Renal Outer Medulla Research Articles

Fumarase Overexpression Abolishes Hypertension Attributable to endothelial NO synthase Haploinsufficiency in Dahl Salt-Sensitive Rats.

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats

BackgroundHuman blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.ObjectiveWe examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.Methods and ResultsSS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.ConclusionsFumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.Support or Funding InformationThis work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell‐autonomous expression of membrane proteins in kidney thick ascending limb epithelium is vasopressin‐dependent

The thick ascending limb (TAL) of the mammalian kidney exhibits functionally and structurally distinct patterns along its course from the border of the inner medulla to its transition to the distal convoluted tubule. While reabsorption of 25% of the filtered load is the principal task shared by all TAL cells, the transport of K+ and the divalent cations Ca++ and Mg++ is subject to axial and cellular heterogeneity and occurs along trans‐ or paracellular pathways. Differential expression of the renal outer medullary K+ channel (ROMK) has been associated with net K+ secretion or reabsorption, and claudin (Cldn) 10 and 16 with selective paracellular cation permeability. Earlier we had detected basolateral vasopressin (AVP) V2 receptor expression and substantial activating responsiveness of transport parameters along TAL upon adding AVP. Here we tested the hypothesis whether AVP modulates cell‐autonomous expression of membrane proteins involved in TAL epithelial ion transport.We analyzed perfusion‐fixed mouse and rat kidneys. Human kidney biopsies were studied for comparison. The effect of AVP was tested in AVP‐deficient Brattleboro rats using V2R‐specific desmopressin (dDAVP; 7 and 72 hours). NKCC2, ROMK, Kir4.1, Cldn‐10 and Cldn‐16 signals were analyzed using sensitive in situ hybridization, IHC, EM, and Western blot (WB).TAL morphological cell heterogeneity was obvious between kidney zones, but not at the cell‐to‐cell level within any zone. Morphologically heterogeneous “smooth” and “rough” patterns of neighboring TAL cells were not discovered, but instead, ROMK und Kir4.1 expression showed conspicuous heterogeneity in a mutually exclusive pattern in tubules of outer medulla and cortex of rodent and human kidneys. Most tight junctions of inner stripe TAL were Cldn‐10 positive. In its outermost layer, Cldn‐10‐ and ‐16 signals were co‐expressed; ROMK‐positive cells displayed Cldn‐10‐positive tight junctions between the adjacent ROMK‐positive cells whereas between ROMK‐negative cells, junctions were Cldn‐16‐positive. 72h dDAVP administration significantly increased the number of ROMK‐positive cells in outer medullary TAL. Kir4.1‐positive cells increased in parallel. Cldn‐10‐positive junctions increased at the expense of Cldn‐16‐positive junctions. WB showed 2.2‐fold increase for ROMK and 1.3‐fold for Kir4.1 (p<.05). Sensitive ISH revealed differential quantities of ROMK mRNA abundances in TAL, with increases in the number of cells expressing ROMK mRNA strongly upon dDAVP.These results demonstrate cell‐autonomous regulation of crucial TAL cell membrane proteins in response to activating stimuli in the absence of major cell morphological changes. Variable expression of ROMK, Kir4.1, and Cldn‐10 and ‐16 signals likely reflected vasopressindependent adaptive features in renal solute handling in the renal outer medulla.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Renal Pathophysiological Processes Induced by an Iodinated Contrast Agent in a Diabetic Rabbit Model Using Intravoxel Incoherent Motion and Blood Oxygenation Level-Dependent Magnetic Resonance Imaging.

