Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medulla-specific inhibition of class I HDACs in the rat during high-salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide deficiency. Three new inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single-nucleus RNA-sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders, such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high-salt diets, and regulation of homeostatic mechanisms to remain in fluid-electrolyte balance.
Deranged histone deacetylase (HDAC) activity causes uncontrolled proliferation, inflammation, fibrosis, and organ damage. It is unclear whether deranged HDAC activity results in acute kidney injury in the renal hypoperfusion model of bilateral ischemia-reperfusion injury (IRI) and whether in vivo inhibition is an appropriate therapeutic approach to limit injury. Male mice were implanted with intraperitoneal osmotic minipumps containing vehicle, the class I HDAC inhibitor, MS275, or the pan-HDAC inhibitor, trichostatin A (TSA), 3 days before sham/bilateral IRI surgery. Kidney cortical samples were analyzed using histological, immunohistochemical, and Western blotting techniques. HDAC-dependent proliferation rate was measured in immortalized rat epithelial cells and primary mouse or human proximal tubule (PT) cells. There were dynamic changes in cortical HDAC localization and abundance following IRI including a fourfold increase in HDAC4 in the PT. HDAC inhibition resulted in a significantly higher plasma creatinine, increased kidney damage, but reduced interstitial fibrosis compared with vehicle-treated IRI mice. HDAC-inhibited mice had reduced interstitial α-smooth muscle actin, fibronectin expression, and Sirius red-positive area, suggesting that IRI activates HDAC-mediated fibrotic pathways. In vivo proliferation of the kidney epithelium was significantly reduced in TSA-treated, but not MS275-treated, IRI mice, suggesting class II HDACs mediate proliferation. Furthermore, HDAC4 activation increased proliferation of human and mouse PTs. Kidney HDACs are activated during IRI with isoform-specific expression patterns. Our data point to mechanisms whereby IRI activates HDACs resulting in fibrotic pathways but also activation of PT proliferation and repair pathways. This study demonstrates the need to develop isoform-selective HDAC inhibitors for the treatment of renal hypoperfusion-induced injury.
The collecting duct (CD) concentrates the urine, thereby maintaining body water volume and plasma osmolality within a normal range. The endocrine hormone arginine vasopressin acts in the CD to increase water permeability via the vasopressin 2 receptor (V2R)-aquaporin (AQP) axis. Recent studies have suggested that autocrine factors may also contribute to the regulation of CD water permeability. Nitric oxide is produced predominantly by nitric oxide synthase 1 (NOS1) in the CD and acts as a diuretic during salt loading. The present study sought to determine whether CD NOS1 regulates diuresis during changes in hydration status. Male and female control and CD NOS1 knockout (CDNOS1KO) mice were hydrated (5% sucrose water), water deprived, or acutely challenged with the V2R agonist desmopressin. In male mice, water deprivation resulted in decreased urine flow and increased plasma osmolality, copeptin concentration, and kidney AQP2 abundance independent of CD NOS1. In female control mice, water deprivation reduced urine flow, increased plasma osmolality and copeptin, but did not significantly change total AQP2; however, there was increased basolateral AQP3 localization. Surprisingly, female CDNOS1KO mice while on the sucrose water presented with symptoms of dehydration. Fibroblast growth factor 21, an endocrine regulator of sweetness preference, was significantly higher in female CDNOS1KO mice, suggesting that this was reducing their drive to drink the sucrose water. With acute desmopressin challenge, female CDNOS1KO mice failed to appropriately concentrate their urine, resulting in higher plasma osmolality than controls. In conclusion, CD NOS1 plays only a minor role in urine-concentrating mechanisms.
Histone deacetylase enzymes (HDACs) modify transcriptional activity by removing an acetyl group from histone tails, generally resulting in a repression of transcription. Administration of HDAC inhibitors has been shown to reduce inflammation and apoptosis in both cancer and cardiovascular disease models. Cisplatin is a chemotherapy agent; however, due to its accumulation in the kidney, it can be nephrotoxic. Administering cisplatin serves as a model for acute kidney injury in mice. Previous studies with male mice have shown that whole kidney Hdac expression increases 24 and 72 h post a 30 mg/kg cisplatin injection, and inhibiting HDACs may prevent kidney injury. The goals of this study were 1) to determine if in females there is also an increase in Hdac expression following cisplatin treatment, especially in the cortex where cisplatin is known to accumulated in proximal tubules, and 2) to determine if class I HDAC inhibition prevents kidney injury in female mice. We hypothesized that females will receive the same protection from HDAC inhibitors as males. Mice were administered either acetic acid as vehicle or the class I‐selective HDAC inhibitor, MS275, at 20 mg/kg/day, via s.c. osmotic mini pump. Three days later the mice were injected with either saline or cisplatin (15 mg/kg i.p. once), and samples collected 3 days later. Male, vehicle, saline (MVS) injected mice had similar plasma creatinine to male, MS275, saline (MMS) mice (MVS 0.13 ± 0.01 mg/dl, MMS 0.14 ± 0.01 mg/dl) and were significantly lower than male vehicle cisplatin (MVC) and male, MS275, cisplatin (MMC) mice (MMS 0.27 ± 0.07 mg/dl, MMC 0.20 ± 0.05 mg/dl, PMS275= 0.8, Pcisplatin= 0.02, PMxC= 0.6, n=5–7). In females, there were no statistically significant differences among any group (FVS 0.14 ± 0.01, FMS 0.13 ± 0.2, FVC 0.16 ± 0.03, FMC 0.28 ± 