Outer 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.

The Protective Role of Tubular Epithelial Cells Produced Fms-like Tyrosine Kinase Receptor 3 Ligand in Acute Kidney Injury

Introduction: Dendritic cells (DC) accumulate in the kidneys of patients with acute kidney injury (AKI), but the Fms-like tyrosine kinase receptor 3 ligand (Flt3L)-dendritic cell axis is poorly defined in kidneys with AKI. Meanwhile it has not been reported whether Flt3L is produced by kidney cells under ischemic-reperfusion injury (IR) induced microenvironment. Methods: (1) Blood specimens and urine were collected from two patient cohorts: a) patients with pre-renal AKI and healthy controls for analysis of serum Flt3L. b) patients with acute myocardial infarction (AMI) developed with or without AKI on admission for analysis of serum and urine Flt3L. (2) Wild type mice and T cell-deficient mice ( Tcra -/- mice) were applied. IR induced AKI mice and hypoxia-reoxygenation stimulated HK-2 cell line (human tubular epithelial cell line) were constructed. The expression of Flt3L in kidney, tubular epithelial cells, and T cells were measured by RT-PCR, ELISA, immunohistochemistry, immunofluorescence staining, RNAscope, and flow cytometry. (3) Wild type mice and type I conventional dendritic cell (cDC1)-deficient mice ( Irf8 -/- mice) were treated with 15 μg rFlt3L or 10mg/kg gilteritinib (Flt3 inhibitor). They were subjected to IR. Serum and kidney tissues were collected. Kidney function, tubular injury, and DC accumulation were quantified. Results: Initially, among patients with pre-renal AKI, we observed an increased level of serum Flt3L within two days after admission. Another patient cohort revealed that this increase was specific for AMI patients with AKI rather than without AKI or healthy controls. Furthermore, among these AMI patients, serum Flt3L levels correlated with Creatinine increase (R 2 = 0.4532, p = 0.0002) and BUN increase (R 2 = 0.3417, p = 0.0017) within 48 hours after admission. Similarly, urine Flt3L levels among these AMI patients also steadily elevated with Creatinine increase (R 2 = 0.6395, p &amp;lt; 0.0001) and BUN increase (R 2 = 0.5115, p = &amp;lt; 0.0001) within 48 hours after admission. Flt3L was also enhanced in wild type mice with IR-AKI but not affected in T cell-deficient mice ( Tcra -/-) with IR-AKI. Among kidney tissue, Flt3L-positive cells scattered within tubules in outer stripe of the outer medulla (OSOM) and mRNA and protein increased within 24 hours after IR-AKD. In vitro, FLT3L mRNA was markedly induced in HK-2 cells during hypoxia and FLT3L protein remained enhanced during reoxygenation. Phenotypically, the numbers of renal cDC1 and CD64 +DC were significantly decreased in gilteritinib-treated IR-AKI mice, which were associated with more severe tubular injury. With reduced kidney cDC1, Irf8 -/- mice also showed worsen kidney injury and aggravated functional failure upon IR-AKI. Therapeutic administration of rFlt3L significantly increased kidney cDC1 and CD64 +DC upon IR-AKI in wild type but not Irf8 -/- mice. This was associated with significantly reduced tubular injury of wild type mice .In addition, pre-treatment of wild type mice with rFlt3L increased kidney cDC1 and protected the mice from severe IR-AKI. Conclusion: Flt3L is upregulated in human and mice and produced by tubular epithelial cells during IR-AKI. It fosters the accumulation of kidney cDC1 and CD64 +DC, thereby limiting the severity of kidney injury in mice. The current study implies the possibility of DC-based immunotherapy for treatment of AKD.

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.

Site-specific genotoxicity of rubiadin: localization and histopathological changes in the kidneys of rats.

