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Sara Rees Wararat Kittikulsuth Karl Roos Kevin A. Strait Alfred Van Hoek Donald E. Kohan 《Journal of the American Society of Nephrology : JASN》2014,25(2):232-237
cAMP is an important mediator of cystogenesis in polycystic kidney disease (PKD). Several adenylyl cyclase (AC) isoforms could mediate cAMP accumulation in PKD, and identification of a specific pathogenic AC isoform is of therapeutic interest. We investigated the role of AC6 in a mouse model of PKD that is homozygous for the loxP-flanked PKD1 gene and heterozygous for an aquaporin-2-Cre recombinase transgene to achieve collecting duct-specific gene targeting. Collecting duct-specific knockout of polycystin-1 caused massive renal cyst formation, kidney enlargement, and severe kidney failure, with a mean survival time of 2 months. In contrast, coincident collecting duct-specific knockout of polycystin-1 and AC6 (also homozygous for the floxed ADCY6 gene) markedly decreased kidney size and cystogenesis, improved renal function, reduced activation of the B-Raf/ERK/MEK pathway, and greatly increased survival. Absence of collecting duct AC6 did not alter urinary cAMP excretion or kidney cAMP concentration. In conclusion, AC6 is a key mediator of cyst formation and renal injury in a model of PKD.Polycystic kidney disease (PKD) is the fourth leading cause of ESRD in the United States. Renal cyst development and expansion in PKD critically depend on vasopressin (AVP).1 Crossing Brattleboro rats, which have no AVP, with autosomal recessive PKD rats markedly inhibits cystogenesis, whereas an AVP analog restores the cystic phenotype.2 The recent Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes trial found that the AVP V2 receptor antagonist tolvaptan slowed the increase in kidney volume and the decline in kidney function in PKD patients over a 3-year period.3 The cystic effect of AVP is likely caused in large part by stimulation of cAMP.1 In cells from PKD kidneys, cAMP agonism stimulates cell growth, whereas in normal cells, cAMP inhibits cell growth.1 The mechanism of cAMP-stimulated cell proliferation has been largely ascribed to protein kinase A activation of the B-Raf/mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway.1,4 Intracellular Ca2+ may be important in determining the effect of cAMP on cell proliferation: under normal conditions, Akt, a serine-threonine protein kinase, inhibits B-Raf (another serine-threonine protein kinase); however, in PKD cells, intracellular Ca2+ is reduced, which decreases Akt activity, permitting cAMP activation of B-Raf.1 In addition, cyst fluid secretion is driven by chloride transport stimulated by cAMP.1,4 Thus, cAMP is a key factor in cyst formation and enlargement, and AVP is important in driving cAMP formation.The renal collecting duct (CD) is the major source of cysts in humans and animal models of autosomal dominant PKD and autosomal recessive PKD.1 Given that cAMP plays a central role in the pathogenesis of PKD, it would be important to define which adenylyl cyclase (AC) isoforms are involved in AVP-mediated cyst formation in the CD. The CD contains intercalated and principal cells. Only principal cells give rise to cysts in mouse models of CD PKD1 deficiency, and only AC3, AC4, and AC6 are expressed in mouse principal cells.5 It is unknown which of these three AC isoforms is involved in AVP-stimulated cyst formation in the CD; however, AC3 and/or AC6 may be particularly important, because their expression has been localized to primary cilia (albeit in nonprincipal cells6,7), the cellular organelle found to be critically important in controlling cyst development.8To begin to evaluate the role of individual AC isoforms in PKD renal disease, we have now studied mouse models of polycystin-1 deficiency with or without AC6 deficiency. Given that no specific AC isoform inhibitors exist (although this area represents an active area of drug development9), a genetic engineering approach was used. We previously reported a