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A detailed knowledge of the lipid composition of components of nephrons is crucial for understanding physiological processes and the development of kidney diseases. However, the lipidomic composition of kidney tubular segments is unknown. We manually isolated the proximal convoluted tubule (PCT), the cortical thick ascending limb of Henle's loop, and the cortical collecting duct from 5 lean and obese mice and subjected the samples to shotgun lipidomics analysis by high-resolution mass spectrometry acquisition. Across all samples, more than 500 lipid species were identified, quantified, and compared. We observed significant compositional differences among the 3 tubular segments, which serve as true signatures. These intrinsic lipidomic features are associated with a distinct proteomic program that regulates highly specific physiological functions. The distinctive lipidomic features of each of the 3 segments are mostly based on the relative composition of neutral lipids, long-chain polyunsaturated fatty acids, sphingolipids, and ether phospholipids. These features support the hypothesis of a lipotype assigned to specific tubular segments. Obesity profoundly impacts the lipotype of PCT. In conclusion, we present a comprehensive lipidomic analysis of 3 cortical segments of mouse kidney tubules. This valuable resource provides unparalleled detail that enhances our understanding of tubular physiology and the potential impact of pathological conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

The controlled self-assembly of peptide- and protein-based pharmaceuticals is of central importance for their mode of action and tuning of their properties. Peptide YY 3-36 (PYY 3-36 ) is a 36-residue peptide hormone that reduces food intake when peripherally administered. Herein, we describe the synthesis of a PYY 3-36 analogue functionalized with a metal-ion-binding 2,2'-bipyridine ligand that enables self-assembly through metal complexation. Upon addition of Cu II , the bipyridine-modified PYY 3-36 peptide binds stoichiometric quantities of metal ions in solution and contributes to the organization of higher-order assemblies. In this study, we aimed to explore the size effect of the self-assembly in vivo by using non-invasive quantitative single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. For this purpose, bipyridine-modified PYY 3-36 was radiolabeled with a chelator holding 111 In III , followed by the addition of Cu II to the bipyridine ligand. SPECT/CT imaging and biodistribution studies showed fast renal clearance and accumulation in the kidney cortex. The radiolabeled bipyridyl-PYY 3-36 conjugates with and without Cu II presented a slightly slower excretion 1 h post injection compared to the unmodified-PYY 3-36 , thus demonstrating that higher self-assemblies of the peptide might have an effect on the pharmacokinetics., (© 2020 Wiley-VCH GmbH.)

High-resolution magic-angle-spinning 1 H NMR spectroscopy (HR-MAS NMR) is a well-established technique for assessing the biochemical composition of intact tissue samples. In this study, we utilized a method based on HR-MAS NMR spectroscopy with slice localization (SLS) to achieve spatial resolution of metabolites. The obtained 7 slice spectra from each of the model samples (i.e., chicken thigh muscle with skin and murine renal biopsy including medulla (M) and cortex (C)) showed distinct metabolite compositions. Furthermore, we analyzed previously acquired 1 H HR-MAS NMR spectra of separated cortex and medulla samples using multivariate statistical methods. Concentrations of glycerophosphocholine (GPC) were found to be significantly higher in the renal medulla compared to the cortex. Using GPC as a biomarker, we identified the tissue slices that were predominantly the cortex or medulla. This study demonstrates that HR-MAS SLS combined with multivariate statistics has the potential for identifying tissue heterogeneity and detailed biochemical characterization of complex tissue samples.

Heavy metals, including cadmium (Cd), lead (Pb) and mercury (Hg) act as nephrotoxic agents, particularly in the renal cortex. The aim of the study was to determine the concentrations of Cd, Pb and Hg in kidneys removed from patients due to lesions of various etiologies and from patients after the rejection of transplanted kidneys. Additionally, we determined the influence of selected biological and environmental factors on the concentrations of toxic metals. The study material consisted of kidneys with tumor lesions (n = 27), without tumors (n = 7) and its extracted grafts (n = 10) obtained from patients belongs to the north-western areas of Poland. The determined metal concentrations in the renal cortex and medulla may be arranged in the following descending order: Cd > Pb > Hg. The highest concentrations of Cd and Hg were found in the cortex, while the maximum content Pb was observed in the medulla. Significant correlations were found in the concentrations of the same metals between cortex and medulla and between Pb and Hg in the renal medulla. Pb content was higher in the renal medulla of men than in the cortex of the elderly (above 60 years of age). The highest concentrations of Pb and Hg were found in the cortex and medulla, of the kidneys had not neoplastic changes, and lower content of these metals were found in the extracted kidney grafts. In summary, renal grafts accumulate less heavy metals than cancerous kidneys, what could have been caused by immunosuppressors taken by the graft recipients. Moreover, sex, age and smoking are key factors responsible for xenobiotics concentrations.

In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver ( n = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex ( n = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney-specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo., (Copyright © 2017 by The Author(s).)

