Nonselective Cyclooxygenase Inhibition Retards Cyst Progression in a Murine Model of Autosomal Dominant Polycystic Kidney Disease.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p<0.05). Basal Cyclooxygenase-1 levels were unchanged in both immunohistochemistry and real-time PCR. Administration of Sulindac to Pkd2WS25/- mice and to control mice for 8 months resulted in reduced kidney weights and volume in cystic mice. Renal function and electrolytes were not significantly different between groups. Conclusion: Thus treatment of a murine model of polycystic kidney disease with Sulindac results in decreased kidney cyst burden. These findings provide additional implications for the use of Cyclooxygenase inhibition as treatment to slow the progression of cyst burden in patients with polycystic kidney disease.

Increased dietary sodium induces COX2 expression by activating NFκB in renal medullary interstitial cells.

High salt diet induces renal medullary cyclooxygenase 2 (COX2) expression. Selective blockade of renal medullary COX2 activity in rats causes salt-sensitive hypertension, suggesting a role for renal medullary COX2 in maintaining systemic sodium balance. The present study characterized the cellular location of COX2 induction in the kidney of mice following high salt diet and examined the role of NFκB in mediating this COX2 induction in response to increased dietary salt. High salt diet (8 % NaCl) for 3 days markedly increased renal medullary COX2 expression in C57Bl/6 J mice. Co-immunofluorescence using a COX2 antibody and antibodies against aquaporin-2, ClC-K, aquaporin-1, and CD31 showed that high salt diet-induced COX2 was selectively expressed in renal medullary interstitial cells. By using NFκB reporter transgenic mice, we observed a sevenfold increase of luciferase activity in the renal medulla of the NFκB-luciferase reporter mice following high salt diet, and a robust induction of enhanced green fluorescent protein (EGFP) expression mainly in renal medullary interstitial cells of the NFκB-EGFP reporter mice following high salt diet. Treating high salt diet-fed C57Bl/6 J mice with selective IκB kinase inhibitor IMD-0354 (8 mg/kg bw) substantially suppressed COX2 induction in renal medulla, and also significantly reduced urinary prostaglandin E2 (PGE2). These data therefore suggest that renal medullary interstitial cell NFκB plays an important role in mediating renal medullary COX2 expression and promoting renal PGE2 synthesis in response to increased dietary sodium.

Fatigue and recovery in 12-hour dayshift hospital nurses.

AIM: The study investigated the status of acute fatigue, chronic fatigue and inter-shift recovery among 12-hour shift nurses and how they differed by organisational and individual factors. BACKGROUND: While the 12-hour shift has been a widely accepted staffing solution in hospitals, the fatigue-recovery process in nurses working 12-hour shifts remains unclear. METHODS: A cross-sectional survey was completed by 130 full-time nurses working 12-hour dayshifts in three hospitals to assess the perceived levels of acute fatigue, chronic fatigue and inter-shift recovery, as well as their associations with selected organisational and individual factors. RESULTS: Nurses experienced a moderate to high level of acute fatigue and moderate levels of chronic fatigue and inter-shift recovery. Fatigue and recovery levels differed by the interaction between hospital and unit after controlling for individual factors. Lack of regular exercise and older age were associated with higher acute fatigue. CONCLUSIONS: An unhealthy fatigue-recovery process was found for nurses working a 12-hour shift during the day. IMPLICATIONS FOR NURSING MANAGEMENT: There appears to be a need to establish fatigue intervention programmes for 12-hour shift nurses in hospitals. Hospital administration, unit managers and staff nurses need to collaborate to achieve a healthy fatigue-recovery balance when implementing 12-hour shifts.

Antihypertensive effects of selective prostaglandin E2 receptor subtype 1 targeting.

Clinical use of prostaglandin synthase-inhibiting NSAIDs is associated with the development of hypertension; however, the cardiovascular effects of antagonists for individual prostaglandin receptors remain uncharacterized. The present studies were aimed at elucidating the role of prostaglandin E2 (PGE2) E-prostanoid receptor subtype 1 (EP1) in regulating blood pressure. Oral administration of the EP1 receptor antagonist SC51322 reduced blood pressure in spontaneously hypertensive rats. To define whether this antihypertensive effect was caused by EP1 receptor inhibition, an EP1-null mouse was generated using a "hit-and-run" strategy that disrupted the gene encoding EP1 but spared expression of protein kinase N (PKN) encoded at the EP1 locus on the antiparallel DNA strand. Selective genetic disruption of the EP1 receptor blunted the acute pressor response to Ang II and reduced chronic Ang II-driven hypertension. SC51322 blunted the constricting effect of Ang II on in vitro-perfused preglomerular renal arterioles and mesenteric arteriolar rings. Similarly, the pressor response to EP1-selective agonists sulprostone and 17-phenyltrinor PGE2 were blunted by SC51322 and in EP1-null mice. These data support the possibility of targeting the EP1 receptor for antihypertensive therapy.