mouse model of PKD by selectively deleting the Pkd1 gene in CD principal cells.10,11 In this model, mice containing loxP sites within introns 1 and 4 of the Pkd1 gene are bred with mice transgenic for the aquaporin-2 promoter driving expression of Cre recombinase (AQP2-Cre); the AQP2 promoter is expressed in the kidney only within principal cells. In the current study, we found that mice with homozygous Pkd1 gene disruption in the CD (PKD knockout [KO]) have multiple large cysts and markedly enlarged kidneys at 33 days of age (Figure 1A). The mean survival was 59±6 days (Figure 1B), and BUN, used as an indicator of renal function, was greatly elevated (135±8 mg/dl) (Figure 1C). Thus, mice with homozygous Pkd1 gene disruption in the CD had rapid cyst progression, marked renal failure, and early mortality.Open in a separate windowFigure 1.AC6 KO improves survival and lessens kidney disease in PKD KO mice. Coincident AC6 KO reduced kidney and cyst size. A shows representative images from 15 different kidneys of each genotype. B shows activated caspase 3 (apoptosis) or proliferating cell nuclear antigen (PCNA; proliferation), both of which are green, and AQP2 (red; to help show cyst walls, although not all cyst walls are red) staining in representative kidney sections from five different kidneys of each genotype. C shows mouse survival probability (all animals are PKD KO; legend shows AC6 genotype; n=26–52 per data point). D shows BUN at 33 days of age (n=15–27). *P<0.05 versus PKD KO alone.To examine the role of AC6 in PKD, mice were generated with targeted homozygous disruption of both the Pkd1 and adenylyl cyclase 6 (ADCY6) genes. Mice with loxP-flanked exons 3–12 of the ADCY6 gene, which encode the first catalytic domain, were used (we previously reported CD-specific KO of AC6; these mice have a mild urinary concentrating defect and normal renal histology at 1 year of age12). A key feature of these double KO mice (PKD/AC6 KO) is that the Pkd1 and ADCY6 genes are deleted in the same cells (i.e., whenever Cre is expressed); regardless of whether 100% of principal cells are affected, both target genes have an extremely high likelihood of undergoing recombination. Notably, kidney polycystin-1 mRNA levels were similar between PKD KO and PKD/AC6 KO mice (Figure 1, Figure 1). PKD/AC6 KO mice manifested greatly increased survival compared with PKD KO mice (Figure 1). When PKD/AC6 KO mice died, the average age of death was 209±11 days; however, approximately one third of mice were alive at 1 year. The increased survival was associated with markedly reduced BUN (36±2 mg/dl) compared with PKD KO animals (Figure 1). Mice homozygous for PKD KO but heterozygous for AC6 KO had an intermediate survival time (Figure 1).
Open in a separate windowAll animals were euthanized at 33±2 days of age. ΔCT, threshold cycle; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.aP<0.05 versus PKD KO alone.Western analysis of cAMP-regulated signaling pathways involved in PKD disease progression was performed on PKD KO and PKD/AC6 KO mouse kidneys (Figure 2). PKD/AC6 KO mice at 33 days of age had significantly less expression of B-Raf, ERK1/2, phospho-ERK1/2, MEK1/2, and phopho-MEK1/2 compared with PKD KO mice, which is consistent with the notion that AC6-derived cAMP is involved in cell signaling processes involved in cyst formation and growth.Open in a separate windowFigure 2.AC6 KO reduces expression of B-Raf/ERK/MEK in PKD KO mice. A shows Western analysis for B-Raf, phospho–B-Raf, ERK1/2, phospho-ERK1/2, MEK1/2, phosphor-MEK1/2, and β-actin in whole kidneys from 33-day-old PKD KO and PKD/AC6 KO mice (n=6 per group). WT, one wild-type mouse lane. B shows densitometry analysis, normalizing to β-actin, for each blot. *P<0.05 versus PKD KO.Urine volume was similar between PKD1 KO (0.14±0.2 ml/d) and PKD1/AC6 KO (0.15±0.2 ml/d) mice at 32 days of age. Urine cAMP concentration was similar between the two groups (37±8 pmol/ml in PKD KO and 39±7 pmol/ml in PKD/AC6 KO), whereas urinary cAMP excretion was also