American Journal of Physiology-renal Physiology最新文献

Patients with chronic kidney disease (CKD) are at increased risk for adverse cardiovascular events. CKD is associated with increases in arterial stiffness, whereas improvements in arterial stiffness correlate with better survival. However, arterial stiffness is increased early in CKD, suggesting that there might be additional factors, unique to kidney disease, that increase arterial stiffness. Lysyl oxidase (LOX) is a key mediator of collagen cross linking and matrix remodeling. LOX is predominantly expressed in the cardiovascular system, and its upregulation has been associated with increased tissue stiffening and extracellular matrix remodeling. Thus, this study was designed to evaluate the role of increased LOX activity in inducing aortic stiffness in CKD and whether β-aminopropionitrile (BAPN), a LOX inhibitor, could prevent aortic stiffness by reducing collagen cross linking. Eight-week-old male C57BL/6 mice were subjected to 5/6 nephrectomy (Nx) or sham surgery. Two weeks after surgery, mice were randomized to BAPN (300 mg/kg/day in water) or vehicle treatment for 4 wk. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound. Aortic levels of LOX were assessed by ELISA, and cross-linked total collagen levels were analyzed by mass spectrometry and Sircol assay. Nx mice showed increased PWV and aortic wall remodeling compared with control mice. Collagen cross linking was increased in parallel with the increases in total collagen in the aorta of Nx mice. In contrast, Nx mice that received BAPN treatment showed decreased cross-linked collagens and PWV compared with that received vehicle treatment. Our results indicated that LOX might be an early and key mediator of aortic stiffness in CKD.NEW & NOTEWORTHY Arterial stiffness in CKD is associated with adverse cardiovascular outcomes. However, the mechanisms underlying increased aortic stiffness in CKD are unclear. Herein, we demonstrated that 1) increased aortic stiffness in CKD is independent of hypertension and calcification and 2) LOX-mediated changes in extracellular matrix are at least in part responsible for increased aortic stiffness in CKD. Prevention of excess LOX may have therapeutic potential in alleviating increased aortic stiffness and improving cardiovascular disease in CKD.

The Na⁺/K⁺/2Cl^- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. cGMP, the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3 ubiquitin ligase families, the cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesized that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, cullin-2, cullin-3, cullin-4A, and cullin-5 were expressed at the protein level, whereas the other members of the cullin family were expressed at the mRNA level, in rat TALs. CRL complex activity is regulated by neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, a process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2 while increasing the expression of cullin-1 by threefold, but this effect was not seen with other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.NEW & NOTEWORTHY The Na⁺/K⁺/2Cl^- cotransporter (NKCC2) reabsorbs NaCl by the thick ascending limb. Nitric oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs by increasing the second messenger cGMP. The present findings indicate that cGMP increases NKCC2 ubiquitination via a cullin-RING ligase complex and regulates in part surface NKCC2 levels. Identifying the E3 ubiquitin ligases that regulate NKCC2 expression and activity may provide new targets for the development of specific loop diuretics.

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor Wilms tumor-1 (WT1), which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to the formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA repair genes that protect the genome. We analyzed transcript levels for a panel of DNA repair genes in embryonic day 17.5 (E17.5) versus adult mouse kidneys and noted seven genes that were increased >20-fold. We then isolated Cited1⁺ NPCs from E17.5 kidneys and found that only one gene, nei-like DNA glycosylase 3 (Neil3), was enriched. RNAscope in situ hybridization of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone versus mature nephron structures. To determine whether Neil3 expression is WT1 dependent, we knocked down Wt1 in Cited1⁺ NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 interacts with the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased twofold in WT1⁺ versus WT1^- cells. We propose that Neil3 is a WT1-dependent DNA repair gene expressed at high levels in Cited1⁺ NPCs, where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.NEW & NOTEWORTHY We studied the molecular events leading to Wilms tumors as a model for the repair of genomic injury. Specifically, we showed that WT1 activates DNA repair gene Neil3 in nephron progenitor cells. However, our observations offer a much broader principle, demonstrating that the embryonic kidney invests in lineage-specific expression of DNA repair enzymes. Thus, it is conceivable that failure of these mechanisms could lead to a variety of "sporadic" congenital renal malformations and human disease.

