Inner Stripe Of The Outer Medulla Research Articles

Evaluation of Kidney Injury Using Arterial Spin Labeling and Blood Oxygen Level-Dependent MRI: An Experimental Study in Rats With Carbon Tetrachloride-Induced Liver Cirrhosis.

Serum creatinine (Scr) may be not suited to timely and accurately reflect kidney injury related to chronic liver disease. Currently, the ability of arterial spin labeling (ASL) and blood oxygen level-dependent (BOLD) sequences to evaluate renal blood flow (RBF) and blood oxygen in chronic liver disease remains to be verified. To investigate the value of ASL and BOLD imaging in evaluating hemodynamics and oxygenation changes during kidney injury in an animal model of chronic liver disease. Prospective. Chronic liver disease model was established by subcutaneous injection of carbon tetrachloride. Forty-three male Sprague-Dawley rats (8 weeks) were divided into a pathological group (0, 2, 4, 6, 8, 12 weeks, each group: N = 6) and a continuous-scanning group (N = 7). 3-T, ASL, BOLD, and T2W. Regions of interest in the cortex (CO), outer stripe of the outer medulla (OSOM), and inner stripe of the outer medulla (ISOM) are manually delineated. The RBF and T2* values at each time point (0, 2, 4, 6, 8, 12 weeks) are measured and compared. Hematoxylin-eosin score (HE Score, damage area scoring method), alpha-smooth muscle actin (α-SMA), hypoxia-inducible factor-1alpha (HIF-1α), peritubular capillar (PTC) density, Scr, and neutrophil gelatinase-associated lipocalin were harvested. Analysis of variance, Spearman correlation analysis, Kruskal-Wallis tests, and receiver operating characteristic analysis with the area under the curve (AUC). A P-value <0.05 was considered statistically significant. Renal RBF and T2* values of CO, OSOM, and ISOM were significantly different from baseline. Both RBF and T2* were significantly correlated with HE Score, α-SMA, HIF-1α, and PTC density (|r| = 0.406-0.853). RBF demonstrated superior diagnostic capability in identifying severe kidney injury in this model of chronic liver disease (AUC = 0.964). Imaging by ASL and BOLD may detect renal hemodynamics and oxygenation changes related to chronic liver disease early. 5 TECHNICAL EFFICACY: Stage 2.

Multi-Parametric MRI for Evaluating Variations in Renal Structure, Function, and Endogenous Metabolites in an Animal Model With Acute Kidney Injury Induced by Ischemia Reperfusion.

Ischemia reperfusion injury (IRI)-induced acute kidney injury (AKI) may occur after renal ischemic injury. There is a lack of an accurate and comprehensive detection technique for IRI-AKI. To longitudinally evaluate IRI-AKI in rats by renal structure, function, and metabolites using multi-parametric MRI (mpMRI). Prospective. Forty-eight rats undergoing IRI-AKI. 7-T, T1 mapping, and arterial spin labeling (ASL): echo planar imaging (EPI) sequence; blood oxygen level-dependent (BOLD): gradient recalled echo (GRE) sequence; T2 mapping, quantitative magnetization transfer (qMT), and chemical exchange saturation transfer (CEST): rapid acquisition with relaxation enhancement (RARE) sequence. The mpMRI for IRI-AKI was conducted at 0 (control), 1, 3, 7, 14, and 28 days, all included eight rats. The longitudinal mpMRI signal of manually outlined cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla, and medulla plus pelvis were calculated and compared, their diagnosis performance for IRI-AKI also been evaluated. Pearson correlations analysis for correlation between mpMRI signal and renal injury, unpaired t-tests for comparing the signal changes, and receiver operating characteristics (ROC) analysis was used to identify most sensitive indicator of mpMRI. A P-value <0.05 was considered statistically significant. Compared with control kidneys, the T1 and T2 values of the cortex and medulla in IRI kidneys increased and reached their highest values on day 14, and the kidneys also showed the most severe edema and segments blurred. The RBF in the cortex and OSOM showed a significant decline after day 3. The BOLD signal in the OSOM largest increased on day 28. The cortical PSR and the amine-CEST both decreased with IRI-AKI progression, and amine-CEST achieved the highest AUC for the diagnosis (0.899). Multi-parametric MRI may show comprehensive variations in IRI-AKI, and amine-CEST may exhibit the highest accuracy for diagnosis of IRI-AKI. 1 TECHNICAL EFFICACY: Stage 2.

