Urine composition in pigs is regulated in both the ureter and the bladder.

The former perception of the urothelium as an impermeable barrier has been revised during the last decade, as increasing evidence of changes in urine composition during its passage of the urinary tract has been presented. Since differences in urothelial permeability between upper and lower urinary tract have been found, our aim is to demonstrate whether changes in urine composition occur during passage through the ureter. We studied consecutive urine samples from both renal pelvises in six pigs and compared them to samples from the bladder and distal ureter. We further sampled urine during storage in the bladder at a fixed volume. All samples were analysed by measuring osmolality and pH, along with the concentration of the following parameters: Na(+), K(+), Cl(-), creatinine, urea. Urine alkalinity increased significantly during passage of the ureter. Creatinine concentration, pH and K(+) increased significantly during the passage from pelvis to the bladder. All other parameters increased non-significantly during the passage to the bladder. The increase in concentration was more pronounced at low concentrations in the pelvis. During storage in the bladder, there was a significant increase in urea concentration. Changes in the composition of urine occur during its passage from the renal pelvis to the bladder and during storage in the bladder. Despite the brief transit time, significant changes in alkalinity were found already during passage through the ureter.

Organ-specific responses during brain death: increased aerobic metabolism in the liver and anaerobic metabolism with decreased perfusion in the kidneys.

Hepatic and renal energy status prior to transplantation correlates with graft survival. However, effects of brain death (BD) on organ-specific energy status are largely unknown. We studied metabolism, perfusion, oxygen consumption, and mitochondrial function in the liver and kidneys following BD. BD was induced in mechanically-ventilated rats, inflating an epidurally-placed Fogarty-catheter, with sham-operated rats as controls. A 9.4T-preclinical MRI system measured hourly oxygen availability (BOLD-related R2*) and perfusion (T1-weighted). After 4 hrs, tissue was collected, mitochondria isolated and assessed with high-resolution respirometry. Quantitative proteomics, qPCR, and biochemistry was performed on stored tissue/plasma. Following BD, the liver increased glycolytic gene expression (Pfk-1) with decreased glycogen stores, while the kidneys increased anaerobic- (Ldha) and decreased gluconeogenic-related gene expression (Pck-1). Hepatic oxygen consumption increased, while renal perfusion decreased. ATP levels dropped in both organs while mitochondrial respiration and complex I/ATP synthase activity were unaffected. In conclusion, the liver responds to increased metabolic demands during BD, enhancing aerobic metabolism with functional mitochondria. The kidneys shift towards anaerobic energy production while renal perfusion decreases. Our findings highlight the need for an organ-specific approach to assess and optimise graft quality prior to transplantation, to optimise hepatic metabolic conditions and improve renal perfusion while supporting cellular detoxification.

Aquaporin expression in the fetal porcine urinary tract changes during gestation.

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

Brain death increases COX-1 and COX-2 expression in the renal medulla in a pig model.

BACKGROUND: Brain death is linked to a systemic inflammatory response that includes prostaglandins and cytokines among its mediators. The levels of cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) affect graft survival, but it remains unknown whether these enzymes are modified during brain death. The aims of this study were to investigate the organ expression of COX and to analyse the cytokine response in the plasma, cerebrospinal fluid (CSF), and organs in a porcine model of intracerebral haemorrhage and brain death. METHODS: Twenty pigs were randomly assigned to either a brain death group or a control group. Brain death was induced by an intracerebral injection of blood, and the animals were observed over the next 8 h. Tissue samples were tested for COX-1, COX-2 messenger RNA (mRNA) expression (heart, lung, and kidney), haeme oxygenase-1 (HO-1) (kidney), interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10, and tumour necrosis factor-α. These cytokines were also measured at eight time points in the plasma and CSF. RESULTS: At the organ level, the levels of COX-1 and COX-2 mRNA expression were increased only in the renal medulla (P = 0.03 and P = 0.02, respectively). The cytokine levels in the tissue, plasma, and CSF revealed no differences between the groups. HO-1 expression decreased (P = 0.0088). CONCLUSION: Brain death increases the expression of COX-1 and COX-2 mRNA in the renal medulla. The release of cytokines into the plasma and CSF did not vary between the groups.

Changes in phosphorylated heat-shock protein 27 in response to acute ureteral obstruction in rats.

