Renal Outer Medulla Research Articles

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS).

Abstract MP02: Acute Increases Of Renal Perfusion Pressure Activate Mechanistic Target Of Rapamycin Complex 1 And Increase Macrophage Infiltration In The Kidney Of Sprague Dawley Rats.

Studies were carried out to determine whether acute elevation of renal perfusion pressure (RPP) activates the mechanistic target of rapamycin complex 1 (mTORC1) and inflammation-related genes which may trigger a rapid infiltration of immune cells. RPP was elevated by 40 mmHg (HP group) for 30 minutes in male SD rats (n=5, 10-12 weeks of age) while measuring renal blood flow (RBF; Transonic ultrasonic probe) and urine flow rate. Sham rats (Sham group) were studied in the same way, but RPP was not changed. Since initial studies found that the acute increase of RPP resulted in activation of mTORC1 (pS6 S235/6 /S6; P<0.05) but not mTORC2 (pAKT T308 /AKT ), the effects of inhibition of mTORC1 with rapamycin (Rapa) pretreatment (1.5 mg/kg; n=10) were then determined (HP+Rapa group). RBF was well autoregulated in both HP and HP+Rapa treated rats averaging 6.9 ± 0.5 vs 7.0 ± 0.8 ml/min/gkw in HP (p=0.72) and 8.1 ± 0.8 vs 8.4 ± 0.5 in HP+Rapa group (p=0.34) with a 40 mmHg elevation of RPP. Pressure-natriuresis was unexpectedly blunted in HP+Rapa treated rats increasing from 0.40 ± 0.21 to 4.0 ± 1.1 in HP rats compared to 0.21 ± 0.07 to 2.3 ± 0.5 μmol/min/gkw; p<0.05) in the HP+Rapa treated rats. Urine volumes were similarly affected. Elevation of RPP increased the mTORC1 activity (pS6 S235/6 /S6) in renal cortex (2.8 ± 0.4 vs 4.8 ± 0.5 A.U.; p<0.05, n=5) and outer medulla (2.0 ± 0.3 vs 5.0 ± 0.6 A.U.; p<0.05, n=5) of HP rats compared to Sham. Rapa treatment suppressed this activation. rtPCR analysis found increased mRNA expression of lipocalin-2 (Lcn2; involved innate immune responses; p<0.05), heme oxygenase (Hmox1; p<0.05) and cyclooxygenase 2 (Cox2; p=0.08) in HP rats compared to Sham, responses which were generally blunted by Rapa. Importantly, as determined by immunohistochemistry, CD68 positive macrophage staining was significantly increased (p<0.001) with elevation of RPP in HP compared to sham rat kidneys. This was significantly reduced by Rapa treatment (p<0.001). We conclude that the mTORC1 pathway can be activated very quickly following elevations of RPP and appears to be responsible for rapid macrophage infiltration which is prevented by Rapa treatment. So too, inhibition of mTORC1 with Rapa reduced the pressure-diuresis response through yet unknown mechanisms.

Proteinase-activated receptor-2 (PAR2) on blood pressure and electrolyte handling in the late pregnant rat.

What is the central question of this study? Pregnancy requires marked renal sodium and potassium retention and cumulative plasma volume expansion, in the setting of reduced blood pressure. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in virgin and normal pregnant rats. What is the main finding and its importance? PAR2 expression and sensitivity to activation are increased in pregnancy. This implicates a possible role for PAR2 in supporting the renal/vascular adaptations of pregnancy required for normal maternal plasma volume expansion. A healthy pregnancy involves renal and systemic haemodynamic adaptations, which allow renal sodium and potassium retention and cumulative plasma volume expansion, accompanied by a decline in blood pressure attributable to a reduction in the total peripheral vascular resistance. When these adaptations do not occur, pregnancy is compromised. The mechanisms permitting these opposing adaptations are largely unknown. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in female virgin and normal late pregnant (LP) rats. We measured the mRNA expression of PAR2 in the renal cortex, outer medulla and inner medulla of virgin and LP rats using quantitative real-time PCR. We also measured in vivo blood pressure, natriuretic and kaliuretic responses to PAR2-activating peptide (SLIGRL-NH2 ) in anaesthetized virgin and LP rats. We found that PAR2 mRNA was increased in the inner medulla of LP rats. We also found that LP rats had larger decreases in blood pressure and increases in net sodium retention compared with virgin rats. These findings suggest that pregnancy enhances sensitivity to the blood pressure-lowering and sodium-retaining effects of PAR2.