Evaluation of renal cold ischemia-reperfusion injury with intravoxel incoherent motion diffusion-weighted imaging and blood oxygenation level-dependent MRI in a rat model.

Purpose: Cold ischemia-reperfusion injury (CIRI) is one of the most serious complications following renal transplantation. The current study investigated the feasibility of Intravoxel Incoherent Motion (IVIM) imaging and blood oxygenation level-dependent (BOLD) in the evaluation of different degrees of renal cold ischemia-reperfusion injury in a rat model. Methods: Seventy five rats were randomly divided into three groups (N = 25 for each group): T0: sham-operated group, T2/T4: CIRI groups with different cold ischemia hours (2, 4h, respectively). The rat model of CIRI group was established by left kidney cold ischemia with right nephrectomy. All the rats received a baseline MRI before the surgery. Five rats in each group were randomly selected to undergo an MRI examination at 1h, day 1, day 2 and day 5 after CIRI. The IVIM and BOLD parameters were studied in the renal cortex (CO), the outer stripe of the outer medulla (OSOM), and the inner stripe of the outer medulla (ISOM) followed by histological analysis to examine Paller scores, peritubular capillary (PTC) density, apoptosis rate and biochemical indicators to obtain the contents of serum creatinine (Scr), blood urea nitrogen (BUN), superoxide dismutase (SOD) and malondialdehyde (MDA). Results: The D, D*, PF and T2* values in the CIRI groups were lower than those in the sham-operated group at all timepoints (all p < 0.05). The prolonged cold ischemia times resulted in gradually lower D, D*, PF and T2* values (all p < 0.05). The D and T2* values of cortex and OSOM in Group T0 and T2 returned to the baseline level (all p > 0.05) except Group T4. The D* and PF values of cortex, OSOM and ISOM in Group T2 and T4 still remained below the normal levels (all p < 0.05) except Group T0. D, D*, PF and T2* values were strongly correlated with histopathological (Paller scores, PTC density and apoptosis rate) and the biochemistry indicators (SOD and MDA) (|r|>0.6, p < 0.001). D*, PF and T2* values were moderately to poorly correlated with some biochemistry indicators (Scr and BUN) (|r|<0.5, p < 0.05). Conclusion: IVIM and BOLD can serve as noninvasive radiologic markers for monitoring different degrees of renal impairment and recovery after renal CIRI.

Chronic aldosterone administration alters aquaporin expression and localization in the murine kidney

The steroid hormone aldosterone is an important regulator of fluid balance and electrolyte homeostasis. While acute aldosterone signaling is canonically associated with fluid retention due to its role in promoting sodium reabsorption through ENaC in the distal nephron, chronic aldosterone exposure has been shown to exacerbate urine production in a rat model of diabetes insipidus through its actions on aquaporin 2 (AQP2) localization and expression. In our studies, we aimed to characterize not only the effect of chronic aldosterone administration on AQP2 expression and localization, but also its effect on the basolateral water channels, AQP3 and AQP4. Given the large urinary output we observed in our animals following chronic aldosterone treatments, we hypothesized all three of these channels would show reduced expression.Aldosterone was administered to male C57Bl/6 mice at a dose of 200 μg/kg/day for 28 days via subcutaneous minipumps. Aldosterone treatment caused a significant diuresis as compared to control animals by day 15 (Ctl 4.8 ± 1.6 ml; Aldo 8.4 ± 2.4 ml), and this difference persisted for the remainder of the experiment. Mice given aldosterone also consumed more water as compared to their control counterparts (Ctl 4.0 ± 1.2 ml; Aldo 9.6 ± 2.3 ml). Urine osmolality was measured by freeze-point depression, with a significant decrease in urinary concentration being noted during the daytime by day 4 (Ctl 1219.8 ± 473.4 mOsm/kg; Aldo 638.8 ± 143.8 mOsm/kg) and during the night by day 17 (Ctl 905.3 ± 175.1 mOsm/kg; Aldo 522.2 ± 127.3 mOsm/kg). At the end of the 28 days of aldosterone administration, mice receiving aldosterone had a higher whole blood [Na + ] (Ctl 146 ± 1.9mmol/l; Aldo 152 ± 1.3mmol/l) and lower whole blood [K+] (Ctl 4.8 ± 0.56mmol/l; Aldo 3.0 ± 0.34mmol/l) as measured by iSTAT cartridge with terminal cardiac blood draw.Kidneys were examined by Western blot and immunofluorescent staining for expression and localization of the three AQPs. AQP2 expression decreased after both 14 and 28 days of aldosterone administration, with line scan analysis showing a decreased signal present at the apical membrane. AQP3 expression was decreased in the basolateral membranes of principal cells within the cortex of the kidney but increased in cells within the inner medulla after 28 days of aldosterone. AQP4 levels were significantly increased in AQP2-positive cells of the inner stripe of the outer medulla and the inner medulla, suggesting that this channel may be upregulated to compensate for changes in the other two aquaporins. Potassium supplementation to separate the effects of aldosterone administration from hypokalemia did not rescue the expression of these channels.Overall, our studies reveal that chronic aldosterone administration in healthy mice leads to polyuria and polydipsia, with decreased apical expression of AQP2 and variable changes in AQP3 and AQP4 dependent on location within in the kidney. This study was funded by NIH grants R01 DK130901 (to SS), R01 HL147818 and R01 DK129285 (to TRK), and P30 DK079307 (the Pittsburgh Center for Kidney Research). SMM was supported by T32 DK061296, T32 DK007052, and a grant from Relypsa. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

