This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1DK091623 and P30DK079312 (to B.A.M.). We thank the staff at the Genomics Core Facility of the Research Technology Support Facility (RTSF) at Michigan State University for performing the Nanostring analysis.

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</ol>

The authors have no conflicts of interest.

The study of glomerular physiology has seen many different approaches, most notably the use of micropuncture, perfusion of isolated glomeruli, and microscopy. The availability of surface glomeruli in Munich Wistar rats, Fromter and Simonsen strains, has allowed in vivo dynamic studies. One important note to investigators adopting this technology is the need to set acquisition parameters to maintain consistent images between studies, so the autofluorescence in tissue remains consistent. Utilizing a dual-pass fluorescein/rhodamine epifluorescence cube and adjusting gain settings to the green and red emission channels to mimic on the computer screen what is seen through the eyepieces will ensure a consistent color signature in the autofluorescence even between different microscope systems.
The Fromter strain has been used extensively as it has a reduced number of total glomeruli, ~75% normal, and the males spontaneously develop hypertension at around 12 weeks of age, with progressive proteinuria and subsequent focal glomerular sclerosis, eventually dying of kidney failure12. The use of these rats and the addition of 2-photon microscopy with its reduced phototoxicity, improved depth of penetration, and the ability to view multiple fluorescent probes simultaneously paved the way for new discoveries1,4,5. With the development of computer hardware and software, quantitative data are now the standard for all 2-photon laboratories. Multiple quantitative techniques have been developed and applied to glomerular, proximal tubule, vascular, and interstitial processes under physiologic and disease conditions1,4,5,27,28,29,30.
Transgenic mouse generating facilities added a new dimension to the study of kidney physiology and pathology, and it was only a matter of time until this was combined with 2-photon microscopy to further delineate the importance of specific gene products in kidney structure and function. However, mouse glomeruli, except in very young mice, are located over 100 &#181;m from the surface of the kidney9. Two-photon microscopy is best undertaken at a depth of between 20 and 50 &#181;m as resolution, and fluorescence intensity diminishes rapidly thereafter due to light scattering of emitted light and absorption from interaction with hemoglobin. Therefore, it was necessary to induce surface glomeruli. The approach commonly used is a prolonged unilateral obstruction model for 12 weeks. As these models do not allow baseline determinations, separation of the effects of UUO from the process being studied is not possible.
Using MWF rats, one can compare baseline glomerular function with that following UUO. This UUO model is known to induce inflammation and a rapid rate of fibrosis and has been used to study CKD and fibrosis10,11,12. As expected, there was an increase in surface glomeruli in both the MWF and SD rats. Moreover, the quantitative results obtained following UUO for the MWF and SD rats were very comparable. The reduction in blood flow recorded here had been previously reported comparing microscopic data following UUO to micropuncture data3. It was also well known that tubular and interstitial histology is markedly altered, and the PTs are mostly nonfunctional, as reported here, with a lack of albumin endocytosis. The studies in Figure 2 and Figure 3 show a dramatic reduction in RBC flow rate in glomerular and peritubular capillaries and enhanced WBC adhesion. The reductions in flow are likely due to capillary blockage from WBC adhesion and rouleaux formations.
To further evaluate inflammation, we quantified albumin permeability and showed it to increase tenfold. Additionally, isolated glomeruli showed that mRNA expression increased for many genes previously known to be increased in kidney inflammation in a variety of kidney disease states17,19,20,21,22,23,24,25,26. The increases in glomerular surface density and albumin permeability were progressive, as shown by the 12-week UUO data. The present data are the first to directly show that glomeruli undergo significant structural damage, inflammation, and molecular changes in the UUO model. The results are consistent with an earlier study of whole kidney tissue that analyzed sheep kidney biopsies following UUO, finding multiple inflammation markers elevated19. The present results indicate marked inflammation exists within the glomeruli, previously only known for cortical tissue.
The present data differs from earlier studies in mice where no changes were found in adhesion molecule expression, complement deposition, and neutrophil infiltration between 12-week posthydronephrotic and normal glomeruli31. In addition, the Hickey laboratory used the 12-week UUO model to study immune reactions in glomeruli of mice. They found no differences in neutrophil infiltration between four-week-old mouse glomeruli and postobstructive glomeruli32,33. These later studies were conducted after the pelvis of the obstructed kidney was drained of urine. We did not do this as we wanted to determine the effect of UUO on glomerular function as it would be in vivo, without artificially removing the fluid causing the obstruction. Finally, the use of UUO in mice is being replaced by imaging glomeruli at greater than 100 &#181;m beneath the surface. While possible, there is a trade-off of in resolution and intensity, both being reduced significantly as one goes beyond 50 &#181;m34.
The results presented are not surprising if one pieces together the data from the existing literature on histologic changes, formation of atubular glomeruli, inflammation, fibrosis, hemodynamics10,11,12. The data presented, including WBC adhesion, rouleaux formations, glomerular molecular inflammation markers, and increased albumin permeability, further indicate the extensive inflammation that is ongoing in this UUO model even at five weeks and also present at twelve weeks. Clearly, chronic UUO is not a physiologic state, and the use of UUO to induce surface glomeruli represents an injury model. The MWF rats, which have surface glomeruli under physiological conditions, can be longitudinally studied as injury occurs. It is possible to generate transgenic rats, and numerous investigators are creating them with biosensors to ask specific questions. In particular, the Medical College of Wisconsin now has a colony of MWF rats and has made transgenic rats for the purpose of studying glomerular processes under physiologic and pathologic conditions. These MWF rats offer a great opportunity to study glomerular processes in normal, diseased, and genetically altered rats.