Acute Elevation of Renal Perfusion Pressure Activates Mechanistic Target of Rapamycin Complex 1 in Rats

We have recently reported that the chronic elevation of renal perfusion pressure (RPP) drives the infiltration of renal inflammatory cells in two models of hypertension, the Dahl salt-sensitive (SS) rats fed a high-salt diet and Sprague-Dawley (SD) rats infused with Angiotensin II. In other studies, we have found that chronic inhibition of mechanistic target of rapamycin complex 1 (mTORC1) reduces salt-induced hypertension in SS rats and inhibits renal immune cell infiltration. mTOR, a kinase, is comprised of two complexes (mTORC1 and mTORC2) which serve to sense and integrate both intracellular and extracellular signals and signal changes of many cellular activities ranging from cell proliferation/growth to immune responses. However, the direct effect of acute RPP on mTOR signaling is largely unknown. In the present study, the effects of acute step changes of RPP upon mTORC1 and mTORC2 signaling were evaluated. Male SD rats (10-12 weeks of age) were anesthetized with ketamine and thiobutabarbital and aortic and venous catheters inserted for measurement of RPP and infusion of saline containing 2.0% bovine serum albumin. Both ureters were catheterized for urine collection and an ultrasonic flow probe (Transonic) placed around the renal artery to measure renal blood flow (RBF). After 1 hour of equilibrium, control blood pressure (BP), RBF and urine volume was measured for 20 min and BP then raised by ~40 mmHg by aortic occlusion below the left renal artery with ligation of the celiac and mesenteric arteries. After 30 minutes, kidneys were removed for molecular analysis. Sham rats were prepared and studied in the same way, but BP was not changed. RPP was increased from 107 ± 4 to 150 ± 5 mmHg (p<0.05, n=5). RBF remained unchanged averaging 6.9 ± 0.5 vs 7.0 ± 0.8 ml/min/gkw, (p=0.68, n=5) with elevation of RPP. Urine volume was increased from 3.7 ± 0.8 to 21.3 ± 4.9 μl/min/gkw (p< 0.05, n=5). Elevation of RPP increased the functional marker of mTORC1 activity (pS6S235/236/S6) in renal cortex (2.8 ± 0.4 vs 4.8 ± 0.5 A.U.; p<0.05, n=5) and outer medulla (2.0 ± 0.3 vs 5.0 ± 0.6 A.U.; p<0.05, n=5) compared to sham rats. In contrast, pAKTS473/AKT (marker of mTORC2 activity) was not statistically changed in either region. Increased RPP resulted in increases of mRNA expression of Cd68 (marker of monocytes) (5.7 ± 0.4 vs 9.9 ± 1.0 /106 18s.; p<0.05, n=5), Lcn2 (marker of innate immune response) (8.8 ± 2.4 vs 25.6 ± 4.5 /106 18s.; p<0.05, n=5), Cox2 (prostanoid) (3.1 ± 0.8 vs 11.7 ± 1.0 /106 18s.; p<0.05, n=5) and Hmox1 (heme oxygenase) (5.0 ± 5.3 vs 102.9 ± 123.7 /106 18s.; p<0.05, n=5) in outer medulla compared to sham rats, and relatively smaller increase in cortex (Cd68 (2.6 ± 0.5 vs 4.0 ± 0.4 /106 18s.; p<0.05, n=7), Lcn2 (3.2 ± 0.7 vs 5.0 ± 0.5 /106 18s.; p=0.06, n=7), Cox2 (4.0 ± 0.6 vs 7.0 ± 1.0 /106 18s.; p=0.08, n=5) and Hmox1 (14.4 ± 6.9 vs 163.1 ± 101.5 /106 18s.; p=0.16, n=7)). Acute increases of RPP within the autoregulatory range of the kidney resulted in activation of mTORC1 and inflammation-related genes within 30 minutes of the stimulus. How mTORC1 senses changes of RPP remains to be determined but it is clear these responses are rapid and most prominent in the renal outer medulla.

