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Lithium is widely used in the treatment of bipolar affective disorders, but often causes nephrogenic diabetes insipidus (NDI), a condition characterized by a severe urinary concentrating defect. Lithium-induced NDI is associated with dysregulation of the amiloride-sensitive epithelial sodium channel (ENaC), which is essential for renal sodium reabsorption. Sex hormones have been shown to affect the expression of aquaporin-2 (AQP2) and sodium transporters. Therefore, we evaluated whether tamoxifen (TAM), a selective estrogen receptor modulator (SERM), would affect lithium-induced dysregulation of ENaC subunits and natriuresis. Rats were fed with lithium-containing food for 2 weeks to induce NDI and natriuresis. TAM was administered daily via gastric gavage after 1 week of lithium administration. Lithium treatment alone resulted in increased urinary sodium excretion and significant reduction of βENaC and γENaC at both RNA and protein levels. In addition, the plasma sodium level reduced after lithium treatment. Administration of TAM prevented increased urinary sodium excretion as well as attenuated the downregulation of βENaC and γENaC. Consistent with these findings, immunohistochemistry (IHC) showed stronger labeling of βENaC and γENaC subunits in the apical domain of the collecting duct cells in the cortical tissue of lithium-fed rats treated with TAM. Other major sodium transporters including NaPi-2, NKCC2, Na/K-ATPase, and NHE3, are believed not to have an effect on the increased urinary sodium excretion since their expression increased or was unchanged after treatment with lithium. In conclusion, the results demonstrated that TAM rescued the adverse effects of the lithium-induced increase in fractional excretion of sodium after the establishment of lithium-induced NDI. [ABSTRACT FROM AUTHOR]

Background: Organic cation transporters (OCTs) in the renal proximal tubule are important for the excretion of both exo- and endogenous compounds, and chronic kidney disease (CKD) alter the expression of OCT. Metformin is a well-known substrate for OCT, and recently, we demonstrated that positron emission tomography (PET) with 11C-labelled metformin (C-metformin) is a promising approach to evaluate the function of OCT. The aim of this study is therefore to examine renal pharmacokinetics of C-metformin and expression of OCTs in a transgenic (RenTGF-β1) mouse model of CKD. Methods: Age- and sex-matched RenTGF-β1 (Tg) and wildtype (WT) mice were used (5-8/group). Animals received an iv bolus of C-metformin followed by 90-min dynamic PET and MRI scan. PET data were analysed using a one-tissue compartment model. Renal protein abundance of OCT2 (by Western blot) as well as OCT1, OCT2, and MATE1 messenger RNA (mRNA) (by RT-PCR) was examined. Results: Protein expression of the basolateral uptake transporter OCT2 was 1.5-fold lower in Tg mice compared to WT mice while OCT1 and MATE1 mRNA expression did not differ between the two groups. The influx rate constant of C-metformin in renal cortex ( K) was 2.2-fold lower in transgenic mice whereas the backflux rate constant ( k) was similar in the two groups, consistent with protein expression. Total body clearance (TBC) correlated within each group linearly with K. Conclusions: In conclusion, this study demonstrates that both renal OCT2 expression and C-metformin uptake are reduced in CKD mice. This potentially makes C-metformin valuable as a PET probe to evaluate kidney function. [ABSTRACT FROM AUTHOR]

Aim: The objective was to examine the renal effects of long-term increased angiotensin II and vasopressin plasma levels in early-stage heart failure (HF). We investigated the regulations of the V2 vasopressin receptor, the type 1A angiotensin II receptor, the (pro)renin receptor, and the water channels AQP2, AQP1, AQP3, and AQP4 in the inner medulla of rat kidney. Methods: HF was induced by coronary artery ligation. Sixty-eight rats were allocated to six groups: Sham (N = 11), HF (N = 11), sodium restricted sham (N = 11), sodium restricted HF (N = 11), sodium restricted sham + DDAVP (N = 12), and sodium restricted HF + DDAVP (N = 12). 1-desamino-8-D-arginine vasopressin (0.5 ng h-1 for 7 days) or vehicle was administered. Pre- and post-treatment echocardiographic evaluation was performed. The rats were sacrificed at day 17 after surgery, before cardiac remodeling in rat is known to be completed. Results: HF rats on standard sodium diet and sodium restriction displayed biochemical markers of HF. These rats developed hyponatremia, hypo-osmolality, and decreased fractional excretion of sodium. Increase of AQP2 and p(Ser256)-AQP2 abundance in all HF groups was blunted compared with control groups even when infused with DDAVP and despite increased vasopressin V2 receptor and Gsα abundance. This was associated with decreased protein abundance of the AT1A receptor in HF groups vs. controls. Conclusion: Early-stage HF is associated with blunted increase in AQP2 and p(Ser256)-AQP2 despite of hyponatremia, hypo-osmolality, and increased inner medullary vasopressin V2 receptor expression. Decreased type 1A angiotensin II receptor abundance likely plays a role in the transduction of these effects. [ABSTRACT FROM AUTHOR]