ObjectiveTo examine the potential of intravoxel incoherent motion (IVIM) and blood oxygen level-dependent (BOLD) magnetic resonance imaging for detecting renal changes after iodinated contrast-induced acute kidney injury (CI-AKI) development in a diabetic rabbit model.Materials and MethodsSixty-two rabbits were randomized into 2 groups: diabetic rabbits with the contrast agent (DCA) and healthy rabbits with the contrast agent (NCA). In each group, 6 rabbits underwent IVIM and BOLD imaging at 1 hour, 1 day, 2 days, 3 days, and 4 days after an iohexol injection while 5 rabbits were selected to undergo blood and histological examinations at these specific time points. Iohexol was administrated at a dose of 2.5 g I/kg of body weight. Further, the apparent transverse relaxation rate (R2*), average pure molecular diffusion coefficient (D), pseudo-diffusion coefficient (D*), and perfusion fraction (f) were calculated.ResultsThe D and f values of the renal cortex (CO) and outer medulla (OM) were significantly decreased compared to baseline values in the 2 groups 1 day after the iohexol injection (p < 0.05). A marked reduction in the D* values for both the CO and OM was also observed after 1 hour in each group (p < 0.05). In the OM, a persistent elevation of the R2* was detected for 4 days in the DCA group (p < 0.05). Histopathological changes were prominent, and the pathological features of CI-AKI aggravated in the DCA group until day 4. The D, f, and R2* values significantly correlated with the histological damage scores, hypoxia-inducible transcription factor-1α expression scores, and serum creatinine levels.ConclusionA combination of IVIM and BOLD imaging may serve as a noninvasive method for detecting and monitoring CI-AKI in the early stages in the diabetic kidney.

Role of DNA De Novo (De)Methylation in the Kidney in Salt-Induced Hypertension.

Numerous adult diseases involving tissues consisting primarily of nondividing cells are associated with changes in DNA methylation. It suggests a pathophysiological role for de novo methylation or demethylation of DNA, which is catalyzed by DNA methyltransferase 3 and ten-eleven translocases. However, the contribution of DNA de novo (de)methylation to these diseases remains almost completely unproven. Broad changes in DNA methylation occurred within days in the renal outer medulla of Dahl SS rats fed a high-salt diet, a classic model of hypertension. Intrarenal administration of anti-DNA methyltransferase 3a/ten-eleven translocase 3 GapmeRs attenuated high salt-induced hypertension in SS rats. The high-salt diet induced differential expression of 1712 genes in the renal outer medulla. Remarkably, the differential expression of 76% of these genes was prevented by anti-DNA methyltransferase 3a/ten-eleven translocase 3 GapmeRs. The genes differentially expressed in response to the GapmeRs were involved in the regulation of metabolism and inflammation and were significantly enriched for genes showing differential methylation in response to the GapmeRs. These data indicate a significant role of DNA de novo (de)methylation in the kidney in the development of hypertension in SS rats. The findings should help to shift the paradigm of DNA methylation research in diseases involving nondividing cells from correlative analysis to functional and mechanistic studies.

Angiotensin II upregulates CYP4A isoform expression in the rat kidney through angiotensin II type 1 receptor

Angiotensin II (AngII) stimulates the renal production and release of 20-hydroxyeicosatetraenoic acids (20-HETE), which is a major metabolite of arachidonic acid catalyzed by CYP4A isoforms. However, the effects of AngII on CYP4A isoform expression in the kidney and its mechanism remains unclear. To clarify the regulation of CYP4A isoform expression by AngII, we examined the chronic effects of AngII and AngII type 1 receptor (AT1-R) blockade on CYP4A isoform expression. Sprague-Dawley rats were infused with vehicle or AngII for 1 week, and the AngII-infused rats were also treated with or without the AT1-R blocker, candesartan. AngII increased CYP4A isoform protein expression in the renal cortex (CO) and outer medulla (OM) in a dose-dependent manner, and candesartan inhibited the AngII-increased CYP4A expression in a dose-dependent manner. AngII increased the CYP4A isoform mRNA expression in the CO and OM, and candesartan inhibited AngII-increased CYP4A isoform mRNA expression. These results indicated that AngII chronically increased the CYP4A isoform expression in the rat kidney. The AngII-induced CYP4A isoform expression was mediated by AT1-R.

