The apical 70-pS potassium K channel in the thick ascending limb of Henle's loop is a large-conductance Na+-and Cl--activated, KNa1.1-like, channel.

Apical potassium channels are crucial for thick ascending limb (TAL) of Henle's loop transport function. The ROMK (KNCJ1) gene encodes a 30-pS K channel whose loss of function causes the reduced NaCl reabsorption in the TAL associated with Type 2 Bartter's syndrome. In contrast, the molecular basis of a functionally ROMK-related 70-pS K channel is still unclear. The aim of this study was to highlight new specific channel properties that may give insights on its molecular identity. Using the patch-clamp technique on the apical membrane of mouse split-open TAL tubules, we observed that 70-pS K channel activity, but not ROMK channel activity, increases with the internal Na+ and Cl- concentrations, with relative 50 % effective concentrations (EC50) and Hill coefficients (nH) of 40.6 mM (SD 1.65) and 2.4 (SD 0.28) for Na+, and of 29.3 mM (SD 2.35) and 2.2 (SD 0.39) for Cl-. Conversely, 70-pS K channel activity was inhibited by internal K+ with a relative EC50 of 64 mM (SD 13.5) and a nH of 3.5 (SD 2.3), and by internal NH4+ and Ca2+. The reevaluation of channel conductive properties revealed an actual inward conductance of ~ 170 pS, with multiple subconductance levels and an inward rectification, and a substantial permeability to NH4+ ( = 0.2). We conclude that the apical 70-pS K channel in TAL cells is a large-conductance Na+- and Cl--activated potassium channel functionally resembling a KNa1.1 channel and propose that ROMK determines its functional expression possibly at the level of channel protein synthesis or trafficking.

A machine learning approach for quantifying age-related histological changes in the mouse kidney.

The ability to quantify aging-related changes in histological samples is important, as it allows for evaluation of interventions intended to effect health span. We used a machine learning architecture that can be trained to detect and quantify these changes in the mouse kidney. Using additional held out data, we show validation of our model, correlation with scores given by pathologists using the Geropathology Research Network aging grading scheme, and its application in providing reproducible and quantifiable age scores for histological samples. Aging quantification also provides the insights into possible changes in image appearance that are independent of specific geropathology-specified lesions. Furthermore, we provide trained classifiers for H&E-stained slides, as well as tutorials on how to use these and how to create additional classifiers for other histological stains and tissues using our architecture. This architecture and combined resources allow for the high throughput quantification of mouse aging studies in general and specifically applicable to kidney tissues.

A Realistic Multiregion Mouse Kidney Dosimetry Model to Support the Preclinical Evaluation of Potential Nephrotoxicity of Radiopharmaceutical Therapy.

Suborgan absorbed dose estimates in mouse kidneys are crucial to support preclinical nephrotoxicity analyses of α- and ß-particle-emitting radioligands exhibiting a heterogeneous activity distribution in the kidneys. This is, however, limited by the scarcity of reference dose factors (S values) available in the literature for specific mouse kidney tissues. Methods: A computational multiregion model of a mouse kidney based on high-resolution MRI data from a healthy mouse kidney was developed. The model was used to calculate S values for 5 kidney tissues (cortex, outer and inner stripes of outer medulla, inner medulla, and papilla and pelvis) for a wide range of ß- or α-emitting radionuclides (45 in total) of interest for radiopharmaceutical therapy, using Monte Carlo calculations. Additionally, regional S values were applied for a 131I-labeled single-domain antibody fragment with predominant retention in the outer stripe of the renal outer medulla. Results: The heterogeneous activity distribution in kidneys of considered α- and low- to medium-energy ß-emitters considerably affected the absorbed dose estimation in specific suborgan regions. The suborgan tissue doses resulting from the nonuniform distribution of the 131I-labeled antibody fragment largely deviated (from -40% to 57%) from the mean kidney dose resulting from an assumed uniform activity distribution throughout the whole kidney. The absorbed dose in the renal outer stripe was about 2.0 times higher than in the cortex and in the inner stripe and about 2.6 times higher than in inner tissues. Conclusion: The use of kidney regional S values allows a more realistic estimation of the absorbed dose in different renal tissues from therapeutic radioligands with a heterogeneous uptake in the kidneys. This constitutes an improvement from the simplistic (less accurate) renal dose estimates assuming a uniform distribution of activity throughout kidney tissues. Such improvement in dosimetry is expected to support preclinical studies essential for a better understanding of nephrotoxicity in humans. The dosimetric database has added value in the development of new molecular vectors for radiopharmaceutical therapy.

Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization.

Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues. Herein, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI), an ambient liquid extraction-based ionization technique, as a viable alternative to other MSI techniques to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. MSI data acquired over a broad m/z range showed low signals of the drug and its metabolites resulting from the low ionization efficiency and substantial signal suppression on the tissue. Significant improvements in the signal-to-noise were obtained using selected ion monitoring (SIM) with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide and hydroxydiclofenac are localized to the inner medulla and cortex of the kidney, respectively, which is consistent with the previously reported localization of enzymes that process diclofenac into its respective metabolites. In contrast, a uniform distribution of diclofenac and its metabolites was observed in the liver tissue. Concentration ratios of diclofenac and hydroxydiclofenac calculated from nano-DESI MSI data are generally in agreement to those obtained using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collectively, our results demonstrate that nano-DESI MSI can be successfully used to image diclofenac and its primary metabolites and derive relative quantitative data from different tissue regions. Our approach will enable a better understanding of metabolic processes associated with diclofenac and other drugs that are difficult to analyze using commercially available MSI platforms.

A 3D In Vivo Model for Studying Human Renal Cystic Tissue and Mouse Kidney Slices.

(1) Background: Autosomal dominant polycystic kidney disease (ADPKD) is a frequent monogenic disorder that leads to progressive renal cyst growth and renal failure. Strategies to inhibit cyst growth in non-human cyst models have often failed in clinical trials. There is a significant need for models that enable studies of human cyst growth and drug trials. (2) Methods: Renal tissue from ADPKD patients who received a nephrectomy as well as adult mouse kidney slices were cultured on a chorioallantoic membrane (CAM) for one week. The cyst volume was monitored by microscopic and CT-based applications. The weight and angiogenesis were quantified. Morphometric and histological analyses were performed after the removal of the tissues from the CAM. (3) Results: The mouse and human renal tissue mostly remained vital for about one week on the CAM. The growth of cystic tissue was evaluated using microscopic and CT-based volume measurements, which correlated with weight and an increase in angiogenesis, and was accompanied by cyst cell proliferation. (4) Conclusions: The CAM model might bridge the gap between animal studies and clinical trials of human cyst growth, and provide a drug-testing platform for the inhibition of cyst enlargement. Real-time analyses of mouse kidney tissue may provide insights into renal physiology and reduce the need for animal experiments.

Renal Artery Catheterization for Microcapsules' Targeted Delivery to the Mouse Kidney.

The problem of reducing the side effects associated with drug distribution throughout the body in the treatment of various kidney diseases can be solved by effective targeted drug delivery. The method described herein involves injection of a drug encapsulated in polyelectrolyte capsules to achieve prolonged local release and long-term capillary retention of several hours while these capsules are administered via the renal artery. The proposed method does not imply disruption (puncture) of the renal artery or aorta and is suitable for long-term chronic experiments on mice. In this study, we compared how capsule size and dosage affect the target kidney blood flow. It has been established that an increase in the diameter of microcapsules by 29% (from 3.1 to 4.0 µm) requires a decrease in their concentration by at least 50% with the same suspension volume. The photoacoustic method, along with laser speckle contrast imaging, was shown to be useful for monitoring blood flow and selecting a safe dose. Capsules contribute to a longer retention of a macromolecular substance in the target kidney compared to its free form due to mechanical retention in capillaries and slow impregnation into surrounding tissues during the first 1-3 h, which was shown by fluorescence tomography and microscopy. At the same time, the ability of capillaries to perform almost complete "self-cleaning" from capsular shells during the first 12 h leads to the preservation of organ tissues in a normal state. The proposed strategy, which combines endovascular surgery and the injection of polymer microcapsules containing the active substance, can be successfully used to treat a wide range of nephropathies.

In situ localization of lipids on mouse kidney tissues with acute cadmium toxicity using atmospheric pressure-MALDI mass spectrometry imaging.