The Cl^-/[Formula: see text] exchanger pendrin in the kidney maintains acid-base balance and intravascular volume. Pendrin is upregulated in models associated with high circulating aldosterone concentration, such as dietary NaCl restriction or an aldosterone infusion. However, it has not been established if pendrin is similarly regulated by aldosterone with a high-K⁺ diet because the effects of accompanying anions have not been considered. Here, we explored how pendrin is modulated by different dietary potassium salts. Wild-type (WT) and aldosterone synthase (AS) knockout (KO) mice were randomized to control, high-KHCO₃, or high-KCl diets. Dietary KCl and KHCO₃ loading increased aldosterone in WT mice to the same extent but had opposite effects on pendrin abundance. KHCO₃ loading increased pendrin protein and transcript abundance. Conversely, high-KCl diet feeding caused pendrin to decrease within 8 h of switching from the high-KHCO₃ diet, coincident with an increase in plasma Cl^- and a decrease in [Formula: see text]. In contrast, switching the high-KCl diet to the high-KHCO₃ diet caused pendrin to increase in WT mice. Experiments in AS KO mice revealed that aldosterone is necessary to optimally upregulate pendrin protein in response to the high-KHCO₃ diet but not to increase pendrin mRNA. We conclude that pendrin is differentially regulated by different dietary potassium salts and that its regulation is prioritized by the dietary anion, providing a mechanism to prevent metabolic alkalosis with high-K⁺ base diets and safeguard against hyperchloremic acidosis with consumption of high-KCl diets.NEW & NOTEWORTHY Regulation of the Cl^-/[Formula: see text] exchanger pendrin has been suggested to explain the aldosterone paradox. A high-K⁺ diet has been proposed to downregulate a pendrin-mediated K⁺-sparing NaCl reabsorption pathway to maximize urinary K⁺ excretion. Here, we challenged the hypothesis, revealing that the accompanying anion, not K⁺, drives pendrin expression. Pendrin is downregulated with a high-KCl diet, preventing acidosis, and upregulated with an alkaline-rich high-K⁺ diet, preventing metabolic alkalosis. Pendrin regulation is prioritized for acid-base balance.

Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

Sex differences in renal function and blood pressure have been widely described across many species. Blood pressure dips during sleep and peaks in the early morning. Similarly, glomerular filtration rate, filtered electrolyte loads, urine volume, and urinary excretion all exhibit notable diurnal rhythms, which reflect, in part, the regulation of renal transporter proteins by circadian clock genes. That regulation is sexually dimorphic; as such, sex and time of day are not two independent regulators of kidney function and blood pressure. The objective of the present study was to assess the effect of sex and administration time on the natriuretic and diuretic effects of loop, thiazide, and K⁺-sparing diuretics, which are common treatments for hypertension. Loop diuretics inhibit Na⁺-K⁺-2Cl^- cotransporters on the apical membrane of the thick ascending limb, thiazide diuretics inhibit Na⁺-Cl^- cotransporters on the distal convoluted tubule, and K⁺-sparing diuretics inhibit epithelial Na⁺ channels on the connecting tubule and collecting duct. We simulated Na⁺ transporter inhibition using sex- and time-of-day-specific computational models of mouse kidney function. The simulation results highlighted significant sex and time-of-day differences in the drug response. Loop diuretics induced larger natriuretic and diuretic effects during the active phase. The natriuretic and diuretic effects of thiazide diuretics exhibited sex and time-of-day differences, whereas these effects of K⁺-sparing diuretics exhibited a significant time-of-day difference in females only. The kaliuretic effect depended on the type of diuretics and time of administration. The present computational models can be a useful tool in chronotherapy, to tailor drug administration time to match the body's diurnal rhythms to optimize the drug effect.NEW & NOTEWORTHY Sex influences cardiovascular disease, and the timing of onset of acute cardiovascular events exhibits circadian rhythms. Kidney function also exhibits sex differences and circadian rhythms. How do the natriuretic and diuretic effects of diuretics, a common treatment for hypertension that targets the kidneys, differ between the sexes? And how do these effects vary during the day? To answer these questions, we conducted computer simulations to assess the effects of loop, thiazide, and K⁺-sparing diuretics.