Abstract 054: Tissue-Specific Effects of Targeted Mutation of Mir29b1 in Rats

miR-29 is a master regulator of extracellular matrix genes and is generally considered anti-fibrotic, and it improves nitric oxide (NO) production in human and rat arterioles by targeting Lypla1.Targeted mutation in the Mir29b1 gene exacerbates hypertension in a rat model derived from the Dahl salt-sensitive rat. The kidney, particularly the renal outer medulla, plays a key role in the development of salt-sensitive hypertension. In the present study, we examined the effect of the targeted Mir29b1 mutationon tissue fibrosis and NO levels with a focus on kidney regions. The abundance of miR-29b and the co-transcribed miR-29a was substantially lower in the renal cortex, renal outer medulla, heart and liver in mutant rats. In mutant rats on a 0.4% NaCl diet and prior to the development of overt hypertension, tissue fibrosis was significantly increased in the renal outer medulla, but not in the renal cortex, heart or liver, compared with wild-type littermates. Levels of NO metabolites were significantly lower, and Lypla1 protein abundance significantly higher, in the renal outer medulla, but not in the renal cortex. After 14 days of a 4% NaCl diet, tissue fibrosis remained significantly higher in the renal outer medulla and became higher in the heart of mutant rats compared with wild-type littermates, but not in the renal cortex. 24h urine volume was significantly lower in mutant rats on either the 0.4% or 4% NaCl diet. Microalbuminuria was exacerbated by the 4% NaCl diet, but was not significantly different between mutant rats and wild-type littermates. These findings indicate the effects of miR-29 are tissue-specific. The renal outer medulla might be particularly susceptible to the injurious effects of a miR-29 insufficiency, which might contribute to the development of hypertension in Mir29b1 mutant rats.

Abstract 001: Loss of ET B Receptor Function Activates Inflammasome Signaling Pathways and Dendritic Cells in the Renal Outer Medulla During Type 1 Diabetes

Renal inflammation is a hallmark of diabetic kidney disease. Endothelin-1 (ET-1), a potent vasoactive peptide that acts through two receptors (ET A and ET B ), has been implicated in diabetes and is upregulated in patients with diabetic nephropathy (DN) and in animal models of diabetic kidney injury. ET B receptors are highly expressed in renal outer medulla (OM). ET-1 exerts pro-inflammatory actions in the kidney; however, the mechanisms by which ET-1 mediate these effects are unclear. The present studies were designed to determine the role of the ET B receptor in priming inflammasome signaling pathways, as well as its role in the activation of dendritic cells (DCs), in the renal OM during type 1 diabetes. Diabetes was induced in ET B deficient (ET B def) and transgenic (TG) control rats by i.v. injection of streptozotocin (STZ). 10 wk later, 24-h urine and kidneys were collected for analysis of kidney damage. Diabetic ET B def rats presented exaggerated kidney damage compared to diabetic TG controls, with greater excretion of protein, albumin and ET-1, cortical expression of KIM-1 (respectively: 90±12 vs. 45±6 mg/day, 41±8 vs. 12±4 mg/day, 18±1 vs. 15±1 pg/day, 12±1 vs. 7±1 pg/mg prot; n=4- 6/group, p&lt;0.05), and excessive kidney fibrosis, tubular dilation and cell death. Immunohistochemistry for the DC activation marker CD83 also demonstrated exaggerated activation of DCs in renal OM of diabetic ET B def rats. Expression of inflammasome genes in renal OM was assessed by RT-PCR array. Diabetes led to upregulation of NLRP5 (4-fold increase vs. TG controls; n=3/group; p&lt;0.05) and IL-1β (~3-fold increase vs. TG controls; n=3/group; p&lt;0.05) in OM of ET B def rats. In addition, PSTPIP1, a negative regulator of the inflammasome, was decreased in OM of diabetic ET B def rats vs. TG controls. Together, these results demonstrate that dysfunction of the ET B receptor leads to worsening of diabetic kidney injury and overactivation of the inflammasome and DCs specifically in the renal OM. These results highlight the protective role of ET B receptors against the development of diabetes- induced kidney inflammation and damage. Funded by NIH T32 DK007545 to CDM and P01 HL69999, P01HL136267, AHA SFRN 24450002 and U01HL117684 to DMP and JSP.