Rubiadin (Rub) is a genotoxic component of madder color (MC) that is extracted from the root of Rubia tinctorum L. MC induces renal tumors and preneoplastic lesions that are found in the proximal tubule of the outer stripe of the outer medulla (OSOM), suggesting that the renal carcinogenicity of MC is site specific. To clarify the involvement of Rub in renal carcinogenesis of MC, we examined the distribution of Rub in the kidney of male gpt delta rats that were treated with Rub for 28days. We used desorption electrospray ionization quadrupole time-of-flight mass spectrometry imaging (DESI-Q-TOF-MSI), along with the histopathological analysis, immunohistochemical staining, and reporter gene mutation assays of the kidney. DESI-Q-TOF-MSI revealed that Rub and its metabolites, lucidin and Rub-sulfation, were specifically distributed in the OSOM. Histopathologically, karyomegaly characterized by enlarged nuclear and microvesicular vacuolar degeneration occurred in proximal tubule epithelial cells in the OSOM. The ɤ-H2AX- and p21-positive cells were also found in the OSOM rather than the cortex. Although dose-dependent increases in gpt and Spi- mutant frequencies were observed in both the medulla and cortex, the mutant frequencies in the medulla were significantly higher. The mutation spectra of gpt mutants showed that A:T-T:A transversion was predominant in Rub-induced gene mutations, consistent with those of MC. Overall, the data showed that the distribution of Rub and its metabolites resulted in site-specific histopathological changes, DNA damage, and gene mutations, suggesting that the distribution of genotoxic components and metabolites is responsible for the site-specific renal carcinogenesis of MC.

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.

Discrete localization of phospholipase Cβ3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.

Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCβ3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCβ3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCβ3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCβ3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.

The Tubular Cells Produce Fms-like Tyrosine Kinase Receptor 3 Ligand to Recover Kidney Function after Acute Kidney Injury Disease

INTRODUCTION: Dendritic cells (DC) are involved in the recovery process of acute kidney injury disease (AKD). Fms-like tyrosine kinase receptor 3 ligand (Flt3L) is critical for instructing DC generation and maintenance in organs. In healthy kidney, nonhematopoietic cells produce most Flt3L, rather than immune cells. However, it remains unknown which cells produce Flt3L, and which cellular source drives DC development during AKD. Further, We hypothesized that tubular cells probably produce Flt3L, which improves kidney function after AKD by fostering the accumulation of DC. METHODS: Blood specimens were collected from patients of prerenal or renal AKD and healthy controls for analysis of serum Flt3L and DC subsets. Wild type mice and IRF8KO mice were treated with rFlt3L or Flt3 inhibitor gilteritinib. They were subjected to unilateral ischemia-reperfusion (IR) to induce post-ischemic AKD or were injected with cisplatin to induce nephrotoxic AKD. Serum and kidneys were collected and analyzed. Kidney function, tubular injury, primary/proximal tubular cell numbers and Flt3L expression were quantified. RESULTS: Initially, we observed an increased level of serum Flt3L in patients with AKD. Further analysis revealed that this increase was specific for trauma- rather than heart failure- or sepsis-induced AKD. A similar increase in serum Flt3L was found in mice with IR-AKD but not cisplatin-induced AKD. Flow cytometric analysis revealed a reduced expression level of Flt3L among intrarenal CD4-CD8+T, CD4+CD8-T and, CD4+CD8+T cells. However, the level of serum Flt3L among Tcrαko mice showed no difference with wild type mice after IR-AKD. Conversely, Flt3L-positive cells scattered within tubules in outer stripe of the outer medulla (OSOM). Both mRNA and protein increased within 24 hours after IRI-AKD in renal tissue, which were confirmed by immunohistochemistry staining, in situ hybridization staining, PCR and, RNA-sequencing data of IR kidneys. This was accompanied by reduced peripheral blood DC but increased kidney DC in mice after IR-AKD. The numbers of type I conventional dendritic cell (cDC1) and CD64+DC in kidney were significantly decreased in gilteritinib-treated IR-AKD mice, which were associated with more severe tubular injury. With reduced kidney cDC1, IRF8KO mice also showed worsen kidney injury and aggravated functional failure upon IR-AKD. Therapeutic administration of rFlt3L significantly increased kidney cDC1 upon IR-AKD in wild type but not IRF8KO mice. This was associated with significantly reduced tubular injury, enhanced proliferation activity of tubular cells, and decreased expressions of tubular injury markers (Ngal, Timp-2, Igfbp-7 and Hgf) in kidney of wild type mice. In addition, pre-treatment of wild type mice with rFlt3L increased kidney cDC1 and protected the mice from severe IR-AKD. CONCLUSION: Flt3L is upregulated in humans and mice during IR-AKD and probably produced by tubules in OSOM. It fosters the accumulation of kidney cDC1 and, thereby limiting the severity of kidney injury in mice. The current study implies the possibility of Flt3L-dependent DC-based immunotherapy for treatment of AKD.