Background: Individuals without occupational exposure are exposed to mercury (Hg) from diet and dental amalgam. The kidney is a critical organ, but there is limited information regarding the relationship between Hg in kidney (K-Hg), urine (U-Hg), blood (B-Hg), and plasma (P-Hg)., Objectives: The aim was to determine the relationship between K-Hg, U-Hg, B-Hg, and P-Hg among environmentally exposed individuals, estimate the biological half-time of K-Hg, and provide information useful for biomonitoring of Hg., Methods: Kidney cortex biopsies and urine and blood samples were collected from 109 living kidney donors. Total Hg concentrations were determined and the relationships between K-Hg, U-Hg, P-Hg, and B-Hg were investigated in regression models. The half-time of K-Hg was estimated from the elimination constant., Results: There were strong associations between K-Hg and all measures of U-Hg and P-Hg (r p =0.65-0.84, p<0.001), while the association with B-Hg was weaker (r p =0.29, p=0.002). Mean ratios between K-Hg (in μg/g) and U-Hg/24h (in μg) and B-Hg (in μg/L) were 0.22 and 0.19 respectively. Estimates of the biological half-time varied between 30 and 92days, with significantly slower elimination in women. Adjusting overnight urine samples for dilution using urinary creatinine resulted in less bias in relation to K-Hg or U-Hg/24h, compared with other adjustment techniques., Conclusions: The relationship between K-Hg and U-Hg is approximately linear. K-Hg can be estimated using U-Hg and gender. Women have longer half-time of Hg in kidney compared to men. Adjusting overnight urine samples for creatinine concentration resulted in less bias., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are organ and process specific. Here, we have used different tissues from a controlled porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60min) and 4, 11 and 16h post-reperfusion, and renal cortex samples after 24h of reperfusion. Peptides were analyzed on the Q-Exactive™. In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighted by transcription factors and epithelial adherens junction proteins. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that may provide a set of targets for kidney injury. Additionally, we identified 49, being 4 predominantly renal, proteins in urine. As prove of concept, we validated one of the identified biomarkers, dipeptidyl peptidase IV, in a set of patients with diabetic nephropathy. In conclusion, we identified 55 systemic proteins, some of them predominantly renal, candidates for biomarkers of renal disease., Biological Significance: The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. Here we propose a comprehensive, sequential screening procedure able to identify and validate potential biomarkers for kidney disease, using kidney ischemia/reperfusion as a paradigm for a kidney pathological event., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Here we assessed the ability of an automated sample preparation device equipped with disposable microcolumns to prepare mass-limited samples for high-sensitivity quantitative proteomics, using both label-free and isobaric labeling approaches. First, we compared peptide label-free quantification reproducibility for 1.5-150 μg of cell lysates and found that labware preconditioning was essential for reproducible quantification of <7.5 μg digest. Second, in-solution and on-column tandem mass tag (TMT) labeling protocols were compared and optimized for 1 μg of sample. Surprisingly, standard methods for in-solution and on-column labeling showed poor TMT labeling (50-85%); however, novel optimized and automated protocols restored efficient labeling to >98%. Third, compared with a single long gradient experiment, a simple robotized high-pH fractionation protocol using only 6 μg of starting material doubled the number of unique peptides and increased proteome coverage 1.43-fold. To facilitate the analysis of heterogeneous tissue samples, such as those obtained from laser capture microdissection, a modified BCA protein assay was developed that consumes and detects down to 15 ng of protein. As a proof-of-principle, the modular automated workflow was applied to 0.5 and 1 mm 2 mouse kidney cortex and medulla microdissections to show the method's potential for real-life small sample sources and to create kidney substructure-specific proteomes.

The paper describes a novel approach to investigating Wilms' tumour (nephroblastoma) biology at the atomic level. Isotope Ratio Mass Spectrometry (IRMS) was used to directly assess the isotope ratios of nitrogen and carbon in 84 Wilms' tumour tissue samples from 28 cases representing the histological spectrum of nephroblastoma. Marked differences in nitrogen and carbon isotope ratios were found between nephroblastoma histological types and along the course of cancer disease, with a breakout in isotope ratio of the examined elements in tumour tissue found between stages 2 and 3. Different isotopic compositions with regard to nitrogen and carbon content were observed in blastemal Wilms' tumour, with and without focal anaplasia, and in poorly- and well-differentiated epithelial nephroblastoma. This first assessment of nitrogen and carbon isotope ratio reveals the previously unknown part of Wilms' tumour biology and represents a potential novel biomarker, allowing for a highly individual approach to treating cancer. Furthermore, this method of estimating isotopic composition appears to be the most sensitive tool yet for cancer tissue evaluation, and a valuable complement to established cancer study methods with prospective clinical impact.

The kidney peritubular microvasculature is highly susceptible to injury from drugs and toxins, often resulting in acute kidney injury and progressive chronic kidney disease. Little is known about the process of injury and regeneration of human kidney microvasculature, resulting from the lack of appropriate kidney microvascular models that can incorporate the proper cells, extracellular matrices (ECMs), and architectures needed to understand the response and contribution of individual vascular components in these processes. In this study, we present methods to recreate the human kidney ECM (kECM) microenvironment by fabricating kECM hydrogels derived from decellularized human kidney cortex. The majority of native matrix proteins, such as collagen-IV, laminin, and heparan sulfate proteoglycan, and their isoforms were preserved in similar proportions as found in normal kidneys. Human kidney peritubular microvascular endothelial cells (HKMECs) became more quiescent when cultured on this kECM gel compared with culture on collagen-I-assessed using phenotypic, genotypic, and functional assays; whereas human umbilical vein endothelial cells became stimulated on kECM gels. We demonstrate for the first time that human kidney cortex can form a hydrogel suitable for use in flow-directed microphysiological systems. Our findings strongly suggest that selecting the proper ECM is a critical consideration in the development of vascularized organs on a chip and carries important implications for tissue engineering of all vascularized organs., Competing Interests: Statement No competing financial interests exist.

Objective: We aimed to evaluate the feasibility of quantification of liver, pancreas, spleen, vertebral bone marrow, and renal cortex R2* and magnetic resonance imaging-proton density fat fraction (MRI-PDFF) and to evaluate the correlations among them in patients with transfusion-related iron overload., Materials and Methods: A total of 9 patients (5 boys, 4 girls) who were referred to our clinic with suspicion of hepatic iron overload were included in this study. All patients underwent T1-independent volumetric multi-echo gradient-echo imaging with T2* correction and spectral fat modeling. MRI examinations were performed on a 1.5 T MRI system., Results: All patients had hepatic iron overload. Severe hepatic iron overload was recorded in 5/9 patients (56%), and when we evaluated the PDFF maps of these patients, we observed an extensive patchy artifact in the liver in 4 of 5 patients (R2* greater than 671 Hz). When we performed MRI-PDFF measurements despite these artifacts, we observed artifactual high MRI-PDFF values. There was a close correlation between average pancreas R2* and average pancreas MRI-PDFF (p=0.003, r=0.860). There was a significant correlation between liver R2* and average pancreas R2* (p=0.021, r=0.747), liver R2* and renal cortex R2* (p=0.020, r=0.750), and average pancreas R2* and renal cortex R2* (p=0.003, r=0.858). There was a significant negative correlation between vertebral bone marrow R2* and age (p=0.018, r=-0.759)., Conclusion: High iron content of the liver, especially with a T2* value shorter than the first echo time can spoil the efficacy of PDFF calculation. Fat deposition in the pancreas is accompanied by pancreatic iron overload. There is a significant correlation between hepatic siderosis and pancreatic siderosis. Renal cortical and pancreatic siderosis are correlated, too.