Bilateral ureteral obstruction (BUO) is associated with renal damage and impaired ability to concentrate urine and is known to induce alterations in an array of kidney proteins. The aim of this study was to identify acute proteomic alterations induced by BUO. Rats were subjected to BUO for 2, 6, or 24 h. Mass spectrometry-based proteomics was performed on the renal inner medulla, and protein changes in the obstructed group were identified. Significant changes were successfully identified for 109 proteins belonging to different biological classes. Interestingly, proteins belonging to the cytoskeleton and proteins related to cytoskeletal regulation were found to be biologically enriched in BUO using online-accessible tools. Western blots confirmed the selected results, demonstrating acute downregulation of proteins belonging to all three cytoskeletal components. The microfilament protein β-actin and the intermediate filament proteins pankeratin and vimentin were all downregulated. β-Tubulin, an important microtubular protein, was found to be significantly downregulated after 24 h. Also, there was significant upregulation of cofilin, an actin-binding protein known to be upregulated in other nephropathy models. Furthermore, both upregulation and downregulation of cytoskeletal motor and regulatory proteins were observed. These findings were confirmed by immunohistochemistry, which clearly showed alterations in labeling in the inner medulla. Interestingly, we were able to confirm selected results in mpkCCD cells exposed to mechanical stretch. Our findings add to the knowledge of BUO-induced acute changes in the renal cytoskeleton and suggest that these molecular changes are partly mediated by increased stretch of the cells during obstruction. [ABSTRACT FROM AUTHOR]

Oxidative stress resulting from unilateral ureteral obstruction (UUO) may be aggravated by increasedproduction of ROS. Previous studies have demonstrated increasedcyclooxygenase (COX)-2 expression in renal medullary interstitialcells (RMICs) in response to UUO. We investigated, both in vivo andin vitro, the role of ROS in the induction of COX-2 in rats subjectedto UUO and in RMICs exposed to oxidative and mechanical stress. Rats subjected to 3-day UUO were treated with two mechanisticallydistinct antioxidants, the NADPH oxidase inhibitor diphenyleneiodonium(DPI) and the complex I inhibitor rotenone (ROT), to interferewith ROS production. We found that UUO-mediated induction ofCOX-2 in the inner medulla was attenuated by both antioxidants. Inaddition, DPI and ROT reduced tubular damage and oxidative stressafter UUO. Moreover, mechanical stretch induced COX-2 and oxidativestress in RMICs. Likewise, RMICs exposed to H2O2 as aninducer of oxidative stress showed increased COX-2 expression andactivity, both of which were reduced by DPI and ROT. Similarly,ROS production, which was increased after exposure of RMICs to H2O2, was also reduced by DPI and ROT. Furthermore, oxidativestress-induced phosphorylation of ERK1/2 and p38 was blocked byboth antioxidants, and inhibition of ERK1/2 and p38 attenuated theinduction of COX-2 in RMICs. Notably, COX-2 inhibitors furtherexacerbated the oxidative stress level in H2O2-exposed RMICs. Weconclude that oxidative stress as a consequence of UUO stimulatesCOX-2 expression through the activation of multiple MAPKs and thatthe induction of COX-2 may exert a cytoprotective function inRMICs. [ABSTRACT FROM AUTHOR]