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.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and β-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

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.

Mitochondria-targeted antioxidant MitoQ reduced renal damage caused by ischemia-reperfusion injury in rodent kidneys: Longitudinal observations of T2 -weighted imaging and dynamic contrast-enhanced MRI.

PurposeTo investigate the effect of mitochondria‐targeted antioxidant MitoQ in reducing the severity of renal ischemia‐reperfusion injury (IRI) in rats using T2‐weighted imaging and dynamic contrast‐enhanced MRI (DCE‐MRI).MethodsIschemia‐reperfusion injury was induced by temporarily clamping the left renal artery. Rats were pretreated with MitoQ or saline. The MRI examination was performed before and after IRI (days 2, 5, 7, and 14). The T2‐weighted standardized signal intensity of the outer stripe of the outer medulla (OSOM) was measured. The unilateral renal clearance rate kcl was derived from DCE‐MRI. Histopathology was evaluated after the final MRI examination.ResultsThe standardized signal intensity of the OSOM on IRI kidneys with MitoQ were lower than those with saline on days 5 and 7 (P = 0.004, P < 0.001, respectively). Kcl values of IRI kidneys with MitoQ were higher than those with saline at all time points (P = 0.002, P < 0.001, P = 0.001, P < 0.001). Histopathology showed that renal damage was the most predominant on the OSOM of IRI kidneys with saline, which was less obvious with MitoQ (P < 0.001).ConclusionsThese findings demonstrate that MitoQ can reduce the severity of renal damage in rodent IRI models using T2‐weighted imaging and DCE‐MRI. Magn Reson Med 79:1559–1667, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Protective effect of adipose-derived mesenchymal stem cells against acute kidney injury induced by ischemia-reperfusion in Sprague-Dawley rats.

Acute kidney injury (AKI) is a complex clinical condition associated with significant morbidity and mortality and lacking effective management. Ischemia-reperfusion injury (IRI) remains one of the leading causes of AKI in native and transplanted kidneys. The aim of this study was to evaluate the efficacy of adipose-derived mesenchymal stem cells (ADSCs) in the prevention of renal IRI in rats. The study was conducted on male Sprague-Dawley rats (n=72) weighing 250–300 g. Rats were randomly assigned to three main groups: i) Sham-operated control group (n=24); ii) positive control group, in which rats were subjected to IRI and were administered culture media following 4 h of IRI (n=24); and iii) ADSC group (n=24), in which rats were administered 1×106 ADSCs via the tail vein following 4 h of IRI. Each main group was further divided according to the timing after IRI into four equal-sized subgroups. Renal function was tested via the measurement of serum creatinine levels and creatinine clearance. In addition, malondialdehyde (MDA) levels were determined in serum and renal tissue homogenate as an indicator of oxidative stress. Histopathological changes were analyzed in different regions of the kidney, namely the cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM) and inner medulla. In each region, the scoring system considered active injury changes, regenerative changes and chronic changes. The ADSCs were assessed and their differentiation capability was verified. IRI resulted in a significant increase in serum creatinine, serum and tissue MDA levels and a significant reduction in creatinine clearance compared with those in sham-operated rats,. These changes were attenuated by the use of ADSCs. The prominent histopathological changes in the cortex, ISOM and OSOM were reflected in the injury score, which was significantly evident in the positive control group. The use of ADSCs was associated with significantly lowered injury scores at days 1 and 3; however, no significant effect was observed on day 7. These results indicate that the use of ADSCs ameliorates renal injury and dysfunction associated with IRI in rats.

Effect of anesthesia on renal R2 * measured by blood oxygen level-dependent MRI.