AIM: In vivo, renal medullary interstitial cells (RMICs) and collecting duct principal cells (mpkCCD cells) are subjected to inflammatory, oxidative and mechanical stress as a result of unilateral ureteral obstruction (UUO). Because heat-shock protein (HSP) 27 and HSP70 are induced by cellular stresses and play a role in cytoprotection, we hypothesized that HSP27 and HSP70 are increased in rats subjected to acute UUO and in RMICs and mpkCCD cells exposed to inflammatory, oxidative or mechanical stress. METHODS: Rats were subjected to acute UUO for 6 h and 12 h. To examine the expression of HSP27, phosphorylated HSP27 (pHSP27) and HSP70 in response to inflammatory, oxidative and mechanical stress in vitro, we exposed RMICs and mpkCCD cells to interleukin 1ß (IL-1ß), hydrogen peroxide (H2 O2 ), and stretch stimulation over time. RESULTS: The phosphorylated form of HSP27 (pHSP27) was increased in the renal inner medulla (IM) after 6-h and 12-h UUO, while HSP27 and HSP70 were unchanged. Furthermore, after 6 h and 12 h of UUO, the expression of inflammatory (IL-1ß) and oxidative [haem oxygenase 1 (HO-1)] markers was induced. Exposure to inflammatory, oxidative and mechanical stress changed HSP27 and pHSP27 expression in RMICs but not in mpkCCD cells, while HSP70 was not affected by any of the stress conditions. Exposure of RMICs to oxidative and mechanical stress induced HSP27 phosphorylation via a p38-dependent mechanism. CONCLUSION: These data demonstrate that, in response to acute UUO, different forms of cellular stresses modulate HSP27 expression and phosphorylation in RMICs. This may affect the ability of renal cells to mount an effective cytoprotective response.

Cisplatin decreases renal cyclooxygenase-2 expression and activity in rats.

AIM: Cisplatin (CP) induced acute renal failure (ARF) has previously been associated with decreased urinary prostaglandin E2 (PGE2) excretion and reduced aquaporin 2 (AQP2) expression in kidney collecting duct. In this study we examined the expression of cyclooxygenase (COX)-1 and -2 as well as AQP2 and the Na-K-2Cl cotransporter in kidneys from rats with CP induced ARF. METHODS: Rats were treated with either CP or saline and followed for 5 days. Kidneys were dissected into three zones and prepared for immunoblotting, quantitative polymerase chain reaction (QPCR) and immunohistochemistry. Renal content and urinary PGE2 excretion was measured. RESULTS: Cisplatin treatment was associated with polyuria and a significant decreased creatinine clearance. Inner medullary PGE2 content and urinary PGE2 excretion was decreased in CP-treated rats. QPCR and semiquatitative immunoblotting demonstrated that CP treatment reduced COX-2, AQP2 and Na-K-2Cl cotransporter abundance in the different kidney zones, whereas no change in COX-1 was observed. Results were confirmed by immunohistochemistry. CONCLUSION: Cyclooxygenase-2 expression is decreased in inner medulla and cortex. Consistent with this urinary PGE2 levels were reduced. These data suggest that downregulation of COX-2 is responsible for impaired de novo generation of vasodilatory prostaglandins which may play an important role for the CP induced renal vasoconstriction and development of nephropathy.

Cardioprotective effect of L-glutamate in obese type 2 diabetic Zucker fatty rats.

1. Because diabetic hearts have an increased threshold for cardioprotection by ischaemic preconditioning (IPC), we hypothesized that protection by L-glutamate during reperfusion is restricted in Type 2 diabetic hearts. Previously, we found that L-glutamate-mediated postischaemic cardioprotection mimics IPC. 2. Rat hearts were studied in a Langendorff preparation perfused with Krebs'-Henseleit solution and subjected to 40 min global no-flow ischaemia, followed by 120 min reperfusion. L-Glutamate (0, 15 and 30 mmol/L) was added to the perfusate during reperfusion of hearts from non-diabetic (Wistar-Kyoto) and diabetic (Zucker diabetic fatty (ZDF)) rats, studied at 16 weeks of age. The infarct size (IS)/area-at-risk (AAR) ratio was the primary end-point. Expression of L-glutamate excitatory amino acid transporter (EAAT) 1 (mitochondrial) and EAAT3 (sarcolemmal) was determined by quantitative polymerase chain reaction and immunoblotting. 3. The ISS/AAR ratio did not differ between control hearts from Wistar-Kyoto and ZDF rats (0.52 ± 0.03 and 0.51 ± 0.04, respectively; P = 0.90). L-Glutamate (15 mmol/L) significantly reduced the IS/AAR ratio in non-diabetic hearts, but not in diabetic hearts, compared with their respective controls. The higher concentration of L-glutamate (30 mmol/L) reduced infarct size in diabetic hearts to the same degree as in non-diabetic hearts (IS/AAR 0.35 ± 0.03 (P = 0.002) and 0.34 ± 0.03 (P = 0.004), respectively). The mitochondrial L-glutamate transporter EAAT1 was downregulated in hearts from ZDF rats at both the mRNA and protein levels (P < 0.0005 and P < 0.0001, respectively). However, there was no change in EAAT3 expression at the protein level. Myocardial L-glutamate content was increased by 43% in diabetic hearts (P < 0.0001). 4. Hearts from obese diabetic rats have an elevated threshold for metabolic postischaemic cardioprotection by L-glutamate. These findings may reflect underlying mechanisms of inherent resistance against additional cardioprotection in the diabetic heart.

Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to pelvic pressure increase after obstruction.