Nephrogenic adenoma (NA) is an unusual, benign lesion of the urinary tract, generally presenting in the bladder and with less frequency in the renal pelvis, urethra or ureter. It consists of tubules, microcysts and papillae lined by a single layer of low cuboidal epithelium without atypia. Recently, a fibromyxoid variant mimicking an infiltrating mucinous adenocarcinoma has been described. We report hereby the case of a 70-year-old female with a fibromixoid NA protruding in a renal cortical cyst. Only one case of NA in a renal cortical cyst has been found in the literature and it was of the classical type. The development of a NA in a renal cortical cyst lends support to the theory that the NA results from proliferation of secondarily implanted exfoliated renal epithelial cells., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Emerging evidence has linked mitochondrial dysfunction to the pathogenesis of many renal disorders, including acute kidney injury, sepsis and even chronic kidney disease. Proteomics is a powerful tool in elucidating the role of mitochondria in renal pathologies. Since the pig is increasingly recognized as a major mammalian model for translational research, the lack of physiological proteome data of large mammals prompted us to examine renal mitochondrial proteome in porcine kidney cortex and medulla, Methods: Kidneys were obtained from six healthy pigs. Mitochondria from cortex and medulla were isolated using differential centrifugation and proteome maps of cortical and medullar mitochondria were constructed using two-dimensional gel electrophoresis (2DE). Protein spots with significant difference between mitochondrial fraction of renal cortex and medulla were identified by mass spectrometry., Results: Proteomic analysis identified 81 protein spots. Of these spots, 41 mitochondrial proteins were statistically different between renal cortex and medulla (p < 0.05). Protein spots containing enzymes of beta oxidation, amino acid metabolism, and gluconeogenesis were predominant in kidney cortex mitochondria. Spots containing tricarboxylic acid cycle enzymes and electron transport system proteins, proteins maintaining metabolite transport and mitochondrial translation were more abundant in medullar mitochondria., Conclusion: This study provides the first proteomic profile of porcine kidney cortex and medullar mitochondrial proteome. Different protein expression pattern reflects divergent functional metabolic role of mitochondria in various kidney compartments. Our study could serve as a useful reference for further porcine experiments investigating renal mitochondrial physiology under various pathological states.

Background: Hypercalciuria is a frequent characteristic of hypertension. In this report we extend our earlier studies investigating the role of renal interstitial fluid calcium (ISF(Ca))(2+) as a link between urinary calcium excretion and blood pressure in the Dahl salt-sensitive (DS) hypertensive model., Methods: Dahl salt-sensitive and salt-resistant (DR) rats were placed on control (0.45%) and high (8%) salt diets to determine if changes in renal cortical and medullary ISF(Ca)(2+)correlated with changes in urinary calcium excretion and blood pressure., Results: We observed that renal ISFCa(2+) was predicted by urinary calcium excretion (P < 0.05) in DS rats but not DR rats. Renal cortical ISF(Ca)(2+) was negatively associated with blood pressure (P < 0.03) while renal medullary ISF(Ca)(2+) was positively associated with blood pressure in DS rats (P < 0.04). In contrast, neither urinary calcium excretion nor renal ISF(Ca)(2+) was associated with blood pressure in the DR rats under the conditions of this study., Conclusion: We interpret these findings to suggest that decreased renal cortical ISF(Ca)(2+) plays a role in the increase in blood pressure following a high salt diet in salt hypertension perhaps by mediating renal vasoconstriction; the role of medullary calcium remains to be fully understood. Further studies are needed to determine the mechanism of the altered renal ISF(Ca)(2+) and its role in blood pressure regulation., (© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

Previously, we, and others, have demonstrated a rapid and significant post-mortem increase in brain prostanoid (PG) levels analyzed without microwave fixation, and this is not the result of PG trapping or destruction in microwave-irradiated brain tissue. In the present study, we demonstrate a dramatic increase in kidney eicosanoid levels when analyzed without microwave fixation which was mainly accounted for by the 142-, 81-, and 62-fold increase in medullary 6-ketoPGF1α, PGE2, and PGF2α, levels, respectively, while PGD2 and TXB2 levels were increased ~7-fold. Whole kidney and cortex PG were also significantly increased in non-microwaved tissue, but at lesser extent. Arachidonic acid and the lipoxygenase products hydroxyeicosatetraenoic acids (HETE) were also induced in whole kidney, cortex, and medulla 1.5- to 5.5-fold depending upon tissue and metabolite. Cyclooxygenase inhibition with indomethacin decreased PG mass in non-microwaved tissue to basal levels, however HETE and arachidonic acid were not decreased. These data demonstrate the critical importance of kidney tissue fixation to limiting artifacts during kidney eicosanoid analysis., (© 2013 Elsevier Ltd. All rights reserved.)

Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.