Value of T2 Mapping in the Dynamic Evaluation of Renal Ischemia-Reperfusion Injury

To explore the value of T2 mapping in the dynamic quantitative evaluation of renal ischemia- reperfusion injury (IRI). Forty-eight healthy New Zealand rabbits were randomly divided into IRI group (n = 40) and control group (n = 8). Rabbits in the IRI group underwent left renal artery clamping for 60 minutes. Rabbits underwent MRI examinations (T2WI and T2 mapping) before and 1, 12, 24, and 48 hours after IRI. The inter-observer and intra-observer reproducibility of the T2 values were assessed using the intraclass correlation coefficient (ICC) with 95% confidence interval (CI). Correlations between the T2 value of the renal outer medulla and injury scores were assessed by Spearman correlation analysis. The repeated measures analysis of variance was used to compare the differences in T2 values of the IRI and control group across the different time points. Both of the intra-observer (ICC = 0.97, 95% CI 0.95-0.99) and inter-observer reproducibility (ICC = 0.92, 95% CI 0.86-0.96) were excellent for T2 values. The T2 value of the renal outer medulla was moderately positive correlated with tubular epithelial edema (ρ = 0.686, p < 0.001). In IRI group, T2 values of the renal outer medulla were increase at 1 h after IRI (p = 0.001) and were decrease from 1 h to 12 h (p = 0.002). At 1 h after IRI, the T2 values of the renal outer medulla for the IRI group were higher than those for the control group (p < 0.001). T2 mapping can reflect the dynamic changes of renal parenchyma in an animal model of IRI and be used to assess the early renal IRI.

Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

Transfer RNA Fragments in the Kidney in Hypertension.

[Figure: see text].

A 3-Dimentional Framework for Studying the Effects of Structural and Physical Properties of the Renal Outer Medulla on Urine Concentrating Mechanism

Background: Many theories and mathematical simulations have been proposed concerning urine concentrating mechanism (UCM). Due to significant effect of the tubule and vessel architecture in concentrating mechanism, the numerical analysis of UCM through a 3-Dimensional structure might be the answer to find a better consistency between the experimental and theoretical results. Methods: In this paper we have investigated the effects of structural characteristics of the tubules and vessels on the urine concentrating mechanism in the outer medulla (OM) by developing a simple three-dimensional mathematical model. This model is a framework to attain a converged numerical solution for the momentum and species transport equations along with their stiff and coupled boundary conditions based on standard expressions for trans-tubular solutes and water transports on tubule’s membrane. Results: The model structure and the number of the involved tubules have been assumed to be as simple as possible. The effects of slip boundary condition on membrane, Darcy permeability and solute’s diffusivity of the intermediate media on UCM have been studied. It has been shown that this approach can simply simulate preferential interactions and tubule’s confinement by radial diffusion coefficients. Conclusions: All in all, the feasibility of the idea of completely 3-D modeling by employing the concept of diffusion coefficient and Darcy permeability has been explored and validated.

NBCn1 Increases NH4 + Reabsorption Across Thick Ascending Limbs, the Capacity for Urinary NH4 + Excretion, and Early Recovery from Metabolic Acidosis.

