Cellular mechanisms of prostaglandin E2 and vasopressin interactions in the collecting duct

As the final segment of the nephron, the collecting duct is the ultimate regulator of renal salt and water excretion. Balance between intake and renal excretion of salt and water is fine-tuned by the action of several hormones targeted to the collecting duct. Vasopressin is, perhaps, the prototypical example of such a hormone. As total body water decreases and plasma osmolality rises, vasopressin secretion from the posterior pituitary increases [1]. Picomolar concentrations of circulating vasopressin lead to increased water permeability of the apical membrane of the collecting duct cell, resulting in increased water reabsorption and increased total body water [2,3]. There is abundant evidence demonstrating that vasopressin's effect on water reabsorption in the collecting duct is mediated through the classic second messenger, cAMP [3]. V2 selective receptors are linked via a G protein, to stimulation of plasma membrane adenylyl cyclase, resulting in increased cell cyclic AMP levels [4, 5]. The increased cyclic AMP then leads to augmented water permeability of the apical membrane [6, 7].As one might expect with such an important biologic process, other hormones and autocoids provide a counter-regulatory influence to modulate vasopressin mediated increases in osmotic water permeability. There is good evidence that the arachadonic acid metabolite, prostaglandin E2 (PGE2) plays a critical physiologic and pathophysiologic role in inhibiting vasopressin action in the collecting duct [8, 9]. Not only is the collecting duct the major renal site of synthesis for this cyclo-oxygenase product of arachidonic acid but PGE2 production is stimulated by vasopressin itself [10–12]. PGE2 infusion significantly blunts water reabsorption and cycloxygenase inhibition augments vasopressin antidiuresis [9, 13]. Thus, there is good evidence that the autocoid PGE2 plays an important role in regulating vasopressin-stimulated osmotic water flow

Cloning and expression of the rabbit prostaglandin EP2 receptor

BACKGROUND: Prostaglandin E(2) (PGE(2)) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP(2) receptor in regulating fertility, vascular tone and renal function. RESULTS: The full-length rabbit EP(2) receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP(2) exhibited specific [(3)H]PGE(2) binding with a K(d) of 19.1± 1.7 nM. [(3)H]PGE(2) was displaced by unlabeled ligands in the following order: PGE(2)>>PGD(2)=PGF(2α)=iloprost. Binding of [(3)H]PGE(2) 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 EP(2) 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 EP(2) 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 PGE(2) effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules

Mapping the single-cell transcriptomic response of murine diabetic kidney disease to therapies

Diabetic kidney disease (DKD) occurs in ∼40% of patients with diabetes and causes kidney failure, cardiovascular disease, and premature death. We analyzed the response of a murine DKD model to five treatment regimens using single-cell RNA sequencing (scRNA-seq). Our atlas of ∼1 million cells revealed a heterogeneous response of all kidney cell types both to DKD and its treatment. Both monotherapy and combination therapies targeted differing cell types and induced distinct and non-overlapping transcriptional changes. The early effects of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on the S1 segment of the proximal tubule suggest that this drug class induces fasting mimicry and hypoxia responses. Diabetes downregulated the spliceosome regulator serine/arginine-rich splicing factor 7 (Srsf7) in proximal tubule that was specifically rescued by SGLT2i. In vitro proximal tubule knockdown of Srsf7 induced a pro-inflammatory phenotype, implicating alternative splicing as a driver of DKD and suggesting SGLT2i regulation of proximal tubule alternative splicing as a potential mechanism of action for this drug class

Cyclooxygenase-2 expression is associated with the renal macula densa of patients with Bartter-like syndrome

Cyclooxygenase-2 expression is associated with the renal macula densa of patients with Bartter-like syndrome.BackgroundBartter-like syndrome (BLS) is a heterogeneous set of congenital tubular disorders that is associated with significant renal salt and water loss. The syndrome is also marked by increased urinary prostaglandin E2 (PGE2) excretion. In rodents, salt and volume depletion are associated with increased renal macula densa cyclooxygenase-2 (COX-2) expression. The expression of COX-2 in human macula densa has not been demonstrated. The present studies examined whether COX-2 can be detected in macula densa from children with salt-wasting BLS versus control tissues.MethodsThe intrarenal distribution of COX-2 protein and mRNA was analyzed by immunohistochemistry and in situ hybridization in 12 patients with clinically and/or genetically confirmed BLS. Renal tissue rejected for transplantation, from six adult patients not affected by BLS, was also examined.ResultsThe expression of COX-2 immunoreactive protein was observed in cells of the macula densa in 8 out 11 patients with BLS. In situ hybridization confirmed the expression of COX-2 mRNA in the macula densa in 6 out of 10 cases. COX-2 protein was also detected in the macula densa in a patient with congestive heart failure. The expression of COX-2 immunoreactive protein was not observed in cells associated with the macula densa in kidneys from patients without disorders associated with hyper-reninemia.ConclusionThese studies demonstrate that COX-2 may be detected in the macula densa of humans. Since macula densa COX-2 was detected in cases of BLS, renal COX-2 expression may be linked to volume and renin status in humans, as well as in animals