Background Desmopressin (dDAVP) induces a decrease in immunolabelling of aquaporin (AQP) 4 protein in the terminal inner medulla (IM) of the Brattleboro (BB) rat kidney. It is disputed, however, whether the decreased labelling reflects real down-regulation of protein abundance, or whether it is a result of epitope shielding in the AQP4 protein, preventing binding of the antibody as previously suggested. Furthermore, it is unknown if vasopressin regulates AQP4 in the connecting tubule (CNT) and in the cortical collecting duct (CCD). Using BB rats, we aimed to determine (i) whether the dDAVP-induced decrease in AQP4 labelling in the terminal IM reflects down-regulation in protein abundance and (ii) whether dDAVP increases the AQP4 protein abundance in the CNT and the CCD. Methods BB rats received dDAVP or saline (control) via osmotic minipumps pumps for 6 days. Results Immunolight microscopy revealed strong AQP4 labelling in the initial inner medullary collecting duct (IMCD1), weak labelling in the middle IMCD (IMCD2) and weak/absent labelling in the terminal IMCD (IMCD3) after 6 days of dDAVP administration. AQP3 labelling was similar to that of AQP4. Two-hour administration with dDAVP (previously described BB rats) did not change the labelling pattern of AQP4, suggesting that potential acute phosphorylation did not induce epitope shielding, thereby preventing the binding of the antibody. Conclusions In BB rats, long-term administration with dDAVP (i) increased the AQP4 protein abundance in the IMCD1 and decreased the abundance in the IMCD2 and the IMCD3, and (ii) increased the AQP4 protein abundance in the CNT and the CCD. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin (Ub)-protein ligases (E3s) play a role as regulators of protein trafficking and degradation. We aimed to integrate the profile of E3s in rat kidney and examine the changes in protein abundance of the selected E3s in response to 1-deamino-8-d-arginine vasopressin (dDAVP) stimulation/withdrawal. Sprague-Dawley rats were infused with vehicle (n = 13), dDAVP for 5 days (n = 13), or dDAVP was withdrawn for periods (15 min, 30 min, 1, 3, 6, 12, or 24 h) after 5-day infusion (n = 46). Total RNA was isolated from the inner medulla (IM) for transcriptome analysis. Plasma membrane (PM)- or intracellular vesicle (ICV)-enriched fractions of whole kidney were immunoisolated for liquid chromatography-tandem mass spectrometry analysis. dDAVP infusion for 5 days (D5d) significantly increased urine osmolality, which was maintained during 3-h withdrawal of dDAVP after 5-day infusion (D5d-3h). Consistent with this, aquaporin-2 (AQP2) expression in the PM fractions of D5d and D5d-3h increased, whereas AQP2 expression in the ICV fractions of D5d-3h was further increased, indicating internalization of AQP2. Transcriptome analysis revealed 86 genes of E3s and LC-MS/MS analysis demonstrated 16 proteins of E3s. Among these, seven E3s (BRCA1, UBR4, BRE1B, UHRF1, NEDD4, CUL5, and FBX6) were shared. RT-PCR demonstrated mRNA expressions of the seven identified E3s in the kidney, and immunoblotting demonstrated changes in protein abundance of the selected E3s (BRE1B, NEDD4, and CUL5) in response to dDAVP stimulation/withdrawal or lithium-induced nephrogenic diabetes insipidus. The rate of AQP2 degradation was retarded in mpkCCDc14 cells with small interfering RNA-mediated knockdown of NEDD4 or CUL5. Taken together, identified E3s could be involved in the degradation of proteins associated with vasopressin-induced urine concentration. [ABSTRACT FROM AUTHOR]

Background. Chronic kidney disease (CKD) serves as a risk factor in the development of acute kidney injury (AKI) requiring renal replacement therapy. Furthermore, superimposed AKI on CKD is associated with an increased mortality and risk of progression to end-stage renal disease. We aim to examine whether CKD increases the morbidity and mortality of AKI induced by intestinal ischaemia and reperfusion (I-I/R).Methods. A novel two-stage rat model was developed for CKD induced by 5/6 nephrectomy followed by AKI induced by lethal I-I/R in male rats. All rats initially underwent either 5/6 nephrectomy or sham operation. After 2 weeks, half of each group were subjected to clamping of the superior mesenteric artery for 45 min. The rats were placed in metabolic cages for measurements of water intake and urine output.Results. Fourteen days after 5/6 nephrectomy, polyuria, polydipsia, azotaemia and proteinuria were seen. Furthermore, urinary excretion of neutrophil gelatinase-associated lipocalin was increased in rats with CKD. Earlier death was observed in rats with AKI superimposed on CKD compared with rats with AKI superimposed on normal renal function (the average time to death during reperfusion after intestinal ischaemia: 71.0 ± 7.1 vs 112.4 ± 11.0 min, P < 0.05). Shortly after reperfusion of the intestine, mean arterial pressure dropped to pre-shock levels, which were partly compensated, although to a larger extent, in the sham-operated rats compared with the rats with CKD.Conclusions. The results suggest that even mild CKD has a critical impact on survival during the development of multiple organ failure induced by AKI. [ABSTRACT FROM AUTHOR]