The electroneutral Na+/HCO3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking. Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH4 +] was measured in renal biopsies, NH4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. Basolateral Na+/HCO3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na+/HCO3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH4 + gradient, reduced the capacity for urinary NH4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO3 -] from their initial decline. During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO3 - uptake and transepithelial NH4 + reabsorption in mTALs, renal medullary NH4 + accumulation, urinary NH4 + excretion, and early recovery of arterial blood pH and standard [HCO3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H+ released during NH4 + reabsorption across mTALs.

Calorie Restriction Protects against Contrast-Induced Nephropathy via SIRT1/GPX4 Activation.

Calorie restriction (CR) extends lifespan and increases resistance to multiple forms of stress, including renal ischemia-reperfusion (I/R) injury. However, whether CR has protective effects on contrast-induced nephropathy (CIN) remains to be determined. In this study, we evaluated the therapeutic effects of CR on CIN and investigated the potential mechanisms. CIN was induced by the intravenous injection of iodinated contrast medium (CM) iopromide (1.8 g/kg) into Sprague Dawley rats with normal food intake or 40% reduced food intake, 4 weeks prior to iopromide administration. We found that CR was protective of CIN, assessed by renal structure and function. CM increased apoptosis, reactive oxygen species (ROS), and inflammation in the renal outer medulla, which were decreased by CR. The silent information regulator 1 (SIRT1) participated in the protective effect of CR on CIN, by upregulating glutathione peroxidase 4 (GPX4), a regulator of ferroptosis, because this protective effect was reversed by EX527, a specific SIRT1 antagonist. Our study showed that CR protected CIN via SIRT1/GPX4 activation. CR may be used to mitigate CIN.

Renal microvascular oxygen tension during hyperoxia and acute hemodilution assessed by phosphorescence quenching and excitation with blue and red light.

The kidney plays a central physiologic role as an oxygen sensor. Nevertheless, the direct mechanism by which this occurs is incompletely understood. We measured renal microvascular partial pressure of oxygen (PkO2) to determine the impact of clinically relevant conditions that acutely change PkO2 including hyperoxia and hemodilution. We utilized two-wavelength excitation (red and blue spectrum) of the intravascular phosphorescent oxygen sensitive probe Oxyphor PdG4 to measure renal tissue PO2 in anesthetized rats (2% isoflurane, n = 6) under two conditions of altered arterial blood oxygen content (CaO2): 1) hyperoxia (fractional inspired oxygen 21%, 30%, and 50%) and 2) acute hemodilutional anemia (baseline, 25% and 50% acute hemodilution). The mean arterial blood pressure (MAP), rectal temperature, arterial blood gases (ABGs), and chemistry (radiometer) were measured under each condition. Blue and red light enabled measurement of PkO2 in the superficial renal cortex and deeper cortical and medullary tissue, respectively. PkO2 was higher in the superficial renal cortex (~ 60 mmHg, blue light) relative to the deeper renal cortex and outer medulla (~ 45 mmHg, red light). Hyperoxia resulted in a proportional increase in PkO2 values while hemodilution decreased microvascular PkO2 in a linear manner in both superficial and deeper regions of the kidney. In both cases (blue and red light), PkO2 correlated with CaO2 but not with MAP. The observed linear relationship between CaO2 and PkO2 shows the biological function of the kidney as a quantitative sensor of anemic hypoxia and hyperoxia. A better understanding of the impact of changes in PkO2 may inform clinical practices to improve renal oxygen delivery and prevent acute kidney injury.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Abstract 16: Progressive Alterations Of Mitochondrial Bioenergetics In The Kidney During The Development Of Salt-induced Hypertension