Prostanoids exert physiological effects on ureteral contractility that may lead to pressure changes and pain during obstruction. In the present study, we examined whether (1) obstruction changes the expression of the two cyclooxygenase (COX) isoforms, COX-1 and COX-2 in human and rat ureters and (2) administration of a selective COX-2 inhibitor influences the pelvic pressure change after experimental ureteral obstruction. Rats were subjected to bilateral ureter obstruction. Ureters were removed and dissected into a proximal dilated and distal non-dilated segment. RNA and protein were extracted and analyzed for cyclooxygenase expression by quantitative polymerase chain reaction and Western blotting. Human ureter samples were obtained from patients undergoing radical nephrectomy. Rat and human ureteral samples were processed for immunohistochemistry. COX-1, but not COX-2 mRNA, was readily detected in the normal rat ureter. COX-2 mRNA and protein expression was increased in the proximal dilated ureter compared to distal non-dilated ureter. This increased COX-2 expression was associated with increased urinary prostaglandin E2 (PGE2) excretion after release of obstruction. Immunohistochemistry showed increased COX-2 labeling in surface epithelium and smooth muscle layers in both rat and human obstructed ureters compared to control ureters. Furthermore, contractile PGE2-EP1 and thromboxane TP receptors were expressed in ureteral smooth muscle. Systemic treatment with the COX-2 selective inhibitor parecoxib (5 mg/kg/day) attenuated the pelvic pressure increase during obstruction. In summary, COX-2 expression is significantly increased in the ureteral wall in response to obstruction in the rat and human ureter and COX-2 activity contributes to increased pelvic pressure after obstruction.

Rapid non-genomic effects of aldosterone on rodent vascular function.

The main role of aldosterone is to maintain body sodium homeostasis by promoting salt reabsorption in the collecting ducts of the kidney. In the cardiovascular system, aldosterone may be harmful in a number of disease states by inducing fibrosis and vascular dysfunction. The present review describes novel results from several laboratories, which show that aldosterone also has beneficial effects in the cardiovascular system by stimulating the production of nitric oxide (NO) from the endothelium. The effect of aldosterone is seen within minutes, and is not inhibited by blockers of gene transcription, thus pointing to a non-genomic mechanism. Furthermore, this potentially beneficial effect is observed at low physiological concentrations of aldosterone (0.1-10 pm). The effect is mediated by the classical mineralocorticoid receptor, and it involves heat shock protein 90, phosphatidylinositol (PI)-3 kinase, protein kinase B, endothelial nitric oxide synthase, and liberation of NO. It is proposed that in healthy individuals with a functioning NO system, the detrimental effects of aldosterone on cardiovascular function are balanced by activation of the potentially beneficial effect of NO. However, in situations with endothelial dysfunction, such as congestive heart failure and hypertension, the negative effects of aldosterone are unopposed and inhibition of aldosterone is warranted.

Rapid inhibition of vasoconstriction in renal afferent arterioles by aldosterone.

Aldosterone has been suggested to elicit vessel contraction via a nongenomic mechanism. We tested this proposal in microdissected, perfused rabbit renal afferent arterioles. Aldosterone had no effect on internal diameter in concentrations from 10(-10) to 10(-5) mol/L, but aldosterone abolished the ability of 100 mmol/L KCl to induce vascular contraction. The inhibitory effect of aldosterone was observed from 1 pmol/L. The inhibitory effect was significant after 5 minutes and maximal after 20 minutes and was fully reversible. Actinomycin D (10(-6) mol/L) prolonged the effect of aldosterone. The effect was abolished by the mineralocorticoid receptor antagonist spironolactone (10(-7) mol/L) but not by the glucocorticoid receptor antagonist mifepristone (10(-6) mol/L). The K+-mediated increase of intracellular calcium concentration in afferent arterioles was not affected by aldosterone. Mineralocorticoid receptor was detected by reverse transcription-polymerase chain reaction and immunohistochemistry in rat renal vasculature and rabbit endothelial cells. Inhibition of phosphatidylinositol (PI)-3 kinase with LY 294002 (3x10(-6) mol/L) restored sensitivity to K+ in the presence of aldosterone, and afferent arterioles were immunopositive for PI-3 kinase subunit p110alpha. Inhibition of NO formation by L-NAME (10(-4) mol/L) or inhibition of soluble guanylyl cyclase with 1H-(1,2,4)Oxadiazolo[4,3-a]quinoxaline-1-one restored K+-induced vasoreactivity in the presence of aldosterone. Similar to aldosterone, the NO donor sodium nitroprusside inhibited K+-induced vascular contraction. Geldanamycin (10(-6) mol/L), an inhibitor of heat shock protein 90, abolished aldosterone-induced vasorelaxation. We conclude that aldosterone inhibits depolarization-induced vasoconstriction in renal afferent arterioles by a rapid nongenomic mechanism that is initiated by mineralocorticoid receptor activation and involves PI-3 kinase, protein kinase B, and heat shock protein 90-mediated stimulation of NO generation.