Single amino acid substitution in aquaporin 11 causes renal failure.

A screen of recessive mutations generated by the chemical mutagen n-ethyl-n-nitrosourea (ENU) mapped a new mutant locus (5772SB) termed sudden juvenile death syndrome (sjds) to chromosome 7 in mice. These mutant mice, which exhibit severe proximal tubule injury and formation of giant vacuoles in the renal cortex, die from renal failure, a phenotype that resembles aquaporin 11 (Aqp11) knockout mice. In this report, the ENU-induced single-nucleotide variant (sjds mutation) is identified. To determine whether this variant, which causes an amino acid substitution (Cys227Ser) in the predicted E-loop region of aquaporin 11, is responsible for the sjds lethal renal phenotype, Aqp11-/sjds compound heterozygous mice were generated from Aqp11 +/sjds and Aqp11 +/- intercrosses. The compound heterozygous Aqp11 -/sjds offspring exhibited a lethal renal phenotype (renal failure by 2 wk), similar to the Aqp11 sjds/sjds and Aqp11-/- phenotypes. These results demonstrate that the identified mutation causes renal failure in Aqp11 sjds/sjds mutant mice, providing a model for better understanding of the structure and function of aquaporin 11 in renal physiology.

Characterization of susceptibility of inbred mouse strains to diabetic nephropathy.

Differential susceptibility to diabetic nephropathy has been observed in humans, but it has not been well defined in inbred strains of mice. The present studies characterized the severity of diabetic nephropathy in six inbred mouse strains including C57BL/6J, DBA/2J, FVB/NJ, MRL/MpJ, A/J, and KK/HlJ mice. Diabetes mellitus was induced using low-dose streptozotocin injection. Progression of renal injury was evaluated by serial measurements of urinary albumin excretion, glomerular filtration rate (GFR), and terminal assessment of renal morphology over 25 weeks. Despite comparable levels of hyperglycemia, urinary albumin excretion and renal histopathological changes were dramatically different among strains. DBA/2J and KK/HlJ mice developed significantly more albuminuria than C57BL/6J, MRL/MpJ, and A/J mice. Severe glomerular mesangial expansion, nodular glomerulosclerosis, and arteriolar hyalinosis were observed in diabetic DBA/2J and KK/HlJ mice. Glomerular hyperfiltration was observed in all diabetic strains studied except A/J. The significant decline in GFR was not evident over the 25-week period of study, but diabetic DBA/2J mice exhibited a tendency for GFR to decline. Taken together, these results indicate that differential susceptibility to diabetic nephropathy exists in inbred mice. DBA/2J and KK/HlJ mice are more prone to diabetic nephropathy, whereas the most widely used C57BL/6J mice are relatively resistant to development of diabetic nephropathy.

Molecular cloning and characterization of mouse CYP2J6, an unstable cytochrome P450 isoform.

A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72-94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450nm and the appearance of a 420nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze-thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55-57kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.

Cloning and expression of the rabbit prostaglandin EP2 receptor.

BACKGROUND: Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. RESULTS: The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding with a Kd of 19.1 +/- 1.7 nM. [3H]PGE2 was displaced by unlabeled ligands in the following order: PGE2>>PGD2=PGF2alpha=iloprost. Binding of [3H]PGE2 was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP2 transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP2 mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney. CONCLUSION: EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE2 effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.

Generation of a tenascin-C-CreER2 knockin mouse line for conditional DNA recombination in renal medullary interstitial cells.

Renal medullary interstitial cells (RMIC) are specialized fibroblast-like cells that exert important functions in maintaining body fluid homeostasis and systemic blood pressure. Here, we generated a RMIC specific tenascin-C promoter driven inducible CreER2 knockin mouse line with an EGFP reporter. Similar as endogenous tenascin-C expression, the reporter EGFP expression in the tenascin-C-CreER2(+/-) mice was observed in the inner medulla of the kidney, and co-localized with COX2 but not with AQP2 or AQP1, suggesting selective expression in RMICs. After recombination (tenascin-C-CreER2(+/-)/ROSA26-lacZ(+/-) mice + tamoxifen), ß-gal activity was restricted to the cells in the inner medulla of the kidney, and didn't co-localize with AQP2, consistent with selective Cre recombinase activity in RMICs. Cre activity was not obvious in other major organs or without tamoxifen treatment. This inducible RMIC specific Cre mouse line should therefore provide a novel tool to manipulate genes of interest in RMICs.