3D autofluorescence imaging of hydronephrosis and renal anatomical structure using cryo-micro-optical sectioning tomography.

Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion. Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries. Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm3 and 13.01 ± 3.74 mm3 in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation. Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.

Multiparametric magnetic resonance imaging in diagnosis of long-term renal atrophy and fibrosis after ischemia reperfusion induced acute kidney injury in mice.

Tubular atrophy and fibrosis are pathological changes that determine the prognosis of kidney disease induced by acute kidney injury (AKI). We aimed to evaluate multiple magnetic resonance imaging (MRI) parameters, including pool size ratio (PSR) from quantitative magnetization transfer, relaxation rates, and measures from spin-lock imaging ( and ), for assessing the pathological changes associated with AKI-induced kidney disease. Eight-week-old male C57BL/6 J mice first underwent unilateral ischemia reperfusion injury (IRI) induced by reperfusion after 45 min of ischemia. They were imaged using a 7T MRI system 56 days after the injury. Paraffin tissue sections were stained using Masson trichrome and picrosirius red to identify histopathological changes such as tubular atrophy and fibrosis. Histology detected extensive tubular atrophy and moderate fibrosis in the cortex and outer stripe of the outer medulla (CR + OSOM) and more prominent fibrosis in the inner stripe of the outer medulla (ISOM) of IRI kidneys. In the CR + OSOM region, evident decreases in PSR, , , , and showed in IRI compared with contralateral kidneys, with PSR and exhibiting the most significant changes. In addition, the exchange parameter dropped by the largest degree among all the MRI parameters, while increased significantly. In the ISOM of IRI kidneys, PSR increased while kept decreasing. , , and all increased due to more severe fibrosis in this region. Among MRI measures, PSR and showed the highest detectability of renal changes no matter whether tubular atrophy or fibrosis dominated. and could be more specific to a single pathological event than other MRI measures because only increased and decreased consistently when either fibrosis or tubular atrophy dominated, and their correlations with fibrosis scores were higher than other MRI measures. Multiparametric MRI may enable a more comprehensive analysis of histopathological changes following AKI.

Diffusion kurtosis imaging and arterial spin labeling for the noninvasive evaluation of persistent post-contrast acute kidney injury