Hypertension is a complex disease and a leading cause of morbidity and mortality globally. Although oxidative stress and mitochondrial dysfunction have been found in the kidney in various models of hypertension, progressive alteration of mitochondrial oxidative phosphorylation (OxPhos) in the kidney during the development of salt-sensitive hypertension has not been characterized. The present study determined changes of OxPhos in kidneys of Dahl salt-sensitive (SS) rats before (0.4% NaCl diet; LS) and after switching to a high salt diet (4.0% NaCl; HS) during the development of hypertension. Mitochondria were isolated from the outer medulla (OM) and cortex of the kidney of SS rats fed a LS diet since weaning and studied at days 3, 7, 14 &amp; 21 of a HS diet feeding. Oxygen consumption rates (OCR) were measured in mitochondria energized with pyruvate + malate as substrates for three different respiratory states using an Oroboros Oxygraph-2k Instrument. This includes i) leak state (in the absence of ADP), ii) ADP-stimulated state, and iii) uncoupled state (in the presence of an uncoupler FCCP). A biphasic pattern of ADP-stimulated OCR with progressive uncoupling was observed in both the renal OM and cortex. Mitochondrial efficiency for ATP synthesis was increased in the early phases of hypertension (3 &amp; 7 days) but was severely compromised in the established phases of hypertension (14 &amp; 21 days). This decreased mitochondrial efficiency was associated with uncoupling of OxPhos and high levels of oxidative stress which we hypothesized were due to mitochondrial ROS stimulation of membrane NOXs. To test this, experiments were performed in SS rats with double knock out (DKO) of the cytosolic subunit of NOX2 (p67 phox ) and NOX4 (SS p67phox-/-/Nox4-/- ). DKO SS rats were fed a HS diet and OCR of renal cortical and OM mitochondria was determined at days 7 and 14. In contrast to SS rats, the DKO SS rats fed a HS diet showed no significant differences in mitochondrial OCR in the cortex or OM, nor to a control group maintained on a LS diet. HS diet in SS rats initially increases the efficiency of renal cortical and medullary mitochondrial ATP production (days 1-7) followed by an enhanced ROS production with mitochondrial uncoupling and reduced efficiency of ATP production by the third week.

Application of diffusion tensor imaging and blood oxygenation level-dependent magnetic resonance imaging to assess bilateral renal function induced by Iohexol in rabbits

BackgroundBlood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) and diffusion tensor imaging (DTI) are useful methods for investigating the morphology and function of the kidneys, including revealing unilateral renal damage. Nevertheless, these techniques have not yet been applied for bilateral renal function. The aim of this study was to investigate whether the combination of DTI and BOLD could be used to examine different degrees of contrast-induced acute kidney injury (CI-AKI) in bilateral kidneys compared to standard methods such as serum creatinine (SCr) detection.MethodsForty-Two New Zealand white rabbits were divided into two groups: the experimental group and the control group. Physiological saline and iodine contrast agent (iohexol, 1.0 g iodine/kg, 1.0 ml/sec) were injected via the right renal artery. DTI and BOLD-MR data were acquired longitudinally at the baseline and 1, 24, 48, and 72 h after high-pressure syringe injection to measure the apparent diffusion coefficient (ADC), fractional anisotropy (FA) and relative transverse relaxation rate (R2*). After the MR scan at each time point, three rabbits in each group were sacrificed, and changes in SCr and hypoxia-inducible factor-1α (HIF-1α) were analyzed using histopathology and immunochemistry.ResultsTwenty-four hours after iohexol administration, the values of ADC and FA decreased significantly (P < 0.05), while R2* values increased (P < 0.05) in the renal cortex (CO), outer medulla (OM) and inner medulla (IM). Besides, significant negative correlations were observed among ADC, FA, and R2* in CO, OM, and IM (all P < 0.001, r = − 0.654–0.828).ConclusionsDTI and BOLD can simultaneously and non-invasively assess different degrees of CI-AKI in bilateral kidneys.