Blood oxygen level-dependent (BOLD) MRI is increasingly being used to assess renal tissue oxygenation during disease based on the transverse relaxation rate (R2 *). In preclinical small animal models, the requisite use of anesthesia during imaging may lead to functional changes which influence R2 * and confound results. The purpose of this study was to evaluate the effects of four common anesthetic compounds on renal R2 * in healthy mice. Five female ICR mice were imaged with BOLD MRI approximately 25 min after induction with isoflurane (Iso; 1% or 1.5%, delivered in 100% O2 ), ketamine/xylazine (KX), sodium pentobarbital (PB) or 2,2,2-tribromoethanol (TBE). A significant effect of anesthetic agent on R2 * was observed in all tissue layers of the kidney, including the cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM) and inner medulla (IM). Pairwise significant differences in R2 * between specific agents were found in the cortex, OSOM and ISOM, with the largest difference observed in the ISOM between 1.5% Iso (26.6 ± 1.7 s(-1) ) and KX (66.0 ± 7.1 s(-1) ). The difference between 1% Iso and KX in the ISOM was not abolished when KX was administered with supplemental 100% O2 or when 1% Iso was delivered in 21% O2 , indicating that the fraction of inspired oxygen did not account for the observed differences. Our results indicate that the choice of anesthesia has a large influence on the observed R2 * in mouse kidney, and anesthetic effects must be considered in the design and interpretation of renal BOLD MRI studies.

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity

Cisplatin is a commonly used chemotherapeutic drug, the clinical use of which is limited by the development of dose‐dependent nephrotoxicity. Recent studies have suggested that mitochondrial dysfunction in tubular epithelium contributes to cisplatin‐induced nephrotoxicity (PMID: 22120494). Here we extend those findings by describing the role of an important electron transport chain enzyme, cytochrome c oxidase (COX). Immunohistochemistry for COX 1 protein demonstrated that, in response to cisplatin, expression was mostly maintained in focally damaged tubular epithelium. In contrast, COX enzyme activity in proximal tubules (by light microscopy) was decreased. Ultrastructural analysis of the cortex and outer stripe of the outer medulla (OSOM) showed decreased mitochondrial mass, disruption of cristae and extensive mitochondrial swelling in proximal tubular epithelium. Functional electron microscopy (EM) showed that COX enzyme activity was decreased in the remaining mitochondria in the proximal tubules, but maintained in distal tubules. A single systemic dose of mitochondrially targeted anti‐oxidant prevented mitochondrial injury and dysfunction. In summary, cisplatin induced nephrotoxicity is associated with structural and functional damage to the mitochondria. Mitochondrially targeted antioxidants may offer a novel therapeutic approach to prevent cisplatin nephrotoxicity.

Cellular distribution of cell cycle-related molecules in the renal tubules of rats treated with renal carcinogens for 28 days: relationship between cell cycle aberration and carcinogenesis

Some renal carcinogens can induce karyomegaly, which reflects aberrant cell division in the renal tubules, from the early stages of exposure. To clarify the cell cycle-related changes during the early stages of renal carcinogenesis, we performed immunohistochemical analysis of tubular cells in male F344 rats treated with carcinogenic doses of representative renal carcinogens for 28 days. For this purpose, the karyomegaly-inducing carcinogens ochratoxin A (OTA), ferric nitrilotriacetic acid, and monuron, and the non-karyomegaly-inducing carcinogens tris(2-chloroethyl) phosphate and potassium bromate were examined. For comparison, a karyomegaly-inducing non-carcinogen, p-nitrobenzoic acid, and a non-carcinogenic non-karyomegaly-inducing renal toxicant, acetaminophen, were also examined. The outer stripe of the outer medulla (OSOM) and the cortex + OSOM were subjected to morphometric analysis of immunoreactive proximal tubular cells. Renal carcinogens, irrespective of their karyomegaly-inducing potential, increased proximal tubular cell proliferation accompanied by an increase in topoisomerase IIα-immunoreactive cells, suggesting a reflection of cell proliferation. Karyomegaly-inducing carcinogens increased nuclear Cdc2-, γH2AX-, and phosphorylated Chk2-immunoreactive cells in both areas, the former two acting in response to DNA damage and the latter one suggestive of sustained G₂. OTA, an OSOM-targeting carcinogen, could easily be distinguished from untreated controls and non-carcinogens by evaluation of molecules responding to DNA damage and G₂/M transition in the OSOM. Thus, all renal carcinogens examined facilitated proximal tubular proliferation by repeated short-term treatment. Among these, karyomegaly-inducing carcinogens may cause DNA damage and G₂ arrest in the target tubular cells.

Fasting induces the expression of PGC-1α and ERR isoforms in the outer stripe of the outer medulla (OSOM) of the mouse kidney.

BackgroundPeroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a member of the transcriptional coactivator family that plays a central role in the regulation of cellular energy metabolism under various physiological stimuli. During fasting, PGC-1α is induced in the liver and together with estrogen-related receptor a and γ (ERRαand ERRγ, orphan nuclear receptors with no known endogenous ligand, regulate sets of genes that participate in the energy balance program. We found that PGC-1α, ERRα and ERRγ was highly expressed in human kidney HK2 cells and that PGC-1α induced dynamic protein interactions on the ERRα chromatin. However, the effect of fasting on the expression of endogenous PGC-1α, ERRα and ERRγ in the kidney is not known.Methodology/Principal FindingsIn this study, we demonstrated by qPCR that the expression of PGC-1α, ERRα and ERRγ was increased in the mouse kidney after fasting. By using immunohistochemistry (IHC), we showed these three proteins are co-localized in the outer stripe of the outer medulla (OSOM) of the mouse kidney. We were able to collect this region from the kidney using the Laser Capture Microdissection (LCM) technique. The qPCR data showed significant increase of PGC-1α, ERRα and ERRγ mRNA in the LCM samples after fasting for 24 hours. Furthermore, the known ERRα target genes, mitochondrial oxidative phosphorylation gene COX8H and the tricarboxylic acid (TCA) cycle gene IDH3A also showed an increase. Taken together, our data suggest that fasting activates the energy balance program in the OSOM of the kidney.

Na+/K+-ATPase stabilization by Hsp70 in the outer stripe of the outer medulla in rats during recovery from a low-protein diet

A low-protein (LP) diet induces injury from energy depletion in renal epithelial cells. Overexpression of heat-shock proteins has been implicated in the restoration of the cytoskeletal anchorage of Na(+)/K(+)-ATPase. We tested if Hsp70 stabilizes renal Na(+)/K(+)-ATPase attachment to the cytoskeleton from the cortex and the outer stripe of the outer medulla (OSOM) in rats during recovery from a LP diet. Rats were fed with a LP diet (8% protein) for 14 days, and then the rats were recovered with a 24% protein (RP) diet. The control group received a 24% protein (NP) diet. Increased Na(+)/K(+)-ATPase dissociation was demonstrated in soluble fraction from OSOM with lower ATP content as a result of LP diet vs NP. Meanwhile, decreased Hsp70 levels in the same fraction were shown. Translocation of Hsp70 to the cytoskeletal injured fraction associated with stabilization of Na(+)/K(+)-ATPase was shown in OSOM from LP after in vitro co-incubation of the cytoskeletal fraction of LP and non-cytoskeletal fraction of RP. These effects were abolished by the addition of the anti-Hsp70 antibody. Absence of Na(+)/K(+)-ATPase detachment from its cytoskeletal anchorage was demonstrated in proximal duct segments from cortex in LP. Co-immunoprecipitation showed that the amount of Na(+)/K(+)-ATPase co-precipitating with Hsp70 increased in the OSOM as a result of the LP diet. In the cortex tissues from rats fed the LP and the RP diet, the interaction of both proteins were similar to the control groups. Our results indicate that Hsp70 has a critical role in protecting the integrity of the cytoskeletal anchorage of Na(+)/K(+)-ATPase during recovery from ATP-depleted injury resulting from LP in OSOM.

Ochratoxin A: 13-Week Oral Toxicity and Cell Proliferation in Male F344/N Rats

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 microg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 microg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA. Since the mechanism of OTA carcinogenicity is still largely unknown, this study was conducted to investigate early functional and pathological effects of OTA and to determine if sustained stimulation of renal cell proliferation plays a role. Male F344/N rats were treated with OTA for up to 13 weeks under conditions of the National Toxicology Program (NTP) bioassay. Cell proliferation in the renal cortex and outer stripe of the outer medulla (OSOM) was determined using bromodeoxyuridine incorporation and immunohistochemistry. Histopathological examination showed renal alterations in mid- and high-dose-treated animals involving single-cell death and prominent nuclear enlargement within the straight proximal tubules. Treatment with OTA at doses of 70 and 210 microg/kg bw led to a marked dose- and time-dependent increase in renal cell proliferation, extending from the medullary rays into the OSOM. No effects were evident in kidneys of low-dose-treated animals or in the liver, which is not a target for OTA carcinogenicity. A no observed effect level in this study was established at 21 microg/kg bw, correlating with the dose in the NTP 2-year bioassay that did not produce renal tumors. The apparent correlation between enhanced cell turnover and tumor formation induced by OTA indicates that stimulation of cell proliferation may play an important role in OTA carcinogenicity and provides further evidence for an epigenetic, thresholded mechanism.

