and norepinephrine [NE]). Rats (n = 8) were challenged according to the methodology described under Methods section. The bar charts represent analysis (mean ± SD) of A, creatinine clearance (CCr) established by the following formula (UV/P × 1/1440 min); B, urine osmolality; C, fractional sodium excretion (FeNa⁺) established by the following formula (C Na⁺/CCr); D, Electrolyte- free-water clearance (C_H2O) calculated by the following formula (UV × (1 - [Ur_Na]) + [Ur_K]/[Na plasma]). Data are representative of trunk blood and total 18-hr urine from control (saline, white bar), norepinephrine (NE, gray bar), arginine vasopressin (AVP, dark-gray bar), LPS plus saline (black bar), LPS plus NE (horizontal shading gradient bar), and LPS plus AVP (vertical shading gradient bar)-treated rats. The p values are indicative of significant difference(s) vs. control or vs. LPS.

Figure 3

Modulation of renal filtration fraction (FF) and renal blood flow (RBF) after endotoxin (lipopolysaccharide, LPS) challenge in the presence or absence of vasopressors (arginine vasopressin [AVP] and norepinephrine [NE]). The bar charts represent measurements (mean ± SD) of A, FF(%) and B, RBF, calculated as described in “Methods” section. Data are representative of total 18 hr-urine from groups with similar bar assignment (n = 8), as described in Fig 2. The p values are indicative of significant difference(s) vs. control.

Figure 4

Modulation of urinary biomarkers after endotoxin (lipopolysaccharide, LPS) challenge in the presence or absence of vasopressors (arginine vasopressin [AVP] and norepinephrine [NE]). The bar charts represent measurements in urine of A, total proteins; B, microalbumin; C, kidney injury molecule-1 (Kim-1) (log scale on y-axis) (mean ± SD); D, Clara cell protein (CC-16) (median, 25th—75th percentiles). Data are representative of total 18-hr urine from groups with similar bar assignment (n = 8), as described in Fig 2. The p values are indicative of significant difference(s) vs. control or vs. LPS.

Figure 5

Modulation of renal aquaporin (AQP)-2 apical membrane translocation, mRNA expression and urinary release after endotoxin (lipopolysaccharide, LPS) challenge in the presence or absence of vasopressors (arginine vasopressin [AVP] and norepinephrine [NE]). A, AQP-2 expression in kidney inner medulla (green fluorescence) with nuclear To-Pro-3 contrast (red fluorescence) (low magnification, 100×, bar represents 200 μm). The white-lined square delineates the region of interest for subsequent laser confocal double labeling. B, Internal medulla collecting ducts (IMCD) AQP-2 and lectin double-labeled confocal image analysis with pixel fluorograms. The left portion represents typical fluorograms obtained from the medulla of control, AVP, and LPS plus AVP-treated rats (respectively from top to bottom). Dot fluorograms were obtained by plotting AQP-2 (FITC pixel values) towards the horizontal axis and lectin from Dolichos biflorus (TRITC pixel values) towards the vertical axis. Quadrant markers were placed forming background (lower left), lectin-only (upper left), AQP-2-only (lower right), and overlapping lectin-AQP-2 areas (upper right). The right portion represents typical merged images consistent with fluorogram quantitative analysis. The overlapping signals of green-labeled AQP-2 and red-labeled lectin from Dolichos biflorus appear as yellow (magnification: 630×; scale bar represents 5 μm). C, Quantitation of AQP-2 apical membrane expression in IMCD as described above. D, Urinary release of nonglycosylated AQP-2: representative duplicates of both glycosylated and nonglycosylated (ng) forms run on Western blots are shown, but only ng forms were scan-analyzed. E, Real-time polymerase chain reaction quantification normalized over the housekeeping gene 18S. For all relevant panels, bar assignment (n = 8) is similar to that described in Fig 2. The p values are indicative of significant difference(s) vs. control or vs. LPS.

Figure 6

Modulation of renal V2 receptor (V2R) protein and mRNA expression after endotoxin (lipopolysaccharide, LPS) challenge in the presence or absence of vasopressors (arginine vasopressin [AVP] and norepinephrine [NE]) The bar charts represent quantification (mean ± SD) of A, V2R protein membrane and B, real-time polymerase chain reaction of V2R mRNA expression normalized over the housekeeping gene 18S. Data are representative of kidney medulla with similar bar assignment (n = 8) as that shown in Fig 2. The p values are indicative of significant difference(s) vs. control or vs. LPS.

Figure 7

Effect of V2R antagonist (V2Ra) SR-121463B on lipopolysaccharide (LPS)-induced renal dysfunction with or without arginine vasopressin (AVP) infusion. The bar charts represent analysis (mean ± SD) of A, creatinine clearance (CCr) established by the formula (UV/P × 1/1440 min); B, urine osmolality; C, fractional sodium excretion (FeNa⁺) established by the formula (C Na⁺/CCr); D, Electrolyte free-water clearance (C_H2O) calculated by the formula (UV × (1 - [Ur_Na]) + [Ur_K]/[Na plasma]). Data are representative of trunk blood and total 18-hr urine from (left to right bar) control (saline), AVP and V2Ra (SR-121463B), LPS plus AVP, and LPS plus AVP and V2Ra (SR-121463B)-treated rats (n = 8). The p values are indicative of significant difference(s) vs. control or respective group without V2Ra.