Mg²⁺ is essential for many cellular and physiological processes, including muscle contraction, neuronal activity, and metabolism. Consequently, the blood Mg²⁺ concentration is tightly regulated by balanced intestinal Mg²⁺ absorption, renal Mg²⁺ excretion, and Mg²⁺ storage in bone and soft tissues. In recent years, the development of novel transgenic animal models and identification of Mendelian disorders has advanced our current insight in the molecular mechanisms of Mg²⁺ reabsorption in the kidney. In the proximal tubule, Mg²⁺ reabsorption is dependent on paracellular permeability by claudin-2/12. In the thick ascending limb of Henle's loop, the claudin-16/19 complex provides a cation-selective pore for paracellular Mg²⁺ reabsorption. The paracellular Mg²⁺ reabsorption in this segment is regulated by the Ca²⁺-sensing receptor, parathyroid hormone, and mechanistic target of rapamycin (mTOR) signaling. In the distal convoluted tubule, the fine tuning of Mg²⁺ reabsorption takes place by transcellular Mg²⁺ reabsorption via transient receptor potential melastatin-like types 6 and 7 (TRPM6/TRPM7) divalent cation channels. Activity of TRPM6/TRPM7 is dependent on hormonal regulation, metabolic activity, and interacting proteins. Basolateral Mg²⁺ extrusion is still poorly understood but is probably dependent on the Na⁺ gradient. Cyclin M2 and SLC41A3 are the main candidates to act as Na⁺/Mg²⁺ exchangers. Consequently, disturbances of basolateral Na⁺/K⁺ transport indirectly result in impaired renal Mg²⁺ reabsorption in the distal convoluted tubule. Altogether, this review aims to provide an overview of the molecular mechanisms of Mg²⁺ reabsorption in the kidney, specifically focusing on transgenic mouse models and human hereditary diseases.

Patients with cancer represent a unique patient population with increased susceptibility to kidney disease. Drug-induced acute kidney injury (AKI) in patients with cancer is a common problem. Cisplatin is a highly effective treatment used in many solid-organ cancers and causes AKI in 30% of patients, increasing the risk of chronic kidney disease development. Most preclinical cisplatin toxicity studies have been completed in mice without cancer. We believe that the physiology of patients with cancer is not adequately represented in preclinical models, and the objective of this study was to determine how lung cancer will alter the nephrotoxicity of cisplatin. A genetically engineered mouse model and a syngeneic xenograft model of lung cancer were used. Mice were divided into the following four groups: 1) noncancer/vehicle, 2) noncancer/cisplatin, 3) cancer/vehicle, and 4) cancer/cisplatin. Mice were administered cisplatin via intraperitoneal injection once a week for 4 wk. Animals were euthanized 72 h following their final cisplatin injection. Mice with lung cancer had increased renal toxicity, injury, and fibrosis following repeated low doses of cisplatin. In addition, lung cancer alone induced kidney injury and fibrosis in the kidney before cisplatin treatment. In conclusion, this is the first study that we are aware of that assesses the impact of cancer on the kidney in conjunction with the nephrotoxicity of cisplatin. We believe that cancer is providing the first hit to the kidney and the subsequent damage from repeated doses of cisplatin becomes unsurmountable, leading to AKI and progression to chronic kidney disease.NEW & NOTEWORTHY Patients with cancer have impaired kidney function and increased susceptibility to nephrotoxic agents. Cisplatin is a commonly used chemotherapeutic with nephrotoxicity as the dose-limiting side effect. Cisplatin nephrotoxicity is almost exclusively studied in mice without cancer. Our current preclinical models do not adequately represent the complexity of patients with cancer. This study demonstrates increased renal toxicity, injury, and fibrosis in mice with lung cancer, which is exacerbated with cisplatin treatment. These results highlight the necessity of using preclinical models that more accurately capture the altered physiology of patients with cancer treated with cisplatin.

The diverse functions of each nephron segment rely on the coordinated action of specialized cell populations that are uniquely defined by their transcriptional profile. In the collecting duct, there are two critical and distinct cell populations: principal cells and intercalated cells. Principal cells play key roles in the regulation of water, Na⁺, and K⁺, whereas intercalated cells are best known for their role in acid-base homeostasis. Currently, there are no in vitro systems that recapitulate the heterogeneity of the collecting ducts, which limits high-throughput and replicate investigations of genetic and physiological phenomena. Here, we demonstrated that the transcription factor Foxi1 is sufficient to alter the transcriptional identity of M-1 cells, a murine cortical collecting duct cell line. Specifically, overexpression of Foxi1 induces the expression of intercalated cell transcripts including Gpr116, Atp6v1b1, Atp6v1g3, Atp6v0d2, Slc4a9, and Slc26a4. These data indicate that overexpression of Foxi1 differentiates M-1 cells toward a non-A, non-B type intercalated cell phenotype and may provide a novel in vitro tool to study transcriptional regulation and physiological function of the renal collecting duct.NEW & NOTEWORTHY Transfection of M-1 cells with the transcription factor Foxi1 generates cells that express V-ATPase and Gpr116 as well as other genes associated with renal intercalated cells. This straightforward and novel in vitro system could be used to study processes including transcriptional regulation and cell specification and differentiation in renal intercalated cells.