Cadmium-induced nephrotoxicity has been one of the major concerns for public health over the past century. Lipid peroxidation is a principal mechanism in its pathological process. Atmospheric pressure-MALDI mass spectrometry imaging (AP-MALDI MSI) enables direct mapping of lipids in the biological tissue sections. Considering the spatial visualization of lipids on mouse kidney tissues with acute cadmium toxicity is lacking, this study dedicates to filling the gap by using AP-MALDI MSI. Of the tested matrices, the optimized matrix for labeling lipids was 2,5-dihydroxyacetophenone (DHAP). A set of lipids including phosphatidylcholines (PC), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidic acid (PA), triglyceride (TG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), etc. were identified and visualized. Accordingly, PC, PG, LPC, SM, PA and TG were down-regulated while PE and PI were up-regulated in the renal cortex or medulla regions in kidney tissues of the mouse with acute cadmium toxicity. Such in situ locations of lipids on mouse kidney tissues with acute cadmium toxicity could help discover tissue-specific nephrotoxic biomarkers and provide new insights into its renal toxicological mechanism.

Naturally Acquired Mouse Kidney Parvovirus Infection Produces a Persistent Interstitial Nephritis in Immunocompetent Laboratory Mice.

Mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV), is an emerging, highly infectious agent that has been isolated from laboratory and wild mouse populations. In immunocompromised mice, MKPV produces severe chronic interstitial nephropathy and renal failure within 4 to 5 months of infection. However, the course of disease, severity of histologic lesions, and viral shedding are uncertain for immunocompetent mice. We evaluated MKPV infections in CD-1 and Swiss Webster mice, 2 immunocompetent stocks of mice. MKPV-positive CD-1 mice (n = 30) were identified at approximately 8 weeks of age by fecal PCR (polymerase chain reaction) and were subsequently housed individually for clinical observation and diagnostic sampling. Cage swabs, fecal pellets, urine, and blood were evaluated by PCR at 100 and 128 days following the initial positive test, which identified that 28 of 30 were persistently infected and 24 of these were viremic at 100 days. Histologic lesions associated with MKPV in CD-1 (n = 31) and Swiss mice (n = 11) included lymphoplasmacytic tubulointerstitial nephritis with tubular degeneration. Inclusion bodies were rare; however, intralesional MKPV mRNA was consistently detected via in situ hybridization within tubular epithelial cells of the renal cortex and within collecting duct lumina. In immunocompetent CD-1 mice, MKPV infection resulted in persistent shedding of virus for up to 10 months and a mild tubulointerstitial nephritis, raising concerns that this virus could produce study variations in immunocompetent models. Intranuclear inclusions were not a consistent feature of MKPV infection in immunocompetent mice.

Spin-lock relaxation rate dispersion reveals spatiotemporal changes associated with tubulointerstitial fibrosis in murine kidney.

PURPOSE: To develop and evaluate a reliable non-invasive means for assessing the severity and progression of fibrosis in kidneys. We used spin-lock MR imaging with different locking fields to detect and characterize progressive renal fibrosis in an hHB-EGFTg/Tg mouse model. METHODS: Male hHB-EGFTg/Tg mice, a well-established model of progressive fibrosis, and age-matched normal wild type (WT) mice, were imaged at 7T at ages 5-7, 11-13, and 30-40 weeks. Spin-lock relaxation rates R1ρ were measured at different locking fields (frequencies) and the resultant dispersion curves were fit to a model of exchanging water pools. The obtained MRI parameters were evaluated as potential indicators of tubulointerstitial fibrosis in kidney. Histological examinations of renal fibrosis were also carried out post-mortem after MRI. RESULTS: Histology detected extensive fibrosis in the hHB-EGFTg/Tg mice, in which collagen deposition and reductions in capillary density were observed in the fibrotic regions of kidneys. R2 and R1ρ values at different spin-lock powers clearly dropped in the fibrotic region as fibrosis progressed. There was less variation in the asymptotic locking field relaxation rate R1ρ∞ between the groups. The exchange parameter Sρ and the inflection frequency ωinfl changed by larger factors. CONCLUSION: Both Sρ and ωinfl depend primarily on the average exchange rate between water and other chemically shifted resonances such as hydroxyls and amides. Spin-lock relaxation rate dispersion, rather than single measurements of relaxation rates, provides more comprehensive and specific information on spatiotemporal changes associated with tubulointerstitial fibrosis in murine kidney.

Lead bioavailability in different fractions of mining- and smelting-contaminated soils based on a sequential extraction and mouse kidney model.