Introduction: Cadmium (Cd) is toxic to the kidney and a major part of the body burden occurs here. Cd in urine (U-Cd) and blood (B-Cd) are widely-used biomarkers for assessing Cd exposure or body burden. However, empirical general population data on the relationship between Cd in kidney (K-Cd), urine, and blood are scarce. Our objectives were to determine the relationship between cadmium in kidney, urine, and blood, and calculate the elimination half-time of Cd from the kidney., Methods: Kidney cortex biopsies, urine, and blood samples were collected from 109 living kidney donors. Cd concentrations were determined and the relationships between K-Cd, U-Cd, and B-Cd were investigated in regression models. The half-time of K-Cd was estimated from the elimination constant., Results: There was a strong association between K-Cd and U-Cd adjusted for creatinine (rp=0.70, p<0.001), while the association with B-Cd was weaker (rp=0.44, p<0.001). The relationship between K-Cd and U-Cd was nonlinear, with slower elimination of Cd at high K-Cd. Estimates of the K-Cd half-time varied between 18 and 44years. A K-Cd of 25μg/g corresponds to U-Cd of 0.42μg/g creatinine in overnight urine (U-Cd/K-Cd ratio: about 1:60). Multivariate models showed Cd in blood and urinary albumin as determinants for U-Cd excretion., Discussion: In healthy individuals with low-level Cd exposure, there was a strong correlation between Cd in kidney and urine, especially after adjustment for creatinine. Urinary Cd was also affected by Cd in blood and urinary albumin. Previous estimates of the U-Cd/K-Cd ratio may underestimate K-Cd at low U-Cd., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

Lead acetate (300 mg/L) and/or cadmium chloride (50 mg/L) were administered as drinking water to Sprague-Dawley rats for 9 weeks to investigate the effects of concurrent exposure to lead and cadmium on the distribution patterns of five essential trace elements. Inductively coupled plasma mass spectrometry was used to determine the concentrations of zinc, copper, manganese, selenium and iron in the urine at different exposure times, as well as their levels in the renal cortex and serum at the end of treatment. Compared with the control group, exposure to lead and/or cadmium resulted in a significant increase in the urinary excretion of these five elements during the experiment, whereas significant decreased levels of these elements were found in kidney and serum. In conclusion, increased urinary loss of antioxidant trace elements due to lead and/or cadmium exposure induced the deficiency of antioxidants in the body, which could result in further oxidative damage. Moreover, there was an obvious synergistic effect of lead combined with cadmium on the distribution patterns of these essential trace elements, which may be related to the severity of co-exposure to these two metals.

We describe a method for ratio estimations in (18)O-water labeling experiments acquired from low resolution isotopically resolved data. The method is implemented in a software package specifically designed for use in experiments making use of zoom-scan mode data acquisition. Zoom-scan mode data allow commonly used ion trap mass spectrometers to attain isotopic resolution, which makes them amenable to use in labeling schemes such as (18)O-water labeling, but algorithms and software developed for high resolution instruments may not be appropriate for the lower resolution data acquired in zoom-scan mode. The use of power spectrum analysis is proposed as a general approach that may be uniquely suited to these data types. The software implementation uses a power spectrum to remove high-frequency noise and band-filter contributions from coeluting species of differing charge states. From the elemental composition of a peptide sequence, we generate theoretical isotope envelopes of heavy-light peptide pairs in five different ratios; these theoretical envelopes are correlated with the filtered experimental zoom scans. To automate peptide quantification in high-throughput experiments, we have implemented our approach in a computer program, MassXplorer. We demonstrate the application of MassXplorer to two model mixtures of known proteins and to a complex mixture of mouse kidney cortical extract. Comparison with another algorithm for ratio estimations demonstrates the increased precision and automation of MassXplorer.

Maternal undernutrition can cause reduced nephron number and glomerular hypertrophy, consequently leading to adult kidney disease. We intended to elucidate whether NO deficiency evolves to kidney disease vulnerability in offspring from mothers with caloric restriction diets and whether maternal L-citrulline (L-Cit) supplementation can prevent this. Using a rat model with 50% caloric restriction, four groups of 3-month-old male offspring were sacrificed to determine their renal outcome: control, caloric restriction (CR), control treated with 0.25% L-citrulline solution during the whole period of pregnancy and lactation (Cit), and CR treated in the same way (CR+Cit group). The CR group had low nephron numbers, increased glomerular diameter, and an increased plasma creatinine level compared with the control group. Maternal L-Cit supplementation prevented these effects. The CR+Cit and Cit groups developed hypertension beginning at 4 and 8weeks of age, respectively. Plasma asymmetric and symmetric dimethylarginine (ADMA and SDMA) levels were increased, but L-arginine/ADMA ratios (AAR) were decreased in the CR group vs the control group. This was prevented by maternal L-Cit supplementation. Renal cortical neuronal NOS-alpha (nNOSalpha) protein abundance was significantly decreased in the Cit and CR+Cit groups. Collectively, reduced nephron number, reduced renal nNOSalpha expression, increased ADMA, and decreased AAR contribute to the developmental programming of adult kidney disease and hypertension. Although maternal L-Cit supplementation prevents caloric restriction-induced low nephron number and renal dysfunction, it also induces hypertension., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMV(CTX)) and from purified proximal convoluted tubules (BBMV(PCT)). Both proteomic data and Western blot analysis indicate that the BBMV(CTX) contain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMV(PCT). Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMV(CTX), and 57 proteins unique to BBMV(PCT). Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values

Attenuated total reflection (ATR) Fourier transform infrared (FT-IR) spectroscopy has been applied to study the short and long term postmortem metabolic processes in rat and human kidney cortexes. The goals of this project were as follows: (1) to investigate the changes of ATR spectra in different rat and human tissues after death, (2) to explore the best mathematical model with different band absorption ratio changes to determine the postmortem interval (PMI), and (3) to establish a preliminary human postmortem ATR spectra database. There were three different types of metabolic changes after death based on the spectral results: (1) the intensities of some bands increased continuously (e.g., C-H stretching region), (2) the intensities of other bands decreased continuously (e.g., PO(2)(-) symmetric stretching), and (3) other bands remained relatively stable (e.g., C-OH bending, CO-O-C antisymmetric stretching). The band absorbance ratios for rats were found to display either a significant increase or decrease with increasing time after death. Of the absorbance ratios of the various bands investigated to find the best fit with the cubic model function in rats, the A(1652)/A(1396) ratio showed the strongest correlation (R(2) = 0.937). Comparison of the rat kidney cortex spectra with selected human postmortem cases showed similar postmortem metabolic changes. In conclusion, ATR FT-IR spectroscopy was shown to be a convenient and reliable method of determining short and long term postmortem intervals by simultaneously monitoring several specific parameters, although these observations have yet to be applied at forensic scenes by further field studies.