Understanding glomerular processes, especially podocyte biology, has been a goal for over 50 years. Munich Wistar rats with surface glomeruli have played a central role in these studies, including micropuncture studies, to understand numerous aspects of physiologic and pathologic processes1,2,3. The utilization of microscopy to study glomerular components intravitally was limited due to the effects of phototoxicity until the advent of 2-photon microscopy that minimized this toxic exposure and increased the depth of penetration1,2. Along with rapid advancements in computer hardware and software, this has allowed for three-dimensional (3D) and four-dimensional (time) studies for hours in a single setting1,4,5.
The quantification of glomerular capillary blood flow, vasoconstriction and dilatation in response to drugs, permeability, and the effects of charge on permeability and inflammation are just some of the glomerular processes that have been studied. In addition, the S1 segment of the proximal tubule is identifiable, and the differences in the behavior of S1 and S2 tubular epithelium can be quantified1,4. Studies in mice, especially with the universal availability of mouse transgenic facilities, have led to rapid advances in the understanding of the molecular biology of glomerular disease processes. Individual proteins are responsible for glomerular dysfunction in knockout studies, especially with regard to proteinuria6,7,8. However, the utilization of mouse models for glomerular imaging studies has been limited as glomeruli are more than 100 &#181;m below the surface in the numerous strains studied9.
This has led investigators to develop and utilize mouse models resulting in surface glomeruli that can be studied. The most common model is the use of complete UUO10,11,12. At the end of the extended UUO period, there are numerous surface glomeruli in mice kidneys that can be and have been studied13,14. There has been no baseline or control study in these mouse studies to determine the effects of prolonged UUO on glomerular biology. As this is a severe and prolonged model of injury resulting in rapid fibrosis and cortical destruction10,11,12, we hypothesized there would be effects on glomerular processes and function. To answer this question, Munich Wistar Fromter (MWF) rats with surface glomeruli were used to study control/baseline parameters, and the baseline finding was compared with glomerular studies in MWF rats following five weeks of UUO. We also studied Sprague Dawley (SD) rats that do not have surface glomeruli following UUO. The findings indicate that 5 weeks of UUO in MWF and SD rats do indeed increase the number of surface glomeruli. However, these were abnormal glomeruli with marked changes in glomerular blood flow, inflammation, and macromolecule permeability and size.

<table><tbody><tr><td>70 &amp;micro;m sterile cell strainer</td><td>Corning</td><td>#421751</td><td></td></tr><tr><td>100 &amp;micro;m sterile cell strainer</td><td>Corning</td><td>#421752</td><td></td></tr><tr><td>CA Micro scissors Model 1C300</td><td>Electron Microscopy Sciences</td><td>Cat# 72930</td><td></td></tr><tr><td>Electric heating pad</td><td>Sunbeam</td><td>Kroger</td><td></td></tr><tr><td>Handling Forceps</td><td>Electron Microscopy Sciences</td><td>Cat# 72962</td><td></td></tr><tr><td>Kelly Hemostatic Forceps (straight)</td><td>Electron Microscopy Sciences</td><td>Cat#72930</td><td></td></tr><tr><td>Leica Dive SP-8 Multi-Photon Inverted Microscope</td><td>Leica Microsystems</td><td>Note: Version 7.1r1</td><td></td></tr><tr><td>MaiTai DeepSee titanium-sapphire laser</td><td>Spectra-Physics</td><td>NA</td><td></td></tr><tr><td>Mayo Dissecting Scissors</td><td>Electron Microscopy Sciences</td><td>Cat# 78180-1C3</td><td></td></tr><tr><td>Metamorph Image processing Software</td><td>Molecular Dynamics</td><td>Cat# 78266-04</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft Corportation</td><td>2007 version</td><td></td></tr><tr><td>Quant-iT RNA Assay Kit</td><td>Invitrogen/ThermoFisher</td><td>Q33140</td><td></td></tr><tr><td>Reptitherm Undertank Heater</td><td>Zoomed</td><td>Amazon</td><td></td></tr><tr><td>RNeasy MinElute Cleanup Kit (Spin columns)</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>RPE buffer</td><td>Qiagen</td><td>1018013</td><td></td></tr><tr><td>Strate-Line Autoclave Tape</td><td>Fisher Scientific</td><td>Cat# 11-889-1</td><td></td></tr><tr><td>TRI Reagent</td><td>Sigma</td><td>T9424</td><td></td></tr><tr><td>Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip)</td><td>Electron Microscopy Sciences</td><td>Cat# 70665-07</td><td></td></tr></tbody></table>

using 2-photon microscopy to quantify the effects of chronic unilateral ureteral obstruction on glomerular processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

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Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

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Research

JoVE Journal

Medicine

2.2K Views.  Indiana University School of Medicine. In de gegevens die in deze studie worden gepresenteerd, tonen de dramatische effecten van unilaterale ureterale obstructie op de nierfunctie, ontsteking en fibrose. Het helpt ons om de pathofysiologische mechanismen van unilaterale ureterale obstructie te begrijpen. Het belangrijkste voordeel van deze techniek is het vermogen om cel-celinteracties, subcellulaire gebeurtenissen te bestuderen en structuur en functie op een dynamische manier te associëren binnen een functionerende nier.H...

Video: Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Genetics

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Biology

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://www.jove.com/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

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