ObjectiveWe investigated whether diffusion kurtosis imaging (DKI) and arterial spin labeling (ASL) facilitated the assessment of serial alterations in persistent post-contrast acute kidney injury (PC-AKI). Materials and methodsWe randomly divided 24 rats into four PC-AKI groups (days 1, 3, 7, and 13, n = 6/group), with an additional six control animals. We conducted functional magnetic resonance imaging (MRI), diffusion kurtosis imaging (DKI), and arterial spin-labeling (ASL) analyses. Mean kurtosis (MK), axial kurtosis (Ka), mean diffusivity (MD), fractional anisotropy (FA), radial kurtosis (Kr), and renal blood flow (RBF) maps were normalized to baseline (prior to contrast injection) to calculate adjusted △RBF, △MK, △Ka, △MD, △FA, and △Kr values. We also investigated urinary neutrophil gelatinase associated lipocalin (NGAL), serum cystatin C (CysC), aquaporin-2 (AQP2), hypoxia-inducible factor-1 (HIF-1α), and histological indices. ResultsIn the inner stripe of the outer medulla, when compared with controls, decreased △FA and △MD levels were observed on days 1, 3, and 7, and a distinct elevation in △MK and △Kr on days 1–13, and a persistent decrease in △RBF on days 1–13, and a prominent increase in △Ka on days 7 and 13 in PC-AKI animals (all p < 0.05). △Ka and △MK were positively correlated with AQP-2 (r = 0.8086, p < 0.0001 and r = 0.7314, p < 0.0001, respectively), and △RBF was highly correlated with HIF-1α (r = −0.7592, p < 0.0001). Moreover, both CysC and NGAL were significantly elevated in PC-AKI animals when compared with controls from days 1–13 (all p < 0.05). Renal histological data indicated severe tubular and glomerular injury at days 1–13 in all PC-AKI groups. ConclusionASL and DKI may be noninvasively and longitudinally used to detect PC-AKI and predict further outcomes.

Renal denervation inhibits the renin–angiotensin–aldosterone system in spontaneously hypertensive rats

ABSTRACT This study was conducted to explore the effect of renal denervation (RDN) on the renin–angiotensin–aldosterone system (RAAS) in spontaneously hypertensive rats (SHRs). Our experimental rats were randomly divided into the RDN group conducted by painting 10% phenol on the bilateral renal nerves (RDNX), the shamoperation group simply painting with saline (Sham), and the normotension control group (WKY) following all the animal blood and tissues of kidney, hypothalamus, and adrenal gland collected and examined 2 weeks after RDN operation. We found that the aldosterone (ALD) levels in serum and tissues all decreased in the RDNX group compared with the Sham group (p < .05). Meantime, the expression of angiotensin II type1 receptor (AT1R) mRNA also exhibited significantly reduced by 2.22-fold in the RDNX group compared to the Sham group identical to the expression of AT1R protein in the renal cortex and outer stripe of the outer medulla (OSOM) subjected to denervation surgery, which manifested the lower ATIR protein expression than the Sham group (p < .05). Besides, the expression of angiotensin II (Ang II) protein in the cortex , OSOM, and inner stripe of the outer medulla were all attenuated by RDN in comparison with the Sham group (p < .05). RDN reduced intrarenal RAAS and circulating RAAS to lower blood pressure and repair renal function.

Diffusion-weighted magnetic resonance imaging in rat kidney using two-dimensional navigated, interleaved echo-planar imaging at 7.0T.

The purpose of this study was to investigate the feasibility of two-dimensional (2D) navigated, interleaved multishot echo-planar imaging (EPI) to enhance kidney diffusion-weighted imaging (DWI) in rats at 7.0T. Fully sampled interleaved four-shot EPI with 2D navigators was tailored for kidney DWI (Sprague-Dawley rats, n = 7) on a 7.0-T small bore preclinical scanner. The image quality of four-shot EPI was compared with T2 -weighted rapid acquisition with relaxation enhancement (RARE) (reference) and single-shot EPI (ss-EPI) without and with parallel imaging (PI). The contrast-to-noise ratio (CNR) was examined to assess the image quality for the EPI approaches. The Dice similarity coefficient and the Hausdorff distance were used for evaluation of image distortion. Mean diffusivity (MD) and fractional anisotropy (FA) were calculated for renal cortex and medulla for all DWI approaches. The corticomedullary difference of MD and FA were assessed by Wilcoxon signed-rank test. Four-shot EPI showed the highest CNR among the three EPI variants and lowest geometric distortion versus T2 -weighted RARE (mean Dice: 0.77 for ss-EPI without PI, 0.88 for ss-EPI with twofold undersampling, and 0.92 for four-shot EPI). The FA map derived from four-shot EPI clearly identified a highly anisotropic region corresponding to the inner stripe of the outer medulla. Four-shot EPI successfully discerned differences in both MD and FA between renal cortex and medulla. In conclusion, 2D navigated, interleaved multishot EPI facilitates high-quality rat kidney DWI with clearly depicted intralayer and interlayer structure and substantially reduced image distortion. This approach enables the anatomic integrity of DWI-MRI in small rodents and has the potential to benefit the characterization of renal microstructure in preclinical studies.