Lead bioavailability in contaminated soils varies considerably depending on Pb speciation and sources of contamination. However, little information is available on bioavailability of Pb associated with different fractions. In this study, the Tessier sequential extraction was used to fractionate Pb in 3 contaminated soils to exchangeable (F1), carbonate-bound (F2), Fe/Mn oxides-bound (F3), organic-bound (F4), and residual fractions (F5). In addition, soil residues after F1-F2 extraction (F345), F1-F3 extraction (F45), and F1-F4 extraction (F5) were measured for Pb relative bioavailability (RBA) using a mouse kidney model. Based on the mouse model, Pb-RBA in the soils was 44-93%, which decreased to 43-89%, 28-75%, and 15-68% in the F345, F45, and F5 fractions, respectively. Based on Pb-RBA in the soil residues, Pb-RBA in different fractions was calculated based on a mass balance. The data showed that Pb-RBA was the highest (∼100%) in the exchangeable and carbonate fraction, and the lowest (15-68%) in the residual fraction. In addition, Pb in the first three fractions (F1-F3) contributed most (83-89%) to bioavailable Pb in contaminated soils. Our study shed light on oral bioavailability of Pb in contaminated soils of different fractions based on sequential extraction and provide important information for soil remediation.

Decellularizing and Recellularizing Adult Mouse Kidneys.

Decellularization is the process by which resident cells are lysed and cellular debris is removed from the tissue, leaving behind the extracellular matrix scaffold. The extracellular matrix scaffold can be used for three-dimensional culturing of cells. Here we describe methods of decellularizing whole and thick sections of mouse kidneys using a 0.1% sodium dodecyl sulfate (SDS) detergent solution and strategies to repopulate whole and thick sections of decellularized mouse kidneys with cells.

Selenium-Rich Yeast Protects Against Aluminum-Induced Renal Inflammation and Ionic Disturbances.

The aim of this study was to evaluate the protective effects of SeY (selenium-rich yeast) against Al (aluminum)-induced inflammation and ionic imbalances. Male Kunming mice were treated with Al (10 mg/kg) and/or SeY (0.1 mg/kg) by oral gavage for 28 days. The degree of inflammation was assessed by mRNA expression of inflammatory biomarkers. Ionic disorders were assessed by determining the Na+, K+, and Ca2+ content, as well as the alteration in ATP-modifying enzymes (ATPases), including Na+K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, Ca2+Mg2+-ATPase, and the mRNA levels of ATPase's subunits in kidney. It was observed here that SeY exhibited a significant protective effect on the kidney against the Al-induced upregulation of pro-inflammatory and downregulation of anti-inflammatory cytokines. Furthermore, a significant effect of Al on the Na+, K+, Ca2+, and Mg2+ levels in kidney was observed, and Al was observed to decrease the activities of Na+K+-ATPase, Mg2+-ATPase, and Ca2+Mg2+-ATPase. The mRNA expression of the Na+K+-ATPase subunits and Ca2+-ATPase subunits was regulated significantly by Al. Notably, SeY modulated the Al-induced alterations of ion concentrations, ATPase activity, and mRNA expression of their subunits. These results suggest that SeY prevents renal toxicity caused by Al via regulation of inflammatory responses, ATPase activities, and transcription of their subunits.

Effects of different storage solutions on renal ischemia tolerance after kidney transplantation in mice.

storage is the most prevalent method for graft preservation in kidney transplantation (KTX). The protective effects of various preservation solutions have been studied extensively in both clinical trials and experimental animal models. However, a paucity of studies have examined the effect of different preservation solutions on graft function in mouse KTX; in addition, the tolerance of the transplanted grafts to further insult has not been evaluated, which was the objective of the present study. We performed mouse KTX in three groups, with the donor kidneys preserved in different solutions for 60 min: saline, mouse serum, and University of Wisconsin (UW) solution. The graft functions were assessed by kidney injury markers and glomerular filtration rate (GFR). The grafts that were preserved in UW solution exhibited better functions, reflected by 50 and 70% lower plasma creatinine levels as well as 30 and 55% higher plasma creatinine levels in GFR than serum and saline groups, respectively, during the first week after transplants. To examine the graft function in response to additional insult, we induced ischemia-reperfusion injury (IRI) by clamping the renal pedicle for 18 min at 4 wk after KTX. We found that the grafts preserved in UW solution exhibited ~30 and 20% less injury assessed by kidney injury markers and histology than in other two preservation solutions. Taken together, our results demonstrated that UW solution exhibited a better protective effect in transplanted renal grafts in mice. UW solution is recommended for use in mouse KTX for reducing confounding factors such as IRI during surgery.