Sirt1 activation protects the mouse renal medulla from oxidative injury.

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1+/- mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress-induced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1+/- mice displayed reduced UUO-induced COX2 expression in vivo. Conversely, Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE2 markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.

Axial heterogeneity of vasopressin-receptor subtypes along the human and mouse collecting duct.

Vasopressin and vasopressin antagonists are finding expanded use in mouse models of disease and in clinical medicine. To provide further insight into the physiological role of V1a and V2 vasopressin receptors in the human and mouse kidney, intrarenal localization of the receptors mRNA was determined by in situ hybridization. V2-receptor mRNA was predominantly expressed in the medulla, whereas mRNA for V1a receptors predominated in the cortex. The segmental localization of vasopressin-receptor mRNAs was determined using simultaneous in situ hybridization and immunohistochemistry for segment-specific markers, including aquaporin-2, Dolichos biflorus agglutinin, epithelial Na channels, Tamm Horsfall glycoprotein, and thiazide-sensitive Na(+)-Cl(-) cotransporter. Notably, V1a receptor expression was exclusively expressed in V-ATPase/anion exchanger-1-labeled alpha-intercalated cells of the medullary collecting duct in both mouse and human kidney. In cortical collecting ducts, V1a mRNA was more widespread and detected in both principal and intercalated cells. V2-receptor mRNA is diffusely expressed along the collecting ducts in both mouse and human kidney, with higher expression levels in the medulla. These results demonstrate heterogenous axial expression of both V1a and V2 vasopressin receptors along the human and mouse collecting duct. The restricted expression of V1a-receptor mRNA in intercalated cells suggests a role for this receptor in acid-base balance. These findings further suggest distinct regulation of renal transport function by AVP through V1a and V2 receptors in the cortex vs. the medulla.

A sensitized screen of N-ethyl-N-nitrosourea-mutagenized mice identifies dominant mutants predisposed to diabetic nephropathy.

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2(Akita)). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2(Akita) controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2(Akita) G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2(Akita) G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Differential, inducible gene targeting in renal epithelia, vascular endothelium, and viscera of Mx1Cre mice.

The Cre/loxP transgenic system may be used to achieve temporally and/or spatially regulated gene deletion. The Mx1Cre mouse expresses Cre recombinase under control of the IFN-inducible Mx1 promoter. Mx1Cre mice were crossed with a reporter strain (ROSA26tm1Sor) in which beta-galactosidase activity is expressed only after Cre-mediated recombination to determine the cellular pattern of Cre-mediated genetic recombination in the kidney and other tissues. Widespread recombination was observed in vascular endothelium as well as in the liver and spleen. Recombination was restricted to subsets of stromal cells in uterus, duodenum, colon, aorta, and kidney. In the cortex, chi-galactosidase activity was detected in a subset of tubules and all glomerular cells, including endothelium, mesangium, and podocytes. No chi-galactosidase activity was detected in proximal tubules. Costaining of kidneys with segment-specific markers demonstrated induction of chi-galactosidase activity in collecting duct, with sporadic labeling of the thick ascending limb but no significant labeling of distal convoluted tubules. We conclude that Mx1-driven gene recombination is spatially as well as temporally restricted. The Mx1Cre transgene should prove a useful reagent to achieve temporally regulated recombination in endothelial, glomerular, and distal renal epithelia in mice.

Expression of the prostaglandin F receptor (FP) gene along the mouse genitourinary tract.

PGF(2alpha) is one of the major prostanoids produced by the kidney. The cellular effects of PGF(2alpha) are mediated by a G protein-coupled transmembrane receptor designated the FP receptor. Both in situ hybridization and beta-galactosidase knocked into the endogenous FP locus were used to determine the cellular distribution of the mouse FP receptor. Specific labeling was detected in the kidney, ovary, and uterus. Abundant FP expression in ovarian follicles and uterus is consistent with previous reports of failed parturition in FP-/- mice. In the kidney, coexpression of the mFP mRNA with the thiazide-sensitive cotransporter defined its expression in the distal convoluted tubule (DCT). FP receptor was also present in aquaporin-2-positive cortical collecting ducts (CCD). No FP mRNA was detected in glomeruli, proximal tubules, or thick ascending limbs. Intrarenal expression of the FP receptor in the DCT and CCD suggests an important role for the FP receptor regulating water and solute transport in these segments of the nephron.