0.1 mg/dl, PMS275= 0.4, Pcisplatin= 0.2, PMxC= 0.3, n=4–7). Analysis of qPCR revealed that female mice had higher cortical expression of all Hdacs except Hdac2, and that there was a sex dependent effect of cisplatin on Hdac2, and Hdac5 expression. Immunohistochemistry also confirmed sex‐specific abundance of HDAC2 and HDAC5 in the cortex. In both sexes, cisplatin resulted in kidney injury as assessed by histological analyses, but there was no significant effect of MS275 on the number of dilated tubules, interstitial fibrosis, or loss of proximal tubule brush border. Thus, although there are sex‐dependent differences in cortical kidney HDAC expression and abundance, preventative class I HDAC inhibition did not significantly affect kidney injury, or improve kidney function in our acute nephrotoxic cisplatin model. Future experiments will determine if this is also true at more chronic time points post cisplatin injection, or whether inhibition of other HDAC classes can prevent kidney injury in males or females.Support or Funding InformationSupported by the NIH NIDDK K01DK105038, the UAB‐UCSD O'Brien Center P30 grant (DK 079337), and the UAB Pittman Scholarship to KAH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
We recently reported that there are > 400 proteins in the inner medullary collecting duct that are post‐translationally lysine acetylated (ac‐K). Although historically, ac‐K proteins were thought to be predominantly histone proteins in the nucleus, we now know that all cellular compartments are enriched with ac‐K proteins. Pathway analyses determined that ac‐K proteins are enriched in various physiological processes such as glycolysis and vasopressin‐mediated water reabsorption. We recently identified the basolateral water channel, aquaporin‐3 (AQP3), as undergoing lysine acetylation (AQP3 K282). Thus, the purpose of this study was to determine if ac‐K AQP3 affects water permeability. We developed antibodies to specifically detected ac‐K AQP3 or total AQP3. In both male and female mice, ac‐K AQP3 was expressed in the basolateral membrane of the cortical and outer medullary collecting duct. However, following 24 h of water deprivation, ac‐K AQP3 was also found in the inner medullary collecting duct. Next, we developed AQP3 K point mutation plasmids, with K282Q representing an acetylated mimic, and K282 representing a deacetylated mimic. These constructs were stably expressed in vasopressin‐responsive mouse cortical collecting duct cells (AQP3mpkCCDs). Using the cell volume sensitive dye, calcein, water permeability of these mutant cells was determined following an osmotic stimulus. We found the acetylated mimic (K282Q) had the highest water permeability followed by the AQP3 wild type K282 cells and the deacetylated mimic (K282R). Finally, using CRISPR/CAS we engineered whole body point mutation mice. The K282Q mutant mice and littermate controls (K282) all developed normally. As adults, K282Q mice presented with elevated plasma osmolality (307 ± 2 mOsm/Kg H2O vs 298 ± 4 mOsm/kg H2O, P = 0.04) at baseline. Following 3 days of hydration with 5% sucrose water, K282Q produced twice as much urine as controls during their active period (2.16 ± 0.5 ml/12h vs 0.88 ± 0.2, P = 0.03). Moreover, when challenged with a 2 ml sterile water i.p., K282Q mice excreted majority of the water load within the first 3 h, while controls reached peak urine flow after 3 h. Finally, we quantified AQP2 (the apical water channel) and AQP3 mRNA expression and protein abundance. AQP2, AQP3 and AQP4 mRNA expression was similar between control and mutant mice. Surprisingly, K282Q mutant mice presented with significantly less AQP2 and AQP3 protein abundance compared to controls. Thus, mutating K282 in vivo resulted in a loss of AQP3 (a potentially AQP2) stability, and highlights the importance of this lysine residue. To conclude, lysine acetylation of AQP3 is a novel posttranslational modification that may regulate AQP function and water permeability in the collecting duct. Support or Funding Information K01DK105038, R03DK120503, the University of Alabama at Birmingham Pittman Scholarship, NIH P30 DK074038
The nitric oxide (NO)‐generating enzyme, NO synthase‐1β (NOS1β), is essential for sodium (Na+) homeostasis and blood pressure control. We previously showed that collecting duct principal cell NOS1β is critical for inhibition of the epithelial sodium channel (ENaC) during high Na+ intake. Previous studies on freshly isolated cortical collecting ducts (CCD) demonstrated that exogenous NO promotes basolateral potassium (K+) conductance through basolateral channels, presumably Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16). We, therefore, investigated the effects of NOS1β knockout on Kir4.1/Kir5.1 channel activity. Indeed, in CHO cells overexpressing NOS1β and Kir4.1/Kir5.1, the inhibition of NO signaling decreased channel activity. Male littermate control and principal cell NOS1β knockout mice (CDNOS1KO) on a 7‐day, 4% NaCl diet (HSD) were used to detect changes in basolateral K+ conductance. We previously demonstrated that CDNOS1KO mice have high circulating aldosterone despite a high‐salt diet and appropriately suppressed renin. We observed greater Kir4.1 cortical abundance and significantly greater Kir4.1/Kir5.1 single‐channel activity in the principal cells from CDNOS1KO mice. Moreover, blocking aldosterone action with in vivo spironolactone treatment resulted in lower Kir4.1 abundance and greater plasma K+ in the CDNOS1KO mice compared to controls. Lowering K+ content in the HSD prevented the high aldosterone and greater plasma Na+ of CDNOS1KO mice and normalized Kir4.1 abundance. We conclude that during chronic HSD, lack of NOS1β leads to increased plasma K+, enhanced circulating aldosterone, and activation of ENaC and Kir4.1/Kir5.1 channels. Thus, principal cell NOS1β is required for the regulation of both Na+ and K+ by the kidney.
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