Cross-sex transplantation alters gene expression and enhances inflammatory response in the transplanted kidneys.

Kidney transplantation (KTX) is a life-saving procedure for patients with end-stage renal disease. Expression levels of many genes in the kidney vary between males and females, which may play an essential role in the sex differences in graft function. However, whether these differences are affected after cross-sex-KTX is unknown. In the present study, we assessed postoperative changes in genotype, function, and inflammatory responses of the grafts in same-sex- and cross-sex-KTX. Single kidney transplants were performed between same and different sex C57BL/6 mice paired into four combination groups: female donor/female recipient (F/F); male donor/male recipient (M/M); female donor/male recipient (F/M); and male donor/female recipient (M/F). The remnant native kidney was removed 4 days posttransplant. Expression levels of genes related to the contractility of the afferent arteriole and tubular sodium reabsorption were assessed. Same-sex-KTX did not significantly alter the magnitude or sex difference pattern of gene expression in male or female grafts. Cross-sex-KTX showed an attenuated sex difference in gene expressions. The measurements of endothelin 1, endothelin ETA receptor, Na+-K--2Cl cotransporter 2 (NKCC2), and epithelial Na+ channels (ENaC) subunits exhibited decreases in M/F compared with M/M and increases in F/M compared with F/F. There were no significant differences in hemodynamics or sodium excretion in response to acute volume expansion for any sex combinations. Cross-sex-KTX stimulated more robust inflammatory responses than same-sex-KTX. IL-6 and KC mRNA levels elevated 5- to 20-fold in cross-sex-KTX compared with same-sex-KTX. In conclusion, cross-sex-KTX alters gene expression levels and induces inflammatory responses, which might play an important role in long-term graft function.

Parathyroid hormone controls paracellular Ca2+ transport in the thick ascending limb by regulating the tight-junction protein Claudin14.

Renal Ca2+ reabsorption is essential for maintaining systemic Ca2+ homeostasis and is tightly regulated through the parathyroid hormone (PTH)/PTHrP receptor (PTH1R) signaling pathway. We investigated the role of PTH1R in the kidney by generating a mouse model with targeted deletion of PTH1R in the thick ascending limb of Henle (TAL) and in distal convoluted tubules (DCTs): Ksp-cre;Pth1rfl/fl Mutant mice exhibited hypercalciuria and had lower serum calcium and markedly increased serum PTH levels. Unexpectedly, proteins involved in transcellular Ca2+ reabsorption in DCTs were not decreased. However, claudin14 (Cldn14), an inhibitory factor of the paracellular Ca2+ transport in the TAL, was significantly increased. Analyses by flow cytometry as well as the use of Cldn14-lacZ knock-in reporter mice confirmed increased Cldn14 expression and promoter activity in the TAL of Ksp-cre;Pth1rfl/fl mice. Moreover, PTH treatment of HEK293 cells stably transfected with CLDN14-GFP, together with PTH1R, induced cytosolic translocation of CLDN14 from the tight junction. Furthermore, mice with high serum PTH levels, regardless of high or low serum calcium, demonstrated that PTH/PTH1R signaling exerts a suppressive effect on Cldn14. We therefore conclude that PTH1R signaling directly and indirectly regulates the paracellular Ca2+ transport pathway by modulating Cldn14 expression in the TAL. Finally, systemic deletion of Cldn14 completely rescued the hypercalciuric and lower serum calcium phenotype in Ksp-cre;Pth1rfl/fl mice, emphasizing the importance of PTH in inhibiting Cldn14. Consequently, suppressing CLDN14 could provide a potential treatment to correct urinary Ca2+ loss, particularly in patients with hypoparathyroidism.

Nephron morphometry in mice and rats using tomographic microscopy.

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

Quercetin inhibited cadmium-induced autophagy in the mouse kidney via inhibition of oxidative stress.

The objective of the current study was to explore the inhibitory effects of quercetin on cadmium-induced autophagy in mouse kidneys. Mice were intraperitoneally injected with cadmium and quercetin once daily for 3 days. The LC3-II/ß-actin ratio was used as the autophagy marker, and autophagy was observed by transmission electron microscopy. Oxidative stress was investigated in terms of reactive oxygen species, total antioxidant capacity, and malondialdehyde. Cadmium significantly induced typical autophagosome formation, increased the LC3-II/ß-actin ratio, reactive oxygen species level, and malondialdehyde content, and decreased total antioxidant capacity. Interestingly, quercetin markedly decreased the cadmium-induced LC3-II/ß-actin ratio, reactive oxygen species levels, and malondialdehyde content, and simultaneously increased total antioxidant capacity. Cadmium can inhibit total antioxidant capacity, produce a large amount of reactive oxygen species, lead to oxidative stress, and promote lipid peroxidation, eventually inducing autophagy in mouse kidneys. Quercetin could inhibit cadmium-induced autophagy via inhibition of oxidative stress. This study may provide a theoretical basis for the treatment of cadmium injury.