Klotho Ameliorates Renal Fibrosis in a Murine Model of ADTKD‐UMOD by Enhancing UMOD secretion and Downregulation of TGFBI

Heterozygous Uromodulin (UMOD) mutations cause autosomal dominant tubulo‐interstitial kidney disease (ADTKD). ADTKD‐UMOD is a model for renal fibrosis.Currently, no therapies are available for ADTKD‐UMOD. The antiaging hormone Klotho improves glomerular disease, hypertension, uremic cardiomyopathy, and acute kidney injury (AKI). We tested if Klotho ameliorates renal fibrosis and tubular disease in a murine model of ADTKD‐UMOD.To generate a more pronounced phenotype we used homozygous UmodC93F/C93F mice. Mice were crossed with mice overexpressing Klotho (TgKl). We compared the phenotype with wild‐type (WT), TgKl, and UmodC93F/C93F mice (8 animals per group). We studied blood pressure, urine and blood samples at ages of 6 and 13 months. In different animals of the four groups, we harvested kidneys at 3 months to perform quantitative proteome analysis via LC‐MS/MS on renal outer medulla.Compared to UmodC93F/C93F mice, TgKl/UmodC93F/C93F had better renal function as indicated by significantly lower BUN, creatinine, and cystatin C values, and less renal fibrosis. PTH and FGF23 improved significantly in TgKl/UmodC93F/C93F vs. UmodC93F/C93F mice. Mutant UMOD is retained in the endoplasmic reticulum. We found that relative urinary UMOD secretion was higher in TgKl/UmodC93F/C93F vs. UmodC93F/C93F. No major serum electrolyte differences were found among the four groups. Urinary studies in TgKl/UmodC93F/C93F mice showed less urinary wasting of Na+, Ca2+, Mg2+ and Phosphorus than in UmodC93F/C93F mice. Compared to UmodC93F/C93F, Klotho‐overexpressing animals had significantly lower systolic and diastolic blood pressures. Markers of cardiac hypertrophy such as ANP, BNP, and MYH7 mRNA were significantly lower in TgKl/UmodC93F/C93F than in UmodC93F/C93F animals. We confirmed impaired unfolded protein response (UPR) and autophagy in UmodC93F/C93F compared to WT mice. However, improved renal function and fibrosis in Klotho‐overexpressing mice were not due to changes in UPR or autophagy. Therefore, we performed an unbiased proteomics approach. We identified 262 downregulated and 179 upregulated proteins in kidneys. Analyzing proteins by signaling pathways, we identified in TgKl/UmodC93F/C93F vs. UmodC93F/C93F animals downregulation of Transforming growth factor‐beta‐induced protein (TGFBI) together with collagens Col1a1, Col4a3, Col4a4, Col4a5, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, prolargin, biglycan, and osteoglycin/mimecan. We confirmed lower Col1a1 mRNA expression with qPCR.We show that Klotho improves renal outcome in ADTKD‐UMOD by increasing urinary UMOD secretion and ameliorating renal fibrosis by downregulation of TGFBI and collagen. TGFBI is secreted by mesenchymal stromal cells and infiltrating macrophages and stimulates collagen synthesis. Infiltrating macrophages are an important part of AKI and ADTKD‐UMOD. Our data reveal a new specific target for treatment of renal fibrosis.Support or Funding InformationNIH R03 DK111776, NIH P30 DK079328, and CCRAC Senior Investigator Bridge Award, Children’s Health DallasAt 6 and 13 months TgKl/UmodC93F/C93F show significantly lower BUN, creatinine, and cystatin C values.Figure 1STRING analysis showing networks formed by downregulated proteins in renal outer medulla of TgKl/UmodC93F/C93F compared to UmodC93F/C93F mice. Red circle outlines downregulated TGFBI and collagens.Figure 2

Pharmacologic Blockade of 15-PGDH Protects Against Acute Renal Injury Induced by LPS in Mice.