Abstract 118: DNA de novo (de)Methylation in the Kidney Contributes to Salt-Induced Hypertension

Numerous adult diseases involving tissues that consist primarily of non-dividing cells are associated with changes in DNA methylation. It suggests a role for de novo methylation or demethylation of DNA, which is catalyzed by DNA methyltransferase 3 (Dnmt3) and ten-eleven translocases (Tet). However, the contribution of DNA de novo (de)methylation to these diseases remains nearly completely unproven. Broad changes in DNA methylation occurred within days in the renal outer medulla of Dahl SS rats fed a high-salt diet, a classic model of hypertension. Intra-renal administration of anti-Dnmt3a/Tet3 GapmeR’s attenuated high salt-induced hypertension in SS rats (mean arterial pressure 153+/-4 vs. 141+/-2 mmHg, n=9-11, p&lt;0.05). The high salt diet induced differential expression of 1,712 genes in the renal outer medulla. Remarkably, the differential expression of 76% of these genes were prevented by anti-Dnmt3a/Tet3 GapmeR’s. The genes differentially expressed in response to the GapmeR’s were involved in the regulation of metabolism and inflammation and were significantly enriched for genes showing differential methylation in response to the GapmeR’s. These data indicate DNA de novo (de)methylation in the kidney contributes to the development of hypertension in SS rats. The findings should help to shift the paradigm of DNA methylation research in diseases involving non-dividing cells from correlative analysis to functional and mechanistic studies.

Abstract 117: Inhibition of DNA Methyltransferase Attenuates Salt-Sensitive Hypertension &amp; Renal Disease in Dahl Salt Sensitive Rats

DNA methylation plays a key role in the regulation of gene expression. In the present study, we investigated the impact of changes in DNA methylation in Dahl Salt Sensitive (SS) rats fed a casein-based AIN-76A chow containing low salt (LS) or high salt (HS). In addition to the hypertension and renal damage which occurs following 14 days of HS feeding, we assessed 5-Methylcytosine levels at single-base resolution by reduced representation bisulfite genome sequencing (RRBS) of DNA from the renal outer medulla of SS rats. Several hundred differentially methylated regions (DMRs) were identified between the SS rats on the LS or HS diet. Approximately a quarter of the DMRs were located in transcriptional start site regions, a third in intragenic regions, and the remainder in intergenic regions. To investigate the functional role of methylation in SS hypertension, we administered decitabine (DNA methyltransferase inhibitor, 5mg/kg, ip) or vehicle at Days 0, 3, 5, and 7 during a 14 day HS period. While there was no difference in MAP during the LS period (127±3 vs. 126±6 mmHg, vehicle vs. decitabine, n=9), at the end of 2 weeks of HS there was a significant increase in MAP in the vehicle SS rats compared to the decitabine-treated rats (163±6 vs 141±4 mmHg, p&lt;0.0001 ). This protective effect in MAP in the decitabine-treated group also exhibit a reduction in albumin excretion rate compared to controls (119±19 vs. 182±24 mg/day, p&lt;0.01 ). Upon analysis of the immune cell infiltration profile, there was a reduction in number of CD45+ total leukocytes, CD11b/c+ monocytes/ macrophages, and CD45R+ B cells in the decitabine-treated animals compared to controls. Decitabine administration also significantly reduced renal histological damage as indicated by the presence of outer medullary protein casts compared to vehicle-treated rats (6.8±1.0% vs. 10.6±1.3%, p &lt;0.05). Lastly, methylation rate was significantly reduced in decitabine-treated rats by 40% compared to the control levels. In summary, the inhibition of methylation by decitabine attenuated MAP and markers of renal damage in response to a HS diet suggesting that DNA methylation could be a potential therapeutic target for the treatment of hypertension and renal damage.

Tissue-specific effects of targeted mutation of Mir29b1 in rats.

BackgroundmiR-29 is a master regulator of extracellular matrix genes, but conflicting data on its anti-fibrotic effect have been reported. miR-29 improves nitric oxide (NO) production in arterioles by targeting Lypla1. Mir29b1 targeted mutation exacerbates hypertension in a model derived from the Dahl salt-sensitive rat. We examined the effect of Mir29b1 mutation on tissue fibrosis and NO levels with a focus on kidney regions.MethodsMir29b1 targeted mutant rats on the genetic background of SS-Chr13BN rats were studied. Masson trichrome staining, molecular and biochemical assays, metabolic cage studies, and bioinformatic analysis of human genomic data were performed.FindingsThe abundance of miR-29b and the co-transcribed miR-29a was substantially lower in mutant rats. Tissue fibrosis was significantly increased in the renal outer medulla, but not in the renal cortex, heart or liver in mutant rats on a 0.4% NaCl diet. Lypla1 protein abundance was significantly higher and NO levels lower in the renal outer medulla, but not in the renal cortex. After 14 days of a 4% NaCl diet, 24 h urine volume and urinary sodium excretion was significantly lower in mutant rats, and tissue fibrosis became higher in the heart. NO levels were lower in the renal outer medulla and heart, but not in the renal cortex. Human miR-29 genes are located in proximity with blood pressure-associated single nucleotide polymorphisms.InterpretationThe renal outer medulla might be particularly susceptible to the injurious effects of a miR-29 insufficiency, which might contribute to the development of hypertension in Mir29b1 mutant rats.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.