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Diabetic nephropathy is the leading cause of renal failure in the United States. The obese Zucker rat (OZR; fa/fa) is a commonly used model of type 2 diabetes and metabolic syndrome (MetS), and of the nephropathy and renal oxidative stress commonly seen in these disorders. Heterozygous lean Zucker rats (LZRs; fa/+) are susceptible to high-fat diet (HFD)-induced obesity and MetS. The present study was designed to investigate whether 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable radical scavenger, could alleviate the renal effects of MetS in OZR and LZR fed a HFD, which resembles the typical "Western" diet. OZR and LZR were fed a HFD (OZR-HFD and LZR-HFD) or regular diet (OZR-RD and LZR-RD) and allowed free access to drinking water or water containing 1 mmol/l TEMPOL for 10 weeks. When compared to OZR-RD animals, OZR-HFD animals exhibited significantly higher levels of total renal cortical reactive oxygen species (ROS) production, plasma lipids, insulin, C-reactive protein, blood urea nitrogen (BUN), creatinine (Cr), and urinary albumin excretion (P < 0.05); these changes were accompanied by a significant decrease in plasma high-density lipoprotein levels (P < 0.05). The mRNA expression levels of desmin, tumor necrosis factor-alpha (TNF-alpha), nuclear factor kappaB (NFkappaB), and NAD(P)H oxidase-1 (NOX-1) were significantly higher in the renal cortical tissues of OZR-HFD animals; NFkappaB p65 DNA binding activity as determined by electrophoretic mobility shift assay was also significantly higher in these animals. The same trends were noted in LZR-HFD animals. Our data demonstrate that TEMPOL may prove beneficial in treating the early stages of the nephropathy often associated with MetS.

Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells x staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.

This study aims to investigate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on rat kidney cortex histology. Oral exposure of rats to 2,4-D for 28 days resulted in decreases in body weight gain and kidney weight. Histological examination showed degeneration in renal corpuscles and podocytes; vacuolization in the glomerulus with disintegration of the basal membrane; tissue edema; vacuolization, cystic dilation and invagination of the basal laminae in the tubular structures; dilation and congestion in renal corpuscular vessels and marked decrease in glomerular and stromal fibronectin reaction; suggesting that subacute 2,4-D administration induces dose-dependent histopathological degenerative effects in rat kidney cortex.

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-beta1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (approximately 260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg x kg(-1) x day(-1) (D0.01, N = 14), 1 mg x kg(-1) x day(-1) (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-beta1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P < or = 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-beta1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Background: Recombinant human erythropoietin (rhEPO) increases blood pressure (BP) and the vascular production of endothelin-1 in renal failure rats. This study was designed to investigate the effect of rhEPO on BP and on the ET-1/ET(B)R system in rats with normal renal function. To further characterize the effect of rhEPO on the ET-1/ET(B)R system, we also studied heterozygous (+/-) ET(B)R knockout (KO) mice., Methods: The animals received either the vehicle or rhEPO (100 U/kg subcutaneously three times per week). ET(B)R(+/-) mice were compared with ET(A)R(+/-) and wild-type (WT) mice. In rats, the ET(B)R mRNA expression was assessed in blood vessels as well as the vascular ET(B)R density using immunohistochemistry. In mice, ET-1 concentration was measured in the thoracic aorta., Results: RhEPO administration increased hematocrit levels in all treated animals. This therapy had no effect on BP in normal rats, but it did increase vascular and renal cortex ET(B)R mRNA expression. Immunohistochemistry confirmed that the ET(B)R density was increased in blood vessel endothelium in these normal rats. In contrast, rhEPO increased BP in ET(B)R(+/-) mice and this pressor effect was associated with higher ET-1 concentrations in the thoracic aorta., Conclusions: RhEPO exerts a pleotropic effect on the endothelial ET-1/ET(B)R system. The increase in endothelial ET(B)R expression may contribute to maintaining normal BP during rhEPO administration in normal animals. Conversely, conditions with deficient ET(B)R expression, such as in ET(B)R(+/-) mice, may lead to hypertension while receiving the same therapy.

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.

Despite similar molecular structures, the growth-related sodium/phosphate cotransporter NaPiIIc is regulated differently than the main NaPiIIa phosphate transporter. Using two-hybrid systems and immunoprecipitation, we identified several proteins that interact with NaPiIIc that might account for this differential regulation. NaPiIIc interacted with the PDZ domain-containing sodium-hydrogen exchange-regulating factor (NHERF) 1 and NHERF3 through novel binding motifs in its C terminus. NaPiIIc from brush-border membranes coprecipitated with both NHERF1 and NHERF3, with more NHERF3 co-precipitated in rats fed a low-phosphorus diet. NaPiIIc colocalizes with both NHERF1 and NHERF3 in brush-border membranes of rats fed either a low- or high-phosphorus diet. When mouse NaPiIIc was transfected into opossum kidney cells, it was localized mainly in apical microvilli and the trans-Golgi. Both confocal and total internal reflection microscopy show that NaPiIIc colocalizes with NHERF1 and NHERF3 in the apical microvilli, and this was not altered by truncation of the last three amino acids of NaPiIIc. Interactions of NaPiIIc with NHERF1 and NHERF3 were modulated by the membrane-associated 17 kDa protein (MAP17) similarly to NaPiIIa, but only the MAP17-NaPiIIc-NHERF3 complexes were internalized to the trans-Golgi. Our study shows that NaPiIIc interacts with a limited number of PDZ domain proteins, and the mechanisms and consequences of such interactions differ from those of NaPiIIa.

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.