Pathological and Transcriptome Changes in the ReninAAV db/db uNx Model of Advanced Diabetic Kidney Disease Exhibit Features of Human Disease

The ReninAAV db/db uNx model of diabetic kidney disease (DKD) exhibits hallmarks of advanced human disease, including progressive elevations in albuminuria and serum creatinine, loss of glomerular filtration rate, and pathological changes. Microarray analysis of renal transcriptome changes were more similar to human DKD when compared to db/db eNOS−/− model. The model responds to treatment with arterial pressure lowering (lisinopril) or glycemic control (rosiglitazone) at early stages of disease. We hypothesized the ReninAAV db/db uNx model with advanced disease would have residual disease after treatment with lisinopril, rosiglitazone, or combination of both. To test this, ReninAAV db/db uNx mice with advanced disease were treated with lisinopril, rosiglitazone, or combination of both for 10 weeks. All treatment groups showed significant lowering of urinary albumin to creatinine ratio compared to baseline; however, only combination group exhibited lowering of serum creatinine. Treatment improved renal pathological scores compared to baseline values with residual disease evident in all treatment groups when compared to db/m controls. Gene expression analysis by TaqMan supported pathological changes with increased fibrotic and inflammatory markers. The results further validate this model of DKD in which residual disease is present when treated with agents to lower arterial pressure and glycemic control

Circulating αKlotho influences phosphate handling by controlling FGF23 production

The FGF23 coreceptor αKlotho (αKL) is expressed as a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as an endoproteolytic cleavage product of mKL (cKL). Previously, a patient with increased plasma cKL as the result of a translocation [t(9;13)] in the αKLOTHO (KL) gene presented with rickets and a complex endocrine profile, including paradoxically elevated plasma FGF23, despite hypophosphatemia. The goal of this study was to test whether cKL regulates phosphate handling through control of FGF23 expression. To increase cKL levels, mice were treated with an adeno-associated virus producing cKL. The treated groups exhibited dose-dependent hypophosphatemia and hypocalcemia, with markedly elevated FGF23 (38 to 456 fold). The animals also manifested fractures, reduced bone mineral content, expanded growth plates, and severe osteomalacia, with highly increased bone Fgf23 mRNA (>150 fold). cKL activity in vitro was specific for interactions with FGF23 and was FGFR dependent. These results demonstrate that cKL potently stimulates FGF23 production in vivo, which phenocopies the KL translocation patient and metabolic bone syndromes associated with elevated FGF23. These findings have important implications for the regulation of αKL and FGF23 in disorders of phosphate handling and biomineralization

The American Association for the Surgery of Trauma renal injury grading scale: Implications of the 2018 revisions for injury reclassification and predicting bleeding interventions.

BackgroundIn 2018, the American Association for the Surgery of Trauma (AAST) published revisions to the renal injury grading system to reflect the increased reliance on computed tomography scans and non-operative management of high-grade renal trauma (HGRT). We aimed to evaluate how these revisions will change the grading of HGRT and if it outperforms the original 1989 grading in predicting bleeding control interventions.MethodsData on HGRT were collected from 14 Level-1 trauma centers from 2014 to 2017. Patients with initial computed tomography scans were included. Two radiologists reviewed the scans to regrade the injuries according to the 1989 and 2018 AAST grading systems. Descriptive statistics were used to assess grade reclassifications. Mixed-effect multivariable logistic regression was used to measure the predictive ability of each grading system. The areas under the curves were compared.ResultsOf the 322 injuries included, 27.0% were upgraded, 3.4% were downgraded, and 69.5% remained unchanged. Of the injuries graded as III or lower using the 1989 AAST, 33.5% were upgraded to grade IV using the 2018 AAST. Of the grade V injuries, 58.8% were downgraded using the 2018 AAST. There was no statistically significant difference in the overall areas under the curves between the 2018 and 1989 AAST grading system for predicting bleeding interventions (0.72 vs. 0.68, p = 0.34).ConclusionAbout one third of the injuries previously classified as grade III will be upgraded to grade IV using the 2018 AAST, which adds to the heterogeneity of grade IV injuries. Although the 2018 AAST grading provides more anatomic details on injury patterns and includes important radiologic findings, it did not outperform the 1989 AAST grading in predicting bleeding interventions.Level of evidencePrognostic and Epidemiological Study, level III