Transcutaneous measurement of renal function in two rodent models of obstructive nephropathy.

OBJECTIVE: Glomerular filtration rate (GFR) is a key indicator of renal function. In both clinical practice and pre-clinical research, serum levels of endogenous filtration markers, such as creatinine, are often used to estimate GFR. However, these markers often do not reflect minor changes in renal function. In this study, we therefore set out to evaluate the applicability of transcutaneous GFR (tGFR) measurements to monitor the changes in renal function, as compared to plasma creatinine (pCreatinine), in two models of obstructive nephropathy, namely unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction followed by release (BUO-R) in male Wistar rats. RESULTS: UUO animals showed a significant reduction in tGFR compared to baseline; whereas pCreatinine levels were not significantly changed. In BUO animals, tGFR drops 24 h post BUO and remains lower upon release of the obstruction until day 11. Concomitantly, pCreatinine levels were also increased 24 h after obstruction and 24 h post release, however after 4 days, pCreatinine returned to baseline levels. In conclusion, this study revealed that the tGFR method is superior at detecting minor changes in renal function as compared to pCreatinine measurements.

The Release of 24 h Infravesical Obstruction in Mice: Changes in Molecular, Morphological, and Functional Parameters for 14-Day Observation.

Bladder outlet obstruction (BOO) induces bladder dysfunction and altered bladder architecture. Irrespective of the release of the obstruction, persistent bladder dysfunction severely affects the quality of life. A better understanding of the repair process offers an opportunity to enhance postintervention management. We subsequently evaluated the postobstructive repair process in mice subjected to 24 h BOO followed by release. Male and female mice bladders were obstructed for 24 h by placing a clip around the bladder neck. After the release of obstruction, the mice were studied for 3, 7, and 14 days to observe the bladder repair process over time. Voiding frequency and volume were recorded using the voiding spot assay, and the transcutaneous glomerular filtration rate (tGFR) was measured. Fibrogenesis and associated gene expressions and altered protein levels were evaluated in the bladder using histology, quantatative polymerase chain reaction (qPCR), and Western blot analyses. Bladder wall thickness was increased in both genders over time but occurred later in female mice. Moreover, collagen deposition in the smooth muscle layer increased over time in both genders. Male mice showed a decreased average voided volume at 3 days post release, while female mice showed no significant change during the time course. Fibrosis-related molecular events, including upregulation of fibronectin (FN) protein and Collagen-3 (Col-3) mRNA expression, were transient and normalized again at 14 days in both genders. Transforming growth factor-ß (TGF-ß) and bone morphogenic protein (BMP)-7 mRNA expressions were upregulated at 14 days post release in both genders. Transcutaneous GFR remained normal during the time course. Release of 24 h BOO initiated a bladder remodeling process. The animal model enables a wide range of experiments to study bladder remodeling, and gender differences offer potential targets for understanding bladder fibrosis and adaptation with BOO.

Tamoxifen attenuates renal fibrosis in human kidney slices and rats subjected to unilateral ureteral obstruction.

BACKGROUND AND PURPOSE: Renal fibrosis plays an important role in the development and progression of chronic kidney disease (CKD). Clinical studies have shown that CKD progresses differently in males and females, which may be related to circulating levels of sex hormones. In this study, we investigated the effect of tamoxifen (TAM), a selective estrogen receptor modulator (SERM), on renal fibrosis in male and female rats subjected to unilateral ureteral obstruction (UUO) and human precision-cut kidney slices (PCKS). EXPERIMENTAL APPROACH: Female, ovariectomized female (OVX), and male rats were subjected to 7 days of UUO and treated with TAM by oral gavage. Moreover, we studied individual responses to TAM treatment in PCKS prepared from female and male patients. In all models, the expression of fibrosis markers was examined by western blot, qPCR, and immunohistochemistry. KEY RESULTS: TAM decreased the expression of fibronectin, α-smooth muscle actin, and collagen-1 and -3 in female, OVX, and male rats. In addition, TAM mitigated TGF-ß-induced fibrosis in human PCKS, irrespective of sex, yet interindividual differences in treatment response were observed. CONCLUSION AND IMPLICATIONS: TAM ameliorates renal fibrosis in males and females, although we did observe sex differences in drug response. These findings warrant further research into the clinical applicability of TAM, or other SERMs, for the personalized treatment of renal disease.

Automated and Reproducible Detection of Vascular Endothelial Growth Factor (VEGF) in Renal Tissue Sections.

BACKGROUND: Manual analysis of tissue sections, such as for pathological diagnosis, requires an analyst with substantial knowledge and experience. Reproducible image analysis of biological samples is steadily gaining scientific importance. The aim of the present study was to employ image analysis followed by machine learning to identify vascular endothelial growth factor (VEGF) in kidney tissue that had been subjected to hypoxia. METHODS: Light microscopy images of renal tissue sections stained for VEGF were analyzed. Subsequently, machine learning classified the cells as VEGF+ and VEGF- cells. RESULTS: VEGF was detected and cells were counted with high sensitivity and specificity. CONCLUSION: With great clinical, diagnostic, and research potential, automatic image analysis offers a new quantitative capability, thereby adding numerical information to a mostly qualitative diagnostic approach.

Automatic detection of hypoxia in renal tissue stained with HIF-1alpha.

OBJECTIVE: The objective of this study was the identification of the stain HIF-alpha using the Image Cytometry, and to help to count the positive cells (with HIF-alpha) and the negative cells (without HIF-alpha) from the same sample. METHOD: 17 images of renal tissues from male rats of Winstar lineage; overall, there were 12.587 objects (cells) in the images for analysis. The acquired images were then analyzed through the free softwares CellProfiler (version 2.1.1) and CellProfiler Analyst (version 2.0). In the software CellProfiler Anlyst, there was a separation with the classes of the object, using a classifier, and the classes were: 1) class with HIF-alpha and 2) class without HIF-alpha. RESULTS: With the data obtained through Score All, it was possible to calculate the percentage of cells that had HIF-alpha; out of 12.587 objects of the sample, 6.773 (54%) had HIF-alpha and 5.814 (46%) did not have HIF-alpha. Data of sensibility 0.90, specificity 0.84 and standard deviation 0.10 and 0.12. CONCLUSION: The research shows that the free software CellProfiler, through the light microscope, was able to identify the stains, perform the machine's learning, and subsequently count and separate cells from distinct classes (with and without the stain of HIF-alpha).

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue.

OBJECTIVE: The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation. METHODS: A total of 17 images of kidney histological slide samples from male Wistar rats were used. The slides were photographed using an Axio Zeiss Vert.A1 microscope with a 40x objective lens coupled with an Axio Cam MRC Zeiss camera and Zen 2012 software. The images were analyzed using CellProfiler (version 2.1.1) and CellProfiler Analyst open-source software. RESULTS: Out of the 10,378 objects, 4970 (47,9%) were identified as TUNEL positive, and 5408 (52,1%) were identified as TUNEL negative. On average, the sensitivity and specificity values of the machine learning approach were 0.80 and 0.77, respectively. CONCLUSION: Image cytometry provides a quantitative analytical alternative to the more traditional qualitative methods more commonly used in studies.