unchanged (0.51±0.18 pmol/d in PKD KO and 0.58±0.11 pmol/d in PKD/AC6 KO). Although it was not possible to isolate CD in appreciable amounts from diseased kidneys, total kidney cAMP concentration was determined. No difference was detected in renal cAMP concentration between PKD1 KO (222±37 pmol cAMP/mg total protein, n=5 mice, duplicate measurements in each mouse) and PKD1/AC6 KO (182±41 pmol cAMP/mg total protein, n=6 mice, duplicate measurements in each mouse) mice at 32 days of age. Thus, within our ability to detect changes, AC6 KO in the setting of PKD1 KO did not appreciably alter total urinary cAMP excretion or renal cAMP concentration.Deficiency of a given adenylyl cyclase isoform being protective against PKD renal injury is not necessarily a given. Previous studies suggest that subcellular cAMP distribution within discrete microdomains is of importance.13,14 These microdomains typically contain, among other things, G protein–coupled receptors, adenylyl cyclases, cAMP-activated kinases, phosphodiesterases, and cytoskeletal anchoring proteins.13,14 Thus, a given adenylyl cyclase isoform has the potential to uniquely affect cell function, including potentially modulating cyst formation and growth. Another point is that a given agonist may activate more than one adenylyl cyclase isoform, resulting in differing biologic effects. For example, CD AC6 deficiency prevents AVP-stimulated epithelial Na channel activity in the CD15; however, CD water reabsorption is only mildly impaired by CD AC6 absence.12 Although these studies were conducted under different conditions (epithelial Na channel activity was studied in vitro, and water reabsorption was studied in vivo), they raise the possibility that AVP might exert different effects on the CD depending on which adenylyl cyclase isoforms are activated. It would seem most likely that AVP binding per se is not the determinant of which adenylyl cyclase isoforms are activated, but rather, other cellular modulatory systems are involved. The nature of such adenylyl cyclase regulatory systems is unknown; however, it does provide a basis for postulating that AVP activates more than one adenylyl cyclase isoform, but not all of these isoforms are necessarily involved in AVP-induced cyst formation. In a larger context, one must consider that other agonists that increase CD cAMP could also be involved in cyst formation in the setting of PKD and that one or more adenylyl cyclases may be specifically involved.The findings in the current study may well be relevant to human PKD. A recent study found that, although cAMP was elevated in PKD cells obtained from human kidneys (compared with normal human kidney [NHK] cells), AVP causes a greater increase in NHK cell cAMP,16 suggesting that the total amount of cellular cAMP is less important than its localized effects within the cell. Inhibition of calmodulin, which reduces AC6 and AC3 activity, decreased AVP-induced cAMP in PKD but not NHK cells. Targeting AC3 might provide insights, although it should be noted that mice with global AC3 deletion have markedly reduced GFR.17 In contrast, mice with global AC6 deletion seem to develop and grow normally, albeit with some decrease in urine concentrating ability.18These findings raise the possibility of targeting AC6 in PKD as a therapeutic option. Although AVP receptor antagonism slows PKD progression, it is associated with a significant discontinuation rate because of substantially increased urine output.3 In addition, AC6 is very proximal in the cystogenesis signaling system; hence, its blockade could have multiple effects on downstream mediators. Currently, inhibitors of specific AC isoforms do not exist; however, this area remains an area of active drug development.9,19,20 Even if targeting AC6 per se is ultimately not a viable therapeutic option, continued studies examining the signaling pathways that AC6 specifically regulates (which could be done with our mice) may lead to new insights into potential therapeutic targets in PKD. 相似文献
Table 1.
