Romero, Michael F

Significance The tiny insect renal (Malpighian) tubule can transport fluid at unparalleled speed, suggesting unique specializations. Here we show that strategic allocation of major intrinsic proteins (MIPs) to specific cells within the tubule allows the separation of metabolically intense active cation transport from passive chloride and water conductance. This specialized renal architecture is general to at least many higher insects, providing a clue to the unique success of the class Insecta in colonizing an extraordinary range of ecological niches.

Hypokalemia increases ammonia excretion and decreases K+excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+channel α-, β-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+excretion.

Nephrolithiasis is one of the most common kidney diseases, with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrated function of the sex-determining region Y protein-interacting protein-1 ( Sip1) gene, an ortholog of human Na+/H+exchanger regulatory factor ( NHERF1), in Drosophila Malpighian tubules and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNA interference Sip1 knockdown in otherwise normal flies, confirming a renal etiology. This phenotype was abolished in rosy mutant flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct biochemical assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to collocate with Na+/H+exchanger isoform 2 (NHE2) and with moesin in stellate cells. Knockdown of NHE2 specifically to the stellate cells also increased renal uric acid stone formation, and so a model was developed in which SIP1 normally regulates NHE2 activity and luminal pH, ultimately leading to uric acid stone formation. Drosophila renal tubules may thus offer a useful model for urate nephrolithiasis.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

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.