Non-invasive investigation of early kidney damage in streptozotocin-induced diabetic rats by intravoxel incoherent motion diffusion-weighted (IVIM) MRI

BackgroundThe current study investigated the performance of intravoxel incoherent motion diffusion (IVIM) technology in monitoring early renal injury in streptozotocin rats.MethodsForty-eight Sprague-Dawley (SD) rats were divided into a control group and a diabetic mellitus (DM) group. Six rats in each group were randomly selected for MR scans at four different time points (0, 4, 8, and 12 weeks). The IVIM-derived parameters (D, D*, f and ADC values) of the renal cortex (CO), outer and inner stripe of the outer medulla (OS, IS), and internal medulla (IM) were acquired. Changes in each IVIM-derived parameter over time were analyzed, and differences between the two groups at each point were assessed. The associations between the IVIM parameters and IV collagen expression, urine volume (UV), blood urea nitrogen (BUN), and serum creatinine (Scr) were investigated.ResultsThe D and D* values of CO and the ADC values of CO, OS, IS and IM displayed significantly different trends between the two groups over time (P<0.05). In addition, significant correlations were discovered between the D* value of CO and UV and BUN (r=0.527, P=0.033; r=0.617, P=0.005), between the ADC value of IM and BUN (r=0.557, P=0.019) and between the f value of IM and BUN (r=0.527, P=0.033). No correlation was found between IVIM parameters and IV collagen expression and Scr.ConclusionsIVIM is a potential sensitive and noninvasive technology for the simultaneous assessment of early renal cortical and medullary injuries induced by diabetes.

Prevention of Renal Medullary Vascular Congestion is Associated with Reduced Mortality and Preservation of Capillary Density Following Renal Ischemia

The aggregation of red blood cells in the microvascular of the renal medulla is a hallmark of ischemic acute kidney injury (AKI). Our lab has recently shown that pretreatment with low dose lipopolysaccharide (LPS) prior to ischemia-reperfusion (IR) prevents this vascular congestion. It remains unknown, however, whether prevention of medullary vascular congestion improves recovery from IR. The goal of the current study was to test the hypothesis that ‘prevention of vascular congestion with low dose LPS improves functional recovery of the kidney and attenuates rarefaction of the renal medulla following IR’. Male WKY rats (8 weeks) were implanted with radiotelemetry devices (DSI, St. Paul, MN) and allowed to recover for two weeks prior to beginning blood pressure measurement. Rats were then randomized to receive injections (i.p.) of either low dose LPS (1000µg/kg) (n=4) to prevent vascular congestion or saline control (n=4) once daily for 3 days. 1 day following the 3rd injection, all rats underwent a 45 minute, warm bilateral IR and were allowed to recover for 7 days. Blood pressure was measured for the duration of the study. Plasma was collected by tail vein pre-IR and at sacrifice for measurement of creatinine (Cr) and blood urea nitrogen (BUN). Vascular rarefaction was assessed at sacrifice in histological kidney sections via %Threshold Area of CD34+ vascular endothelium (Metamorph). Following recovery of IR, there was a 100% and 50% survival rate in LPS treated and saline control rats, respectively. Saline control rats (with vascular congestion following IR) had a decrease in CD34+ vascular density in the inner stripe of the outer medulla (OM) (Unpaired t-test, p=0.05) and the inner medulla (Unpaired t-test, p=0.009), but there were no differences in CD34+ staining in the cortex (Unpaired t-test, p=0.159) or outer stripe of the OM (Unpaired t-test, p=0.095) (Table 1). There were no differences in 24 hour mean arterial pressure between groups over the course of the study (two-way RM ANOVA, pINTERACTION=0.689). At sacrifice, plasma Cr (pINTERACTION=0.249) and BUN (pINTERACTION=0.345) were not different between groups and had recovered to near pre-IR values. Prevention of vascular congestion in LPS treated rats increased the 7 day survival to 100% following a 45 minute bilateral IR in contrast to control rats which, as expected, had a 50% survival rate. Importantly, this prevention of congestion was associated with preserved capillary density (CD43+) in the renal medulla. These data indicate an important role of vascular congestion to independently drive increased mortality and capillary rarefaction following IR. As renal vascular rarefaction has been implicated as an important factor in the progression to chronic kidney disease following AKI, prevention of vascular congestion may be an important target to prevent this deleterious sequelae.