Descending thin limb of the intermediate loop expresses both aquaporin 1 and urea transporter A2 in the mouse kidney.

A new intermediate type of Henle's loop has been reported that it extends into the inner medulla and turns within the first millimeter beyond the outer medulla. This study aimed to identify the descending thin limb (DTL) of the intermediate loop in the adult C57Bl/6 mouse kidney using aquaporin 1 (AQP1) and urea transporter A2 (UT-A2) antibodies. In the upper part of the inner stripe of the outer medulla (ISOM), AQP1 was expressed strongly in the DTL with type II epithelium of the long loop, but not in type I epithelium of the short loop. The DTL of the intermediate loop exhibited weak AQP1 immunoreactivity. UT-A2 immunoreactivity was not observed in the upper part of any DTL type. AQP1 expression was similar in the upper and middle parts of the ISOM. UT-A2 expression was variable, being expressed strongly in the DTL with type I epithelium of the short loop, but not in type II epithelium of the long loop. In the innermost part of the ISOM, AQP1 was expressed only in type III epithelium of the long loop. UT-A2-positive and UT-A2-negative cells were intermingled in type I epithelium of the intermediate loop, but were not observed in type III epithelium of the long loop. UT-A2-positive DTLs of the intermediate loop extended into the UT-A2/AQP1-negative type I epithelium in the initial part of the inner medulla. These results demonstrate that the DTL of the intermediate loop is composed of type I epithelium and expresses both AQP1 and UT-A2. The functional role of the DTL of the intermediate loop may be distinct from the short or long loops.

Genomic distribution of 5-Hydroxymethylcytosine in mouse kidney and its relationship with gene expression.

Ten-Eleven Translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytonsie (5hmC). Our recent work found a decline in global 5hmC level in mouse kidney insulted by ischemia reperfusion (IR). However, the genomic distribution of 5hmC in mouse kidney and its relationship with gene expression remain elusive. Here, we profiled the DNA hydroxymethylome of mouse kidney by hMeDIP-seq and revealed that 5hmC is enriched in genic regions but depleted from intergenic regions. Correlation analyses showed that 5hmC enrichment in gene body is positively associated with gene expression level in mouse kidney. Moreover, IR injury-associated genes (both up- and down-regulated genes during renal IR injury) in mouse kidney exhibit significantly higher 5hmC enrichment in their gene body regions when compared to those un-changed genes. Collectively, our study not only provides the first DNA hydroxymethylome of kidney tissues but also suggests that DNA hyper-hydroxymethylation in gene body may be a novel epigenetic marker of IR injury-associated genes.

Tissue fixed with formalin and processed without paraffin embedding is suitable for imaging of both peptides and lipids by MALDI-IMS.

Type and quality of sample preparation have significant impact on imaging mass spectrometry results. Though imaging of fresh-frozen tissues is considered to give the best results, they are incompatible with clinical practice, since routine diagnostics is most frequently performed using formalin-fixed tissues, and formalin-fixed paraffin-embedded material is a gold standard in histopathology. We aimed to assess utility of formalin-fixed tissue specimen processed without paraffin embedding (i.e., deep-frozen and cryo-sectioned) for MALDI imaging of both peptides and lipids. Peptide and lipid imaging was performed in fresh-frozen, FFPE and formalin-fixed/frozen samples of a mouse kidney, then composition of the resulting spectra was compared. We demonstrated similarity of spectra registered during peptide imaging in FFPE and formalin-fixed/frozen tissues, and similarity of spectra registered during lipid imaging in fresh-frozen and formalin-fixed/frozen material. Furthermore, molecular images of formalin-fixed/frozen tissue resembled the features of both fresh-frozen and FFPE tissue in the case of peptide imaging, and the features of fresh-frozen tissue in the case of lipid imaging. We conclude that tissue preserved by formalin fixation and processed without paraffin embedding can be considered as an alternative to both fresh-frozen and FFPE material.