Effect of AC6 KO on kidney parameters in PKD KO mice| Parameter | PKD KO | PKD/AC6 KO |
|---|---|---|
| Body weight (g) | 6.8±0.6 (n=27) | 11.0±0.9a (n=15) |
| Kidney weight (g) | 0.6±0.1 (n=27) | 0.3±0.1a (n=15) |
| Kidney/body weight (%) | 8±2 (n=27) | 3±1a (n=15) |
| Kidney area (mm3) | 791±76 (n=27) | 419±80a (n=15) |
| Cysts/kidney section | 161±35 (n=12) | 78±14a (n=12) |
| Cyst size (mm2) | 1.18±0.27 (n=12) | 0.46±0.10a (n=12) |
| Kidney polycystin-1 mRNA (ΔCT PKD1-GAPDH) | 8.24±0.10 (n=5) | 8.69±0.33 (n=5) |
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Sylvie Opatrná Wararat Chiangjong Marie Korabečná Kitisak Sintiprungrat Supachok Sinchaikul Shui-Tein Chen Martin Matějovič Visith Thongboonkerd 《Clinical biochemistry》2010,43(1-2):142-149
ObjectivesCommon treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage.Design and methodsWe performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 μg proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain.ResultsThe normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed.ConclusionsOur data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD. 相似文献
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Poolsup N Suthisisang C Channark P Kittikulsuth W 《The Annals of pharmacotherapy》2005,39(6):1080-1087
OBJECTIVE: To investigate the structural and symptomatic efficacy and safety of glucosamine in knee osteoarthritis (OA). DATA SOURCES: Clinical trials of glucosamine were identified through electronic searches (MEDLINE, EMBASE, BIOSIS, EMB review, the Cochrane Library) using the key words glucosamine, osteoarthritis, degenerative joint disease, degenerative arthritis, osteoarthrosis, gonarthrosis, knee, disease progression, and clinical trial. The bibliographic databases were searched from their respective inception dates to August 2004. We also hand-searched reference lists of relevant articles. STUDY SELECTION AND DATA EXTRACTION: Studies were included if they were double-blind, randomized, controlled trials that evaluated oral glucosamine long-term treatment in knee OA; lasting at least one year; and reporting as outcome measures the symptom severity and disease progression as assessed by joint space narrowing. Two authors interpreted data independently. Disagreements were resolved through discussion. DATA SYNTHESIS: Glucosamine sulfate was more effective than placebo in delaying structural progression in knee OA. The risk of disease progression was reduced by 54% (pooled RR 0.46; 95% CI 0.28 to 0.73; p = 0.0011). The number-needed-to-treat was 9 (95% CI 6 to 20). The pooled effect sizes for pain reduction and improvement in physical function were 0.41 (95% CI 0.21 to 0.60; p < 0.0001) and 0.46 (95% CI 0.27 to 0.66; p < 0.0001), respectively, in favor of glucosamine sulfate. Glucosamine sulfate caused no more adverse effects than placebo. CONCLUSIONS: The available evidence suggests that glucosamine sulfate may be effective and safe in delaying the progression and improving the symptoms of knee OA. Due to the sparse data on structural efficacy and safety, further studies are warranted. 相似文献
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Intestinal parasitic infections and risk factors among Myanmar migrant workers in northeast Thailand
Nathkapach Rattanapitoon Tongjit Thanchomnang Wararat Sangwalee 《Asian Pacific journal of tropical medicine》2021,(1):17-26
Objective: To determine the prevalence and associated factors of intestinal parasitic infections in migrant workers in Nakhon Ratchasima Province, Northeast Thailand. Methods: A cross-sectional study was conducted from August 2017 to July 2018 in 600 Myanmar migrant workers. Questionnaires were employed for collecting the demographic data of participants. Stool samples were collected and examined using the formalinether concentration technique. Risk factors for intestinal parasitic infections were determined using multiple logistic regressions analyses. Results: The overall infection rate of intestinal parasitic infections was 27.67%(166/600). Among the intestinal helminthes observed, hookworm was most abundant(8.67%) followed by Trichuris trichiura(8.50%), Opisthorchis viverrini(4.17%), Ascaris lumbricoides(1.50%), Strogyloides stercoralis(1.17%) and Hymenolepis nana(0.5%). Meanwhile, Entamoeba coli was the most prevalent intestinal protozoa(4.33%, 26/600) followed by Endolimax nana(1.33%), Entamoeba histolytica complex(1.17%), Blastocystis sp.(1.0%) and Giardia duodenalis(0.17%). The study found significant associations between gender and Strogyloides stercoralis infection(ORadj=5.61, 95% CI=1.18–26.70, P=0.03), workers aged 30 years old were likely to have a lower risk of the T. trichiura infection(ORadj=0.45, 95% CI= 0.23–0.89). Moreover, the history of consuming raw or undercooked cyprinoid fish was a risk factor of Opisthorchis viverrini infection(ORadj=2.82, 95% CI=1.22–6.49, P=0.015). Conclusions: There remains a high prevalence of intestinal parasitic infections among Myanmar migrant workers in the study area and therefore health screenings for all migrant workers in Thailand are required. 相似文献