Physiological system analysis of the kidney by high-temporal-resolution monitoring of an oxygenation step response.

Examine the feasibility of characterizing the regulation of renal oxygenation using high-temporal-resolution monitoring of the response to a step-like oxygenation stimulus. For mapping, multi-echo gradient-echo imaging was used (temporal resolution = 9seconds). A step-like renal oxygenation challenge was applied involving sequential exposure to hyperoxia (100% O2 ), hypoxia (10% O2 + 90% N2 ), and hyperoxia (100% O2 ). In vivo experiments were performed in healthy rats (N = 10) and in rats with bilateral ischemia-reperfusion injury (N = 4). To assess the step response of renal oxygenation, a second-order exponential model was used (model parameters: amplitude [A], time delay [Δt], damping constant [D], and period of the oscillation [T]) for renal cortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and inner medulla. The second-order exponential model permitted us to model the exponential recovery and the superimposed oscillation following renal oxygenation stimulus. The in vivo experiments revealed a difference in Douter medulla between healthy controls (D < 1, indicating oscillatory recovery) and ischemia-reperfusion injury (D > 1, reflecting aperiodic recovery). The increase in Douter medulla by a factor of 3.7 (outer stripe of the outer medulla) and 10.0 (inner stripe of the outer medulla) suggests that this parameter might be rather sensitive to (patho)physiological oxygenation changes. This study demonstrates the feasibility of monitoring the dynamic oxygenation response of renal tissues to a step-like oxygenation challenge using high-temporal-resolution mapping. Our results suggest that the implemented system analysis approach may help to unlock questions regarding regulation of renal oxygenation, with the ultimate goal of providing imaging means for diagnostics and therapy of renal diseases.

Differences in acidosis-stimulated renal ammonia metabolism in the male and female kidney.

Renal ammonia excretion is a critical component of acid-base homeostasis, and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We recently reported substantial sex-dependent differences in basal ammonia metabolism that correlate with sex-dependent differences in renal structure and expression of key proteins involved in ammonia metabolism. The purpose of the present study was to investigate the effect of sex on the renal ammonia response to an exogenous acid load. We studied 4-mo-old C57BL/6 mice. Ammonia excretion, which was less in male mice under basal conditions, increased in response to acid loading to a greater extent in male mice, such that maximal ammonia excretion did not differ between the sexes. Fundamental structural sex differences in the nonacid-loaded kidney persisted after acid loading, with less cortical proximal tubule volume density in the female kidney than in the male kidney, whereas collecting duct volume density was greater in the female kidney. To further investigate sex-dependent differences in the response to acid loading, we examined the expression of proteins involved in ammonia metabolism. The change in expression of phosphoenolpyruvate carboxykinase and Rh family B glycoprotein with acid loading was greater in male mice than in female mice, whereas Na+-K+-2Cl- cotransporter and inner stripe of the outer medulla intercalated cell Rh family C glycoprotein expression were significantly greater in female mice than in male mice. There was no significant sex difference in glutamine synthetase, Na+/H+ exchanger isoform 3, or electrogenic Na+-bicarbonate cotransporter 1 variant A protein expression in response to acid loading. We conclude that substantial sex-dependent differences in the renal ammonia response to acid loading enable a similar maximum ammonia excretion response.

Expression of SLC4A11 protein in mouse and rat medulla: a candidate transporter involved in outer medullary ammonia recycling.

SLC4A11 is a multifunctional membrane transporter involved with H+ transport, NH 3 and alkaline pH stimulated H+ transport, and water transport. The role of SLC4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC4A11/LacZ staining is primarily in the long‐looped descending thin limb (DTL) as determined by colocalization with aquaporin 1 (AQP1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC4A11 in the upper DTLs (which are AQP1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTLs (which are AQP‐1‐null). SLC4A11 also colocalized with AQP1 and the urea transporter UT‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTLs. SLC4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC4A11 pathways in mouse and rat and complement previous studies of DTL Na+, urea and water permeability indicating that the upper and lower DTLs of long‐looped nephrons are functionally distinct.