Background/aims: Renal organic anion transporters (OAT1 and OAT3) localized in the basolateral membrane mediate the uptake of organic anions from the blood into proximal tubules. This study aimed to examine the effects of renal ischemia/reperfusion injury (IRI) on the expression of cortical renal OAT1 and OAT3 and the functional impact., Methods: Male rats underwent a right nephrectomy and clamping of the left renal pedicle for 50 min or sham operation, followed by reperfusion for 1, 2, 4 and 6 days. The expression of OAT1 and OAT3 was detected by RT-PCR, immunohistochemistry and Western blot analysis. Na(+)-K(+)-ATPase activity was also estimated., Results: The renal clearance of para-aminohippurate was significantly decreased on day 1 in IRI rats compared with sham-operated rats and returned to normal when the tubular injury recovered. There were significant increases in the mRNA and protein levels of OAT1 and OAT3 in renal cortex homogenates and basolateral membranes on day 1 after IRI, while on days 2 and 4 after IRI, the renal expression of OAT1 and OAT3 decreased gradually but was still significantly higher than that of the sham-operated rats. The Na(+)-K(+)-ATPase activity in renal cortex homogenates decreased significantly on day 1 after IRI but gradually increased on days 2, 4 and 6., Conclusions: Renal para-aminohippurate clearance was depressed in response to IRI; however, the expressions of renal cortex OAT1 and OAT3 were significantly elevated in the early stage of IRI which may have substantial impact on renal excretion of some drugs and toxic compounds., (Copyright 2008 S. Karger AG, Basel.)

To understand the spectrum of proteins affected by diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of db/db mice using 2-DE combined with MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >two-fold by diabetes, of which 170 mapped to gene identifiers representing 147 nonredundant proteins. Gene Ontology classification demonstrated that 80% of these proteins modulated physiological functions, 55% involved metabolism, approximately 25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11-15%). Twenty eight percent of the proteins identified as significantly altered by diabetes were mitochondrial proteins. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARalpha was the most common node of interaction for the numerous enzymes whose expression levels were influenced by diabetes. These differentially regulated proteins create a foundation for a systems biology exploration of molecular mechanisms underlying the pathophysiology of diabetic nephropathy.

Renal cortical cyclooxygenase-2 (COX-2) is restricted to the macula densa and adjacent cortical thick ascending limbs (MD/cTALH). Renal cortical COX-2 increases in response to diabetes and renal ablation, both of which are characterized by hyperfiltration and reduced NaCl delivery to the MD due to increased proximal NaCl reabsorption. High-protein intake also induces hyperfiltration and decreases NaCl delivery to the MD due to increased NaCl reabsorption proximally. We investigated whether high protein induces cortical COX-2 and whether cortical COX-2 contributes to high protein-induced hyperfiltration and increased intrarenal renin biosynthesis. Cortical COX-2 increased after protein loading but decreased after protein restriction. COX-2 inhibition attenuated high protein-induced hyperfiltration but had no effect on high protein-induced intrarenal renin elevation. Therefore, induction of cortical COX-2 contributed to high protein-induced hyperfiltration but not intrarenal renin elevation. In the kidney cortex, neuronal nitric oxide synthase (nNOS) is also localized to the MD, and interactions between intrarenal nNOS and COX-2 systems have been proposed. Cortical COX-2 elevation seen in salt restriction was blocked by nNOS inhibiton. Cortical nNOS expression also increased after protein loading, and inhibition of nNOS activity completely reversed high protein-induced cortical COX-2 elevation and hyperfiltration. These results indicate that NO is a mediator of high protein-induced cortical COX-2 elevation and suggest that both intrarenal nNOS and COX-2 systems appear to regulate afferent arteriolar tone and subsequent hyperfiltration seen in high-protein intake.

The lead poisoning incidents in cattle investigated by the Veterinary Laboratories Agency between 1990 and 2003 are reviewed. Lead poisoning was most commonly encountered in young calves, but cattle of all ages were affected. The lead was derived mainly from lead paint, lead accumulator batteries and lead in soil from old mine workings. Paint was responsible for the majority of cases of poisoning in young calves; yearling animals were most at risk from discarded batteries, and adult cows were most commonly poisoned by geochemical sources of lead. There was a marked seasonal incidence, with most cases occurring after turnout in the spring and early summer.

Ischemia/reperfusion (I/R) injury in the kidney involves hemodynamic and cellular dysfunctions as well as leukocyte infiltration. Functional recovery occurs via cell proliferation and/or migration. To determine the roles of hyaluronan (HA) and its main receptor CD44 in renal postischemic processes, we compared their localization and expression with that of neutrophils, macrophages, and PCNA-positive (regenerative) cells as characterized by immunohistochemistry, up to 28 days after I/R in uninephrectomized rats. Observations covered all kidney zones, i.e. cortex (C), outer and inner stripes of outer medulla (OSOM, ISOM), and inner medulla (IM). In controls, HA was localized to the interstitium of IM and ISOM, and CD44 was mostly present on the basolateral membranes of collecting ducts in ISOM, the thin descending limb of Henle's loop and macula densa cells. After I/R, HA and CD44 staining appeared in C and OSOM at 12 h and persisted throughout the regenerative period, i.e. until day 7. Thereafter, they regressed but remained associated with remodeling areas. CD44 expression was found de novo on the apical pole of regenerating, not fully differentiated tubular cells and on some interstitial cells. It was prominent on all infiltrating neutrophils, as soon as 2 h post-I/R, and on 30% of the macrophages, including those in late HA-rich inflammatory granulomas. CD44 is probably involved in early leukocyte infiltration, in tubular regeneration, and in macrophage activity, while HA modifies the physico-chemical environment of interstitial and migrating cells. Based on its presence in remodeling areas, the HA-CD44 pair may be implicated in persistent postichemic inflammation as observed in chronic allograft nephropathy.