Prostaglandin pathway plays multiple roles in various physiological and pathological conditions. The present study aimed to investigate the effect of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in the degradation of prostaglandins, on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice. In this study, male C57BL/6J mice were injected intraperitoneally with LPS (10 mg/kg). SW033291, a potent small-molecule inhibitor of 15-PGDH, was used to investigate the therapeutic potential of 15-PGDH inhibition on LPS-induced AKI. We discovered that the expression of 15-PGDH protein was upregulated in kidneys of LPS-stimulated mice, and it was mainly localized in the cytoplasm of renal tubular epithelial cells in renal cortex and outer medulla. SW033291 administration improved the survival rates of mice and attenuated renal injury of mice that were challenged by LPS. Additionally, inhibition of 15-PGDH also reversed LPS-induced apoptosis of renal cells, increased expression of anti-apoptotic protein Bcl-2, and downregulated expression of Fas, caspase-3, and caspase-8. Pretreatment of SW033291 enhanced autophagy in kidney cells after LPS stimulation. Our data also showed that inhibition of 15-PGDH relieved the level of lipid peroxidation and downregulated NADPH oxidase subunits induced by LPS in mice kidneys but had no significant effect on the release of inflammatory factors, such as IL-6, IL-1β, TNF-α, and MCP-1. Our study demonstrated that inhibition of 15-PGDH could alleviate LPS-induced AKI by regulating the apoptosis, autophagy, and oxidative stress rather than inflammation in mice.

Can distinction between the renal cortex and outer medulla on ultrasonography predict estimated glomerular filtration rate in canine chronic kidney diseases?

BackgroundQuantitative evaluation of renal cortical echogenicity (RCE) has been tried and developed in human and veterinary medicine.ObjectivesThe objective of this study was to propose a method for evaluating RCE quantitatively and intuitively, and to determine associations between ultrasonographic renal structural distinction and estimated glomerular filtration rate (eGFR) in canine chronic kidney disease (CKD).MethodsData were collected on 63 dogs, including 27 with normal kidney function and 36 CKD patients. Symmetric dimethylarginine and creatinine concentrations were measured for calculating eGFR. RCE was evaluated as 3 grades on ultrasonography images according to the distinction between the renal cortex and outer medulla. The RCE grade of each kidney was measured.ResultsThere was a significant difference in eGFR between the group normal and CKD (p < 0.001). As mean of RCE grades (the mean values of each right and left kidney's RCE grade) increases, the proportion of group CKD among the patients in each grade increases (p < 0.001). Also, severity of RCE (classified as "high" if any right or left kidney evaluated as RCE grade 3, "low" otherwise) and eGFR is good indicator for predicting group CKD (p < 0.001).ConclusionsThe degree of distinction between the renal cortex and the outer medulla is closely related to renal function including eGFR and the RCE grade defined in this study can be used as a method of objectively evaluating RCE.

Molecular Targets of Natriuretic Action of Prolactin in the Rat Model of Cholestasis of Pregnancy.

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.

Effects of empagliflozin on nondiabetic salt-sensitive hypertension in uninephrectomized rats

Impaired pressure natriuresis (PN) underlies salt-sensitive hypertension, and renal inflammation and hypoxia-inducible factor-1 (HIF-1) have been implicated in the modulation of systemic hypertension. Although sodium–glucose cotransporter-2 (SGLT2) inhibitors were reported to lower blood pressure (BP) in type 2 diabetes mellitus, whether they have a role in nondiabetic hypertensive kidney diseases is unclear. The present study was undertaken to investigate whether nondiabetic salt-sensitive hypertension and accompanying renal inflammation are ameliorated by SGLT2 inhibition. Male Sprague-Dawley rats were randomly divided into three groups: sham controls (SCs), uninephrectomized controls (UCs), and empagliflozin-treated rats (ETs). All rats were fed a rodent diet with 8% NaCl throughout the study period. Empagliflozin was orally administered for 3 weeks after uninephrectomy. Systolic blood pressure was recorded weekly, and kidneys were harvested for immunoblotting, immunohistochemistry, and quantitative PCR analysis at the end of the animal experiment. Systolic BP was significantly decreased in ETs that were orally given empagliflozin for 3 weeks after uninephrectomy. Although ETs did not show any increase in weekly measured urine sodium, the right-shifted PN relationship in UCs was improved by empagliflozin treatment. The expression of HIF-1α was increased in the renal outer medulla of ETs. Consistent with this, HIF prolyl-hydroxylase-2 protein and mRNA were decreased in ETs. The abundance of CD3 and ED-1 immunostaining in UCs was reduced by empagliflozin treatment. The increased IL-1ß, gp91phox, and NOX4 mRNA levels in UCs were also reversed. Empagliflozin restored impaired PN in nondiabetic hypertensive kidney disease in association with increased renal medullary expression of HIF-1α and amelioration of renal inflammation.