Sex Differences in Acidosis‐Stimulated Renal Ammonia Metabolism

Renal ammonia excretion is a critical component in maintaining acid‐base homoeostasis and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We reported recently substantial sex‐dependent differences in basal ammonia metabolism that correlate with sex‐dependent structural differences and differences in the expression of key proteins involved in ammonia metabolism. This study's objective was to determine the effect of sex on the renal ammonia response to an exogenous acid load. We compared 4‐month old C57BL/6 male (M) and female (F) mice. Mice were acid‐loaded by adding HCl to powdered chow for 7 days, and control mice had equal amounts of deionized water added. Basal ammonia excretion, factored for body weight, was ~2‐fold greater in female than male mice, but the response to acid‐loading did not differ significantly. There was no significant sex difference in serum Na+, K+, and HCO3− after an acid load. The basal fundamental structural differences between the female and male kidney were maintained after an acid load. After acid‐loading, in the female kidney, proximal tubules account for a lower percentage of the cortical parenchyma than in the male kidney (F, 43±2; M, 61±3%; P &lt; 0.001), whereas the collecting ducts account for a greater percentage of the renal parenchyma (F, 16±1; M, 11±1%; P &lt; 0.01) which are similar to basal conditions. Cortical PCT and PST cell height increased in response to acid‐loading, and the increase was greater in M than F kidney. In the outer medulla (OM), PST cell height was similar in M and F, and sex did not alter the increase with acid‐loading. Similarly, intercalated cell (IC) size in the collecting duct in the OM was greater in F than in M kidney, and increased with acid‐loading in both sexes; sex did not alter the acid‐loading increase. Cortical expression of phosphoenol pyruvate, a major proximal tubule (PT) ammonia generating protein, was greater under control conditions in F, but the relative increase with acid‐loading was greater in M. Expression of glutamine synthetase, which recycles ammonia, significantly decreased in response to an acid load, with no significant sex difference. Expression of total cortical NBCe1, but not NBCe1‐A, increased in response to acid‐loading. Expression of NHE3, believed to be the major mechanism of PT ammonia secretion, increased in response to an acid load in M, but not in F. Cortical NKCC2, which mediates thick ascending limb ammonia reabsorption, was significantly greater in F than M after an acid load. The collecting duct secretes the majority of urinary ammonia and the Rhesus glycoproteins, Rhbg and Rhcg, are the primary collecting duct ammonia transporting proteins. Acid‐loading increased cortical Rhbg expression in M, but not in F in the cortex, whereas in the OM, Rhbg expression increased similarly in both M and F. Rhcg expression in intercalated cells in the inner stripe of the outer medulla was significantly greater in F than in M mice after an acid load. We conclude that there are substantial sex‐dependent differences in the mechanism of renal ammonia response to acid‐loading that enable a similar maximum ammonia excretion response.Support or Funding InformationNIH R01DK045788, R01DK107798 and 5T32DK104721This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis.

Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. Notch signaling viaHes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

Hydrogen Peroxide Increases Intracellular Sodium Concentration in the Medullary Thick Ascending Limb Isolated from Sprague‐Dawley Rats

Elevations of renal medullary hydrogen peroxide (H2O2) produce a salt‐sensitive form of hypertension as shown by chronic infusion of H2O2 into the renal medulla of a single remaining kidney of normotensive Sprague‐Dawley (SD) rats (Makino et al., Hypertension, 2003) and salt‐resistant consomic SS.13BN rats. Conversely, chronic medullary infusion of catalase (H2O2 scavenger) into the renal medulla of Dahl salt‐sensitive rats significantly reduced salt induced hypertension (Taylor and Cowley, Am J Physiol Regul Integr Comp Physiol, 2005). The goal of this study was to assess the effects of H2O2 upon intracellular sodium concentration [Na+]i and the role of mTORC1 and mTORC2 signaling in the medullary thick ascending limb (mTAL) isolated from the outer medulla (OM) of SD rats.Methodsrats were anesthetized, the kidney flushed with saline, removed, and mTALs dissected from the inner stripe of the OM at 4°C under a stereomicroscope. The mTALs were attached to poly‐Lysine coated cover slips and incubated with HBSS containing 10 nM of the Na+‐sensitive fluorescence dye NaGreen and 0.05% Pluronic acid F‐127 for 30 min at room temperature. The mTALs were washed and then placed in a heated chamber (37°C) mounted on the stage of an inverted Nikon TE‐2000U microscope for recording.ResultsIt was found that 100 uM H2O2 resulted in a significant increase in [Na+]i (p&lt;0.05). The responses to pretreatment of mTAL with 10 uM PP242 (mTORC1/2 inhibitor), 200 nM rapamycin (mTORC1 specific inhibitor), or 1 mM furosemide (Na+/K+/2Cl− inhibitor) were then studied. We found that each of these inhibitors nearly abolished H2O2 induced increases in intracellular Na+. Western blot analysis of isolated mTAL from SD rats for each of the treatments found that 100 uM H2O2 increased the activation of AKT (pAKTSer473; a surrogate for mTORC1/2 activation) and S6 (pS6Ser235/236; a surrogate for mTORC1 activation) and were inhibited by their respective inhibitors.ConclusionTogether these observations indicate that H2O2 acts through the mTOR signaling pathways to inhibit NKCC2 activity, which would in turn be expected to lead to sodium retention and hypertension.Support or Funding InformationHL 122662This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Body Mass‐Specific Na, K‐ATPase Activity in the Medullary Thick Ascending Limb – Implications for Species‐Dependent Urine Concentrating Mechanisms