Hydroquinone: An Evaluation of the Human Risks from its Carcinogenic and Mutagenic Properties

The toxicology of hydroquinone has been reviewed on a number of previous occasions. This review targets its potential for carcinogenicity and possible modes of carcinogenic action. The evaluation made by IARC (1999) of its carcinogenic risk to humans was that hydroquinone is not classifiable as to its carcinogenicity to humans (Group 3). This evaluation was based on inadequate evidence in humans and limited evidence in experimental animals. The epidemiological information comes from four cohort studies involving occupational exposures. A cohort of lithographers, some of whom had worked with hydroquinone, had an excess of malignant melanoma based on five cases, but only two of the cases had reported exposure to hydroquinone. In a study of photographic processors the number of exposed individuals was uncertain and the numbers of cases of individual cancer sites were small. In view of the statistical power limitations of these studies for individual diagnostic categories of cancers, they are not considered to be informative with regard to the carcinogenicity of hydroquinone. A cohort of workers with definite and lengthy exposure to hydroquinone, during either its manufacture or its use, had low cancer rates compared with two comparison populations; the reason for the lower than expected rates is unclear. In a motion picture film processing cohort there were significant excess malignancies of the respiratory system among workers engaged in developing, where there was exposure to hydroquinone as well as other chemicals. There was no information on tobacco smoking habits and no dose-response relationship. Hydroquinone has been shown reproducibly to induce benign neoplasms in the kidneys of male F344 rats dosed orally either by gavage (25 and 50 mg/kg body weight) or diet (0.8%). The gavage study has been evaluated in considerable detail. This evaluation showed that all renal tubule adenomas and all cases of renal tubule atypical hyperplasia occurred in areas of severe or end-stage chronic progressive nephropathy and that the neoplasms were not otherwise confined to any particular part of the kidney. It is likely that the mode of carcinogenic action of hydroquinone is exacerbation of this natural disease process. Hydroquinone is mutagenic in vitro and in vivo, having caused genotoxicity or chromosomal aberrations in rodent bone-marrow cells. At least a portion, if not all, of the chromosomal effects are caused by interference by hydroquinone or its metabolites with chromosomal segregation, probably due to interaction with mitotic spindle proteins. However, the dose routes used to demonstrate these effects in almost all of the studies in vivo were intraperitoneal or subcutaneous injection, which were considered inappropriate. There were five studies by the oral route. These included a mouse bone-marrow cell micronucleus test in which a weak, marginally positive response was obtained following a single oral dose of 80 mg/kg body weight. The remaining oral route studies all showed no significant effect. They included a mouse bone-marrow cell micronucleus test in which there was no genotoxic activity after exposure to a diet containing 0.8% hydroquinone for 6 days; two 32P-post-labeling assays, one with targets of Zymbal gland, liver, and spleen in Sprague-Dawley rats, the other with the kidney as target in F344 rats; and the last oral assay was for 8-hydroxydeoxyguanosine adducts in F344 rat kidney DNA. Thus, the evidence (and the database) for any genotoxic effect in vivo is sparse and none has been observed in kidney. While glutathione conjugates could be responsible for the tumor induction, careful histology seems to show that the most actively toxic of several glutathione compounds tested, 2,3,5-triglutathion-S-yl hydroquinone, targets a very specific region of the kidney, the outer stripe of the outer medulla (OSOM), whereas hydroquinone-associated adenomas are more randomly distributed and occur in the cortex as well as the medulla. A nongenotoxic mode of action that involves exacerbation of a spontaneously occurring rodent renal disease, chronic progressive nephropathy (CPN), is proposed and evaluated. This disease is particularly prominent in male rats and the evidence is consistent with an absence of any human counterpart; therefore, the increased incidence of renal tubule adenomas in hydroquinone-dosed male rats is without human consequence.