Ceramide-activated NAD(P)H oxidase has been reported to participate in homocysteine (Hcys)-induced abnormal metabolism of the extracellular matrix (ECM) in rat glomerular mesangial cells. However, it remains unknown whether this ceramide-redox signaling pathway contributes to glomerular injury induced by hyperhomocysteinemia (hHcys) in vivo. The present study was designed to address this question, defining the role of ceramide and activated NAD(P)H oxidase in the development of hHcys-induced glomerular injury. Uninephrectomized Sprague-Dawley rats were fed a folate-free diet for 8 weeks to produce hHcys and the de novo ceramide synthesis inhibitor myriocin or the NAD(P)H oxidase inhibitor apocynin was administrated. Rats with folate-free diet significantly increased plasma Hcys levels, renal ceramide levels, and NAD(P)H oxidase activity accompanied by marked glomerular injury. Treatment of rats with myriocin significantly reduced ceramide levels and improved glomerular injury, as shown by decreased urinary albumin excretion and reduced glomerular damage index. ECM components changed towards to normal levels with decreased tissue inhibitor of metalloproteinase-1 and increased matrix metalloproteinase-1 activity. NAD(P)H oxidase activity and Rac GTPase activity were reduced by 69 and 66%, respectively. In rats treated with apocynin, similar beneficial effects in protecting glomeruli from hHcys-induced injury were observed. These results support the view that de novo ceramide production is involved in Hcys-induced NAD(P)H oxidase activity in the kidney of hHcys rats and indicate the important role of ceramide-mediated redox signaling in hHcys-induced glomerular injury in rats.

Adaptation to hypoxic environment is conferred through hypoxia-inducible transcription factors (HIFs). We have previously shown that the HIF system is transiently activated in vivo in radiocontrast-induced acute renal failure, associated with profound hypoxia in the renal medulla. Medullary thick ascending limbs (mTALs), the most affected nephron segments in this model, were virtually unable to mount an adaptive HIF response. Here, we study correlations between oxygenation, HIF activation, and cell viability in a related ex vivo model, the isolated perfused rat kidney (IPK). In IPKs perfused with cell-free oxygenated medium, severe medullary hypoxic damage developed, affecting 42+/-9% of mTALs in the mid-inner stripe. HIF-1alpha tubular immunostaining was noted with a zonal and tubular pattern largely similar to our findings in vivo: in 34+/-3% of collecting ducts (CDs) within the mid-inner stripe and extensively in the papillary tip, whereas mTALs were all HIF-negative. In IPKs supplemented with RBCs (improved oxygen supply), mTAL damage was totally prevented and CDs' HIF expression was attenuated (22+/-4%). By contrast, although measures designed to reduce medullary hypoxia by decreasing tubular reabsorptive activity (furosemide, ouabain, or high-albumin-non-filtering system) reduced mTAL damage, all paradoxically resulted in increased HIF expression in CDs (51+/-4%), and 17+/-3% of mTALs became immunostained as well. Our data confirm that CDs and mTALs have markedly different HIF responses, which correlate with their viability under hypoxic stress. mTALs transcriptional adaptation occurs within a narrow hypoxic range, and it appears that workload reduction can shift mTALs into this window of opportunity for HIF activation and survival.

We established a new congenic model of hypertension, the mRen(2). Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 +/- 2 vs. 107 +/- 2 mmHg, P < 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1-7) were 270% higher and ANG I concentrations were 40% lower in the mRen2. Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2. Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1-7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand 125I-sarthran. Isolated CORT nuclei exhibited a high density (Bmax >200 fmol/mg protein) and affinity for the sarthran ligand (KD<0.5 nM); the majority of these sites (>95%) were the AT1 receptor subtype. CORT ANG II receptor Bmax and KD values in nuclei were 75 and 50% lower, respectively, for the mRen2. Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 +/- 4 vs. mRen2. Lewis: 21 +/- 5 fmol/mg protein) and affinity (Lewis: 0.31 +/- 0.1 vs. 0.69 +/- 0.1 nM) were lower in the mRen2. Lewis rats. In summary, the hypertensive mRen2. Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT1 sites in the mRen2. Lewis. The downregulation of AT1 sites in the mRen2. Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II.

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.

The precise mechanisms of mercury accumulation and retention are still unclear. Generally, the association of mercury with selenium is used to explain these phenomena. It seems that the presence of coaccumulated endogenous Se can protect cells from the harmful effects of Hg. However, as speculated by some authors, this binding of Se to Hg can also result in a relative deficiency of biologically available Se needed for selenoenzyme syntheses. Deriving from the assumption that Hg deposited in tissues is bound to Se in a 1:1 ratio, the quantity of non-Hg bound Se could be calculated by the difference between the molar contents of the two elements (Se(mol)-Hg(mol)). In this study we applied such an approach to the data from our previous investigation, where Hg and Se concentrations were determined in autopsy samples of mercury exposed retired Idrija mercury mine workers, Idrija residents living in a Hg contaminated environment and a control group with no known Hg exposure from the environment. Based on these data we tried to estimate the influence of Hg exposure on the physiologically available selenium content in selected tissues, particularly endocrine glands and brain tissues. Comparing the calculated values of (Se(mol)- Hg(mol)) it was found that for Idrija residents the values were similar to those of the control group and as expected, diminished values were found in some mercury-loaded organs of retired Idrija miners. It could be speculated that in Idrija residents Hg sequestration of selenium is sufficiently compensated by increased Se levels, but that particularly in active miners and in some organs of retired miners, the activity and/or synthesis of selenoenzymes could be disturbed.

Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, i.m., twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, i.m., twice a day for 9 days, and 14 with 0.15 M NaCl , i.m., twice a day for 9 days and PDTC, 50 mg kg(-1) day(-1), i.p., twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm2. Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

Although renal sinus vein invasion is the most common site of extrarenal involvement in clear cell renal cell carcinoma (CC), CC also spreads by lymphatics. As cortical lymphatics drain into the sinus, some involved sinus structures may be lymphatics, not veins. This possibility was investigated with podoplanin, a specific lymphatic endothelial marker, in 40 CC with sinus vein invasion. Ten blocks of uninvolved kidney, serving as controls, showed lymphatics within the adventitia of midcortical intralobular arteries. Lymphatics became more numerous and enlarged with progression towards the medulla. No lymphatics were among glomeruli or within the medulla unless associated with inflammation. The largest lymphatics occurred within the sinus, and were also noted within pelvic muscularis, and media of large veins. Intralymphatic tumor was observed and divided into two Groups. Group 1 (four cases) involved lymphatics within the invasive edge of tumors lacking a pseudocapsule. The lymphatics were small (0.045-0.19 mm), irregularly shaped, often incomplete, and contained single cells or small clusters of tumor cells. Group 2 (four cases) involved sinus lymphatics separate from tumor. One case each also involved adventitial lymphatics of an intralobular artery, the muscularis of the renal pelvis, and media of a muscular vein. The intralymphatic tumor in Group 2 often appeared discohesive, not endothelial cell invested, and larger than in Group 1 (0.4-0.5 mm). Conversely, tumor within muscular veins was cohesive, contained a capillary plexis, and was endothelial cell invested. In conclusion, intralymphatic tumor can be demonstrated in CC. Lymphatic involvement is less frequent than venous involvement and involves smaller structures. The potential for lymphatic spread may not be equal among involved lymphatics. Small peritumoral lymphatics may be destined for destruction by tumor growth. However, involved lymphatics within sinus and associated with renal pelvis, are likely sources for lymphatic spread and lymph node metastases.

Cold temperatures have adverse effects on the human cardiovascular system. Endothelin (ET)-1 is a potent vasoconstrictor. We hypothesized that cold exposure increases ET-1 production and upregulates ET type A (ETA) receptors. The aim of this study was to determine the effect of cold exposure on regulation of the ET system. Four groups of rats (6-7 rats/group) were used: three groups were exposed to moderate cold (6.7 +/- 2 degrees C) for 1, 3, and 5 wk, respectively, and the remaining group was maintained at room temperature (25 degrees C) and served as control. Cold exposure significantly increased ET-1 levels in the heart, mesenteric arteries, renal cortex, and renal medulla. Cold exposure increased ETA receptor protein expression in the heart and renal cortex. ET type B (ETB) receptor expression, however, was decreased significantly in the heart and renal medulla of cold-exposed rats. Cold exposure significantly increased the ratio of ETA to ETB receptors in the heart. An additional four groups of rats (3 rats/group) were used to localize changes in ETA and ETB receptors at 1, 3, and 5 wk of cold exposure. Immunohistochemical analysis showed an increase in ETA, but a decrease in ETB, receptor immunoreactivity in cardiomyocytes of cold-exposed rats. Increased ETA receptor immunoreactivity was also found in vascular smooth muscle cells of cold-exposed rats. Cold exposure increased ETA receptor immunoreactivity in tubule epithelial cells in the renal cortex but decreased ETB receptor immunoreactivity in tubule epithelial cells in the renal medulla. Therefore, cold exposure increased ET-1 production, upregulated ETA receptors, and downregulated ETB receptors.

New markers/targets for renal cell carcinoma (RCC) are needed to enable earlier detection and monitoring of disease and therapeutic targeting. To identify such molecules, normal and RCC-derived primary cell lines have been used as a simplified model system for studying changes that accompany tumorigenesis. Short-term cultures allow enrichment of relevant cell types from tissue samples, which is balanced against the potential for in vitro changes. Examination of 3 proteins with altered expression in RCC tissue showed the maintenance of normal-tumour differences in culture, although some changes were apparent, including alteration in the isoform of aldolase. Comparative analysis of primary cell lines by 2-DE found 43 proteins up-regulated and 29 down-regulated in at least three out of five tumour cell lines. Many of the observed changes have been previously reported in RCC, including up-regulation of several glycolytic enzymes, vimentin and heat shock protein 27, validating the approach. Additionally, several novel changes in protein expression were found, including up-regulation of several proteins involved in actin cytoskeleton organisation such as radixin and moesin, two members of the septin family, and the actin bundling protein, fascin. Validation studies using Western blotting and immunohistochemistry indicate that several of these molecules may be useful as markers for RCC.

Previous studies revealed that chronic (days) vasopressin treatment stimulates amiloride-sensitive sodium transport in isolated renal cortical collecting ducts and increases the abundance of beta- and gamma-subunits of the epithelial sodium channel (ENaC) in the kidney. The aim of the present work was to investigate in vivo the cellular basis of these effects. The long-term effect of V2 vasopressin agonist (1-deamino-8-D-arginine vasopressin (dDAVP)) on the abundance and subcellular localization of ENaC along the rat renal collecting system was determined by immunohistochemistry and laser confocal microscopy. Moreover, we studied by real-time reverse transcriptase-polymerase chain reaction the effect of vasopressin on proteins implicated in the regulation of ENaC (Nedd4-2, prostasin, Sgk1). After 5 days of administration, dDAVP markedly increased the intracellular pool of the beta- and gamma-ENaC subunits in the principal cells, with an increasing gradient from connecting tubule to the outer medullary collecting duct, but did not increase any subunit at the cell surface. The apical immunostaining of ENaC increased in response to sodium restriction, as expected, but dDAVP did not further enhance this apical labelling. dDAVP increased the gene expression of prostasin in the cortex but not that of Nedd4-2 and Sgk1. These findings suggest that the previously reported increase in sodium transport induced by sustained stimulation of vasopressin V2 receptor is probably mediated by other mechanism than an increase in the apical density of ENaC.

Eighteen cadavers from routine autopsy casework were subject to a study of tissue levels of total mercury in brain, thyroid, and kidney samples by atomic absorption. On these same cadavers, all dental amalgam fillings (the most important source of inorganic mercury exposure in the general population, according to the World Health Organization (WHO) were charted. Total mercury levels were significantly higher in subjects with a greater number of occlusal amalgam surfaces (>12) compared with those with fewer occlusal amalgams (0-3) in all types of tissue (all P < or = 0.04). Mercury levels were significantly higher in brain tissues compared with thyroid and kidney tissues in subjects with more than 12 occlusal amalgam fillings (all P < or = 0.01) but not in subjects with 3 or less occlusal amalgams (all P > or = 0.07).

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.