In general, the mammalian whole body mass‐specific metabolic rate correlates positively with maximal urine concentration (Umax), irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass‐specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na, K‐ATPase activity and Na, K‐ATPase α1 gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na, K‐ATPase activity (~70%) compared to two rat strains (Sprague‐Dawley and Munich‐Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na, K‐ATPase α1 protein (~4–6‐fold) and mRNA (~13‐fold) and higher TAL mitochondrial volume density (~20%) were seen in the kangaroo rat compared to both rat strains. Rat TAL Na, K‐ATPase α1 protein expression is relatively unaffected by body hydration status or, as shown previously, by dietary Na, arguing against confounding effects from two unavoidably dissimilar diets: grain‐based diet without water (kangaroo rat) or grain‐based diet with water (rat). We conclude that higher TAL Na, K‐ATPase activity contributes to relationships between whole body mass‐specific metabolic rate and high Umax. With the combination of 70% greater Na, K‐ATPase activity and 50% greater total kidney mass:body mass ratio for the kangaroo rat compared to the rat, and assuming all other factors remain equal, the contribution of TAL Na, K‐ATPase activity to overall whole body metabolism would be about 2.5‐fold greater in the kangaroo rat compared to the rat. More vigorous TAL Na, K‐ATPase activity in the kangaroo rat than in the rat may contribute to its steeper Na and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.Support or Funding InformationNIDDK DK083338 (TP) and DK110621 (TR), NSF IOS‐0952885 (TP), Joint DMS/NIGMS‐NSF DMS‐1263943 (TP), APS/NSF Fellowships (MA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of aquaporins and apparent diffusion coefficient from ultra-high b-values in a rat model of diabetic nephropathy

BackgroundTo assess kidney damage in a rat model of type-2 diabetic nephropathy based on apparent diffusion coefficient (ADC) data obtained from ultra-high b-values and discuss its relationship to the expression of aquaporins (AQPs).MethodsThis study was approved by the institutional Animal Care and Use Committee. Thirty male Sprague-Dawley rats were randomised into two groups: (1) untreated controls and (2) diabetes mellitus (DM). All rats underwent diffusion-weighted imaging (DWI) with 18 b-values (0–4500 s/mm2). Maps of low ADC (ADClow), standard ADC (ADCst) and ultra-high ADC (ADCuh) were calculated from low b-values (0–200 s/mm2), standard b-values (300–1500 s/mm2) and ultra-high b-values (1700–4500 s/mm2), respectively. The expression of AQPs in the kidneys was studied using immunohistochemistry. Laboratory parameters of diabetic and kidney functions, ADClow, ADCst, ADCuh, and the optical density (OD) of AQP expression in the two groups were compared using an independent t test. Correlations between ADCs and the OD of AQP expression were evaluated by Pearson’s correlation analysis.ResultsADCuh were significantly higher in the cortex (CO), outer stripe of the outer medulla (OS) and inner stripe of the outer medulla (IS), and the OD values of AQ-2 were significantly higher in the OS, IS and inner medulla (IM) in DM animals compared with control animals. ADCuh and OD values of AQP-2 expression were positively correlated in the OS, IS and IM of the kidney.ConclusionsADCuh may work as useful metrics for early detection of kidney damage in diabetic nephropathy and may be associated with AQP-2 expression.