SUN-171 PRETREATMENT WITH LOW DOSE LIPOPOLYSACCHARIDE PREVENTS ISCHEMIA-REPERFUSION INDUCED RENAL VASCULAR CONGESTION IN WKY RATS.

Our lab has previously shown red blood cell (RBC) congestion in the renal outer medulla is critical in determining the recovery of renal function following ischemia-reperfusion (IR). While pre-conditioning with lipopolysaccharide (LPS) has been reported to provide protection against IR-induced tubular injury, the effect of low dose LPS on renal medullary congestion has not been directly examined. We hypothesized prior exposure to low dose LPS would induce mild inflammation, promote rouleaux and increase RBC congestion following IR. Study 1: LPS dose response in 10wk old male Wistar-kyoto rats (WKY). Rats were injected (i.p.) with either 10 (n=3), 100 (n=3), 1000 (n=6) ug/kg LPS or saline (n=5) once daily for 7 days prior to IR. Low dose LPS concentrations are less than 1/10th of the dose reported to cause hemodynamic dysfunction. Study 2: Preconditioning vs acute low dose LPS in male WKY. Rats were treated with either 1) 1 injection (i.p) of LPS (1000ug/kg, n=3) or saline (n=3) 24h prior to IR, 2) daily injection of LPS (1000ug/kg, n=4) or saline (n=4) for 3 days prior to IR, or 3) daily injections of LPS (1000ug/kg, n=5) or saline (n=4) for 7 days prior to IR. Following pretreatment, all animals were anesthetized for 30min (Study 1) or 45min (Study 2) ischemia with 24h reperfusion, and humanely sacrificed. Kidneys were collected for histological analysis of RBC congestion (blinded scoring scale 0-5) and tubular injury (blinded, 0-100%). Blood was collected at baseline, pre-IR, and sacrifice for measurement of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), blood urea nitrogen (BUN), and serum creatinine (SCr). Study 1: CRP and ESR measurements indicated modest inflammation and rouleaux formation in LPS (1000 ug/kg) pretreated animals (ESR: 5.9mm±1.2) compared to control (ESR: 3.2mm±1.4). Most striking, medullary peritubular congestion showed LPS pretreatment dose dependently attenuated congestion when compared to saline treated controls (Fig. 1). Study 2: Preconditioning with low dose LPS (1 injection daily for 3 or 7days) induced mild inflammation and reduced RBC congestion in the renal medulla (7 day congestion score: LPS 1.3±0.94, control 3.5±0.54). In these same animals, tubular injury showed no difference between LPS and control groups (p= 0.397). In contrast, a single, acute injection of low dose LPS 24h prior to IR demonstrated similar renal medullary RBC congestion as saline injected animals (Congestion score: LPS 3.2±0.83, control 2.2±1.09). We conclude that despite promoting inflammation, and rouleaux, 3-7days of pretreatment with low dose LPS attenuates RBC congestion in the renal medulla following IR. The effect appears to be largely independent of tubular injury, suggesting the phenomenon is not secondary to reduced parenchymal damage. As sustained congestion and hypoxia following IR may lead to progressive renal injury in the medulla after renal blood flow and GFR returns towards normal, understanding the mechanisms by which low dose LPS prevents renal congestion may lead to novel therapeutic approaches to improve recovery from ischemic acute kidney injury.