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Maxime G. Blanchard Wararat Kittikulsuth Anil V. Nair Jeroen H.F. de Baaij Femke Latta Jonathan R. Genzen Donald E. Kohan René J.M. Bindels Joost G.J. Hoenderop 《Journal of the American Society of Nephrology : JASN》2016,27(3):804-813
The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg2+ channels participate in transcellular Mg2+ transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg2+ reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na+/Cl− cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg2+ reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal—and low—Mg2+ diet. Increased urinary Mg2+ wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg2+ concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg2+ diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels. 相似文献
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We hypothesized that angiotensin (Ang) II hypertensive rats have impaired natriuresis after renal medullary endothelin (ET) B receptor stimulation that would be more evident in male versus female rats. Acute intramedullary infusion of the ET(B) agonist sarafotoxin 6c in normotensive male rats increased sodium excretion from 0.51±0.11 μmol/min during baseline to 1.64±0.19 μmol/min (P<0.05) after S6c. After 2 weeks of Ang II infusion (260 ng/kg per minute SC), male rats had an attenuated natriuretic response to S6c of 0.62±0.16 μmol/min during baseline versus 0.95±0.07 μmol/min after S6c. In contrast, ET(B)-dependent natriuresis was similar in female hypertensive rats (0.48±0.07 versus 1.5±0.18 μmol/min; P<0.05) compared with normotensive controls (1.05±0.07 versus 2.14±0.24 μmol/min; P<0.05). Because ET(A) receptors also mediate natriuresis in normotensive female rats, we examined ET(A) receptor function in female Ang II hypertensive rats. Intramedullary infusion of ET-1 increased sodium excretion in both hypertensive and normotensive female rats, which was partially blocked by the ET(A) antagonist BQ-123. Maximum ET(B) receptor binding in inner medullary membrane preparations was comparable between vehicle and Ang II hypertensive females; however, maximum ET(B) binding was significantly lower in male hypertensive rats (1952±251 versus 985±176 fmol/mg; P<0.05). These results indicate that renal ET(B) function is impaired in male Ang II hypertension attributed, at least in part, to a reduced number of ET(B) binding sites. Furthermore, renal ET receptor function is preserved in female rats during chronic Ang II infusion, suggesting that renal ET receptor function could serve to limit hypertension in females compared with males. 相似文献
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Hypertension and chronic kidney disease are more common in men than in premenopausal women at the same age. In animal models, females are relatively protected against genetic or pharmacological procedures that produce high blood pressure and renal injury. Overactivation or dysfunction of the endothelin (ET) system modulates the progression of hypertension or kidney diseases with the ETA receptor primarily mediating vasoconstriction, injury and anti-natriuresis, and ETB receptors having opposite effects. The purpose of this review is to examine the role of the ET system in the kidney with a focus on the inequality between the sexes associated with the susceptibility to and progression of hypertension and kidney diseases. In most animal models, males have higher renal ET-1 mRNA expression, greater ETA-mediated responses, including renal medullary vasoconstriction, and increased renal injury. These differences are reduced following gonadectomy suggesting a role for sex hormones, mainly testosterone. In contrast, females are relatively protected from high blood pressure and kidney damage via increased ETB versus ETA receptor function. Furthermore, ETA receptors may have a favourable effect on sodium excretion and reducing renal damage in females. In human studies, the genetic polymorphisms of the ET system are more associated with hypertension and renal injury in women. However, the knowledge of sex differences in the efficacy or adverse events of ETA antagonists in the treatment of hypertension and kidney disease is poorly described. Increased understanding how the ET system acts differently in the kidneys between sexes, especially with regard to receptor subtype function, could lead to better treatments for hypertension and renal disease.
LINKED ARTICLES
This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1 相似文献9.
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