Multiparametric magnetic resonance imaging of experimental chronic kidney disease: A quantitative correlation study with histology.

ObjectivesIn human chronic kidney disease (CKD) the extent of renal tubulointerstitial fibrosis correlates with progressive loss of renal function. However, fibrosis can so far only be assessed by histology of kidney biopsies. Magnetic resonance imaging (MRI) can provide information about tissue architecture, but its potential to assess fibrosis and inflammation in diseased kidneys remains poorly defined.Materials and methodsWe evaluated excised kidneys in a murine adenine-induced nephropathy model for CKD by MRI and correlated quantitative MRI parameters (T1, T2, and T2* relaxation times, apparent diffusion coefficient and fractional anisotropy) with histological hallmarks of progressive CKD, including renal fibrosis, inflammation, and microvascular rarefaction. Furthermore, we analyzed the effects of paraformaldehyde fixation on MRI parameters by comparing kidney samples before and after fixation with paraformaldehyde.ResultsIn diseased kidneys T2 and T2* relaxation times, apparent diffusion coefficient and fractional anisotropy in the renal cortex and/or outer medulla were significantly different from those in control kidneys. In particular, T2 relaxation time was the best parameter to distinguish control and CKD groups and correlated very well with the extent of fibrosis, inflammatory infiltrates, tubular dilation, crystal deposition, and loss of peritubular capillaries and normal tubules in the renal cortex and outer medulla. Fixation with paraformaldehyde had no impact on T2 relaxation time and fractional anisotropy, whereas T1 times significantly decreased and T2* times and apparent diffusion coefficients increased in fixed kidney tissue.ConclusionsMRI parameters provide a promising approach to quantitatively assess renal fibrosis and inflammation in CKD. Especially T2 relaxation time correlates well with histological features of CKD and is not influenced by paraformaldehyde fixation of kidney samples. Thus, T2 relaxation time might be a candidate parameter for non-invasive assessment of renal fibrosis in human patients.

Chronic high fat diet disrupts renal molecular clock

Kidney function follows a strong circadian rhythm that is tightly regulated by clock genes. We previously reported that high salt diet induced dyssynchrony in renal clock gene expression in the cortex and medulla. However, whether changes in other dietary factors pose threats to renal circadian rhythms remain largely unknown. The current study was designed to test the hypothesis that high fat diet disrupts renal clock gene expression with 2 mouse models. First, C57Bl/6J male mice (8 weeks old, n=3) were fed normal fat (NF) or high fat (HF) diet for 20 weeks. During the last 2 weeks of the protocol, mice underwent either time restricted feeding, where food access was allowed only during lights‐off, active period (7 pm–7 am), or sham feeding procedure. Restricted feeding did not cause significant changes in body weight or food consumption. Renal cortex was collected in 4‐hour increments throughout a 24‐hour period and clock gene expression measured by qPCR. We found that Per2 expression has a robust circadian rhythm with a peak expression at zeitgeber time (ZT) 13. We did not observe any significant differences among groups. Second, Period2Luciferase (Per2Luc) mouse model was utilized to monitor real‐time molecular clock rhythm. Male and female Per2Luc mice (n=4) were fed NF or HF diet with sham or time restricted feeding. Kidneys were dissected into three parts (cortex, outer and inner medulla) at the end of study and cultured for 3 days to measure bioluminescent rhythms. We found that HF diet lengthened the period of luciferase rhythm by 2 hours (NF: 23.98 hours vs. HF: 26.17 hours, p=0.02) in the cortex. No significant differences were observed in the groups in renal outer medulla or inner medulla. Restricted feeding did not cause any significant changes in any groups. These data suggest that clock gene expression in the kidney follows a circadian rhythm that can be disrupted by chronic high fat diet.Support or Funding InformationP01 HL136267 Integrating novel mechanisms controlling sodium excretion and blood pressure UAB School of Medicine AMC21 Circadian Disruption and Susceptibility to Target Organ DamageThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Previous Exercise Effects in Cisplatin-Induced Renal Lesions in Rats

