Phenotyping & Pathophysiology Core

The Phenotyping and Pathophysiology Core is designed to address the need for effective phenotyping of renal function in mouse models of selective gene overexpression or deletion.

Designed to provide experimental mouse models of acute or progressive renal injury to assess potential therapeutic interventions.
Helps investigators who may have developed genetic mouse models or potential therapeutic interventions, but