Background: We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats (SHRs) and Sprague-Dawley rats (SDs) one week after tMCAo; (2) to present the practical use of the optical fractionator and 2D nucleator in stereological brain tissue analyses; and (3) to report our experiences with an intraluminal tMCAo model where the occluding filament is advanced 22 mm beyond the carotid bifurcation and the common carotid artery is clamped during tMCAo. Methods: Twenty-three SDs and twenty SHRs were randomized into four groups subjected to 90 minutes tMCAo or sham. BrdU (50 mg/kg) was administered intraperitoneally twice daily on Day 4 to 7 after surgery. On Day 8 all animals were euthanized. NeuN-stained tissue sections were used for brain and infarct volume estimation with the 2D nucleator and Cavalieri principle. Brains were studied for the presence of activated microglia (ED-1) and hippocampal BrdU incorporation using the optical fractionator. Results: We found no significant difference or increase in post-ischemic NPC proliferation between the two strains. However, the response to remote ischemia may differ between SDs and SHRs. In three animals increased post-stroke NPC proliferation was associated with hippocampal ischemic injury. The mean infarct volume was 89.2 ± 76.1 mm3 in SHRs and 16.9 ± 22.7 mm3 in SDs (p < 0.005). Eight out of eleven SHRs had ischemic neocortical damage in contrast to only one out of 12 SDs. We observed involvement of the anterior choroidal and hypothalamic arteries in several animals from both strains and the anterior cerebral artery in two SHRs. Conclusions: We found no evidence of an early hippocampal NPC proliferation one week after tMCAo in both strains. Infarction within the anterior choroidal artery could induce hippocampal ischemia and increase NPC proliferation profoundly. NPC proliferation was not aggravated by the presence of activated microglia. Intraluminal tMCAo in SHRs gave a more reliable infarct with neocortical involvement, but affected territories supplied by the anterior cerebral, anterior choroidal and hypothalamic arteries. [ABSTRACT FROM AUTHOR]

Purpose: Unilateral ureteral obstruction is a common clinical problem that is often associated with a urinary acidification defect caused by decreased net H+ secretion and/or HCO3 − reabsorption. To clarify the molecular mechanisms of these defects we examined expression levels of key acid-base transporters along the renal nephron segments and collecting duct. Materials and Methods: Wistar rats (Møllegard Breeding Centre, Eiby, Denmark) underwent 24-hour unilateral ureteral obstruction, unilateral ureteral obstruction release followed for 4 days or unilateral ureteral obstruction release followed for 4 days plus experimental acidosis induced by NH4Cl oral administration. After sacrifice kidneys were processed for immunoblotting and immunohistochemistry. Results: Semiquantitative immunoblotting revealed that unilateral ureteral obstruction caused significant mean ± SE down-regulation of type 3 Na+/H+ exchanger to 53% ± 9%, electrogenic Na+/HCO3 − cotransporter to 60% ± 9%, type 1 bumetanide sensitive Na+-K+(NH4 +) -2Cl− cotransporter to 64% ± 7%, electroneutral Na+/HCO3 − cotransporter to 43% ± 4% and anion exchanger (pendrin) to 53% ± 10% in the obstructed kidney, which was confirmed by immunohistochemistry. After release of unilateral ureteral obstruction down-regulation of these transporters persisted together with marked down-regulation of H+-adenosine triphosphatase in the obstructed kidney. In rats with unilateral ureteral obstruction release followed for 4 days with experimental acidosis induced by NH4Cl oral administration plasma pH and HCO3 − were dramatically decreased in response to NH4Cl for 2 days compared with those in sham operated rats with acid loading, indicating a defect in H+ excretion and HCO3 − reabsorption after obstruction release. Expression of these transporters did not change in the contralateral nonobstructed kidney of rats with unilateral ureteral obstruction and unilateral ureteral obstruction release followed for 4 days. Conclusions: The expression of renal acid-base transporters is markedly decreased in the obstructed kidney, which may be responsible for the contribution of impaired renal H+ excretion and HCO3 − reabsorption to the urinary acidification defect in response to unilateral ureteral obstruction. [Copyright &y& Elsevier]

Abstract: Animal models of disease are essential for cardiovascular research. However, animal models of right-sided heart failure are few and remain poorly characterized. The aim with this study was to establish a rat model of right-sided heart failure (HF) using pulmonary trunk banding (PTB) and subsequently to characterize the systemic and cardiac changes in this model, including protein expression of SERCA2 and α-sarcomeric actin. Rats underwent banding or sham operation. To evaluate the development of HF over time three groups were included in this study. They were killed 2–3, 5–7 or 16–17 weeks after operation, respectively. PTB rats showed marked hypertrophy of the right ventricle (RV). Catheterization of the RV showed a three- to four-fold increase in right ventricular systolic and diastolic pressures as well as increased dP/dT max and dP/dT min. Plasma analyses revealed increased liver enzymes in most PTB groups and post mortem examination revealed congestion of the liver as well as formation of ascites and hydrothorax in many PTB rats. Immunoblotting of the RV revealed no changes in SERCA2 or α-sarcomeric actin. In conclusion, PTB was an effective method to induce right-sided HF. The presence of HF was confirmed by severe signs of backward failure in conjunction with markedly elevated RV pressures and reduced RV ejection fraction (EF). [Copyright &y& Elsevier]

Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H+]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H+]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 3.4 vs. 62 1.8% of controls (P < 0.05; n = 4) and in inner medullary collecting ducts, 58±1.6 vs. 81 ± 1.3% of controls (P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H+]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus. [ABSTRACT FROM AUTHOR]

The expression of aquaporin-2 (AQP2) is decreased in rats with bilateral ureteral obstruction (BUO) and unilateral ureteral obstruction (UUO). Therefore, the expression of additional renal aquaporins (AQP1-4) and phosphorylated AQP2 (p-AQP2), known to play a role in urinary concentration, was examined in a Wistar rat model with 24 h of UUO. In obstructed kidneys, immunoblotting revealed a significant decrease in the expression of inner medullary AQP2 to 42 ± 4, p-AQP2 to 23 ± 5, AQP3 to 19 ± 6, AQP4 to 11 ± 5, and AQP1 to 64 ± 8% of sham levels. AQP1 expression located in the proximal tubule decreased to 74 ± 4% of sham levels (P < 0.05). Immunocytochemistry confirmed the downregulation of AQP3, AQP4, and p-AQP2. In contralateral nonobstructed kidneys, immunoblotting also revealed significant reductions of AQP1 in the inner medulla, outer medulla, and cortex, whereas expression of AQP2, AQP3, AQP4, and p-AQP2 was unchanged. Furthermore, we collected the urine from both obstructed and nonobstructed kidneys for 2 h, respectively, after 24 h of UUO. Urine collection from obstructed kidneys during 2 h after release of UUO revealed a significant reduction in urine osmolality and solute-free water reabsorption (T[sup c]H[sub 2]O). Moreover, an increase in urine production and T[sup c]H[sub 2]O was observed in contralateral kidneys. To examine whether vasopressin-independent mechanisms are involved in AQP2 regulation, vasopressin-deficient Brattleboro (BB) rats with 24 h of UUO were examined. Immunoblotting revealed downregulation of AQP2, p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation of p-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1) UUO is associated with severe downregulation of AQP2, AQP3, AQP4, and AQP1; thus all of these AQPs may play important roles in the impaired urinary concentrating capacity in the obstructed kidney; 2) the reduced levels of AQP1 in the nonobstructed kidney may... [ABSTRACT FROM AUTHOR]

Background. Acute renal failure (ARF) is a frequent complication of sepsis. Characteristics of ARF in sepsis are impaired urinary concentration, increased natriuresis and decreased glomerular filtration rate (GFR), in which inducible nitric oxide synthase (iNOS) has been revealed to play a role. Aims. We aimed to investigate renal water and sodium excretion and in parallel the segmental regulation of renal AQP2 and major sodium transporters in rats with acute LPS-induced endotoxaemia. Next, we aimed to examine the changes of iNOS expression and activated macrophage infiltration in the kidney and the effects of iNOS inhibition on AQP2 and NKCC2 expression in LPS rats. Methods. Rats were treated with LPS (i.p.) or with LPS + iNOS inhibitor L-NIL, and 6 h later kidneys were subjected to semiquantitative immunoblotting and immunohistochemistry. Results. Polyuria and increased natriuresis were seen 6 h after LPS injection alongside downregulation of both AQP2 and S256-phosphorylated AQP2 in CTX/OSOM and ISOM but not in inner medulla (IM). Thick ascending limb sodium transporters NHE3 and NKCC2 were downregulated in ISOM and NaPi2 was decreased in CTX/OSOM, whereas NCC and ENaC were not consistently downregulated. Immunolabelling intensity of iNOS was increased in vascular structures and transitional epithelium, and an infiltration of activated macrophages was seen in CTX and ISOM. L-NIL co-treatment prevented the downregulation of NKCC2 but not AQP2 in LPS rats. Conclusions. Early downregulation of AQP2 and sodium transporters takes place segmentally in the kidney after LPS administration. In addition, an infiltration of activated macrophages and increased iNOS expression may play a role in the urinary concentrating defect in acute LPS-induced entotoxaemia. [ABSTRACT FROM AUTHOR]