Background/Aims: Physical training has beneficial effects on endothelial function and can influence the regeneration of the endothelial cell. We investigated the effect of physical training on cisplatin (CP)-induced acute kidney injury and assessed the impact of training on endothelial structure and function, and on the inflammatory processes in rats. Methods: We injected male Wistar rats subjected to previous physical training in treadmill running (trained, TR) or not (sedentary, SED) with CP (5 mg/kg) (TR+CP and SED+CP groups, respectively). Five days after the injections, blood and urine samples were collected to evaluate renal function and kidneys were harvested for morphological, immunohistochemical, enzyme-linked immunosorbent assay, and analysis of nitric oxide (NO) levels. Results: Rats treated with CP showed increased levels of plasma creatinine and sodium and potassium fractional excretion. These alterations were associated with increase in tubulointerstitial lesions and macrophage number, reduction of endothelial cells, and increased VEGF, vimentin, and α-smooth muscle actin expression in the outer renal medulla in the SED+CP group. We also found increased levels of renal IL-1β and increased excretion of monocyte chemoattractant protein-1 and transforming growth factor-β compared with controls. These changes were milder in trained rats, associated with increased levels of renal tissue NO, and increased expression of p-eNOS and stromal cell-derived factor-1α (a chemokine involved in kidney repair) in the kidneys of CP-injected trained rats. Conclusions: The protective effect of previous training in CP-treated rats was associated with reduced endothelial cell lesions and increased renal production of NO in trained rats.

Effect of Repeated Injection of Iodixanol on Renal Function in Healthy Wistar Rats Using Functional MRI.

Purpose To determine the optimal time interval of repeated intravenous injections of iodixanol in rat model and to identify the injury location and causes of renal damage in vivo. Materials and Methods Rats were randomly divided into Control group, Group 1 with one iodixanol injection, and Group 2 with two iodixanol injections. Group 2 was subdivided into 3 cohorts according to the interval between the first and second iodixanol injections as 1, 3, and 5 days, respectively. Blood oxygen level-dependent (BOLD) imaging and diffusion weighted imaging (DWI) were performed at 1 hour, 1 day, 3 days, 5 days, and 10 days after the application of solutions. Results Compared with Group 1 (7.2%), Group 2 produced a remarkable R2⁎ increment at the inner stripe of the renal outer medulla by 15.37% (P = 0.012), 14.83% (P = 0.046), and 13.53% (P > 0.05), respectively, at 1 hour after repeated injection of iodixanol. The severity of BOLD MRI to detect renal hypoxia was consistent with the expression of HIF-1α and R2⁎ was well correlated with HIF-1α expression (r = 0.704). The acute tubular injury was associated with urinary NGAL and increased significantly at 1 day. Conclusions Repetitive injection of iodixanol within a short time window can induce acute kidney injury, the impact of which on renal damage in rats disappears gradually 3–5 days after the injections.

Short-term hypoxia and vasa recta function in kidney slices.

Descending vasa recta (DVR) supply the inner part of outer renal medulla an area at risk for hypoxic damages. We hypothesize increased vasoreactivity after hypoxia/re-oxygenation (H/R) in DVR, which might contribute to the reduced medullary perfusion after an ischemic event. Live kidney slices (200μm) from SD rats were used for functional experiments. TUNEL assay and H&E staining were used to estimate slice viability. Kidney slices were treated with carbogen or hypoxia (1% O2) for 60 or 90 min and vasoreactivity to Ang II (10-7 M) was recorded by DIC microscopy after re-oxygenation with carbogen. Expression of NOS and NADPH enzymes mRNA were determined in iron-perfusion isolated VR. Percentage of apoptotic cells increased in control and H/R after 90 min in the medulla. Ang II- induced constriction of DVR was reduced after 90 min in control (compared to 60 min), but not after H/R. NOS enzymes mRNA expression levels decreased over 90 min hypoxia. Increased reactivity of DVR to Ang II after H/R compared to control (90 min) suggest a role of DVR in renal ischemia/reperfusion injury.

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3±1.2 and 11.7±3.2µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4±3.1µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1±9.8µM, 55.5±2.8ΔF/s/mg protein) than BBMV (K m 78.1±13.3µM and 35.5±1.7ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

Abstract 041: New Evidence for G Protein-coupled Estrogen Receptor as a Natriuretic Factor

Premenopausal women have a lower risk of cardiovascular and renal diseases compared to age-matched men. This protection includes fewer salt-dependent complications. We have shown that female rats have a more robust natriuretic capacity compared to males in response to increased dietary salt. The novel estrogen receptor, G protein-coupled estrogen receptor (GPER), is a membrane-bound receptor linked to acute signaling pathways. GPER activation elicits protective effects throughout the cardiovascular system. However, its role in sodium handling is not defined. We hypothesized that activation of GPER in the renal medulla stimulates sodium excretion. In female Sprague Dawley (SD) rats, isosmotic saline was infused into the renal medullary interstitium (500 μl/h) during a 60-80 min equilibration period and 20 min baseline urine collection period. This was followed by infusion of the GPER agonist G1 (50 pmol/kg/min) or vehicle into the renal medulla for two further 20 min periods. Compared with vehicle, G1 significantly increased urinary sodium excretion and urine flow (from 0.5 ± 0.1 to 0.9 ± 0.2 μmol/min and from 5.3±1.1 to 8.3±1.6 μl/min, respectively, n=6, p&lt;0.05). Urinary potassium excretion and mean arterial pressure remained unchanged during the experiments (0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min and 108.6 ± 3.4 vs. 107.2 ± 5.1 mmHg, respectively, n=6). Immunohistochemical analysis of GPER revealed more predominant staining in both inner and outer renal medulla in female SD rats, compared to males. GPER appears to be predominantly expressed in interstitial cells. These data reveal that renal medullary GPER plays an important role in renal sodium handling and may account for the enhanced ability to handle increased dietary salt in females. Funded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP

P 38 Proteinase activated receptor 2 (PAR2) on blood pressure and electrolyte handling in the normal pregnant rat

Objectives Healthy pregnancy is characterized by avid aldosterone-mediated renal sodium retention driving a ∼40% plasma volume expansion in the presence of decreased or normal blood pressure. Incredibly, gestational potassium retention occurs in the face of this high circulating aldosterone, decreased renal NCC abundance/ phosphorylation, and increased renal ENaC activity, all of which are potent kaliuretic factors. The mechanisms permitting these opposing adaptations are largely unknown. Research in non-pregnant rats has shown that activation of proteinase activated receptor type 2 (PAR2) can produce vasodilation, inhibit potassium secretion via the renal outer medullary potassium channel (ROMK) in principal cells, and stimulate thiazide sensitive sodium chloride reabsorption (pendrin/NDCBE-mediated) in intercalated cells of the cortical collecting duct. Here, we investigate renal PAR2 activation in virgin and normal pregnant rats. Methods We measured the mRNA expression of PAR2 in the renal cortex, outer medulla, and inner medulla of virgin (V), mid- (MP), and late pregnant (LP) rats using quantitative real-time PCR. We also measured in vivo blood pressure (BP), natriuretic, and kaliuretic responses to 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg doses of PAR2 agonist peptide (SLIGRL-NH2) in anesthetized V and LP rats. Results We found that PAR2 mRNA was increased in the inner medulla of MP and LP rats. We also found that LP rats had larger decreases in BP compared to baseline than V rats at 0.1 and 0.3 mg/kg doses of agonist peptide. LP rats also had increased net sodium retention compared to virgin rats following 0.3 mg/kg and 1 mg/kg agonist peptide, but decreased net potassium retention compared to virgins with all doses. Conclusions PAR2 mRNA (an index of activity) is increased in the pregnant rat and activation of this receptor causes larger decreases in BP and increases in sodium retention compared to virgin controls. Further studies will determine the mechanism involved as well as the physiological role of increased renal PAR2 in pregnancy.