Romero, Michael F.

BackgroundProstate stiffness and increased collagen content both associate with the presence of urinary symptoms in men but mechanisms responsible, including impact of age and androgens, are unknown. Dogs develop prostate‐related urinary dysfunction similar to humans, but mechanisms are also unknown. Mice have been used to examine how prostatic collagen accumulation affects voiding but whether mouse prostatic collagen organization resembles human or dog has not been evaluated. Here, we have constructed the first comprehensive, comparative maps of collagen architecture in canine, human, and mouse prostate and test whether canine prostatic collagen content is increased by aging and reduced by castration.MethodsComplete transverse prostate sections were stained with picrosirius red and imaged with confocal microscopy to reveal and compare collagen architecture across species. Canine prostatic collagen fiber length, diameter, and density in prostatic urethral, periurethral, peripheral, and capsular regions were quantified and compared among four experimental groups: young intact, young neutered, old intact, and old neutered dogs.ResultsSurprisingly, the majority of collagen was localized to the prostatic urethra in canine, human, and mouse. In canine and human, capsular regions also featured a dense collagen network but it appeared less dense than around prostatic urethra. Older, intact male canines exhibited overall denser prostate collagen fibers and had thicker capsular fibers than young, intact males. Prostatic glandular regions undergo dramatic atrophy and regression following castration, and our finding of neutered animals having increased collagen fiber density in both periurethral and peripheral regions is consistent with glandular contraction and increased proportion of stroma.ConclusionsCollagen architecture in dog appears similar to that in humans when cross sections are compared side‐by‐side. Canine collagen organization is affected by both age and androgen status, suggesting these factors may contribute to collagen accumulation in some males.

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study’s purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

Key points Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid‐extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals. AbstractNeuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named ‘pHerry’, in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity‐induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+/Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid‐extruding protein when deposited on the plasmamembrane.

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.

Most vertebrates, including cartilaginous fishes, maintain their plasma SO42−concentration ([SO42−]) within a narrow range of 0.2–1 mM. As seawater has a [SO42−] about 40 times higher than that of the plasma, SO42−excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO42−via the kidney. However, little is known about the underlying mechanisms for SO42−transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish ( Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO42−transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO42−transporter, while cmSlc26a6 is an apically located, electrogenic Cl−/SO42−exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO42−was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO42−by the kidney of elephant fish. Possible mechanisms for SO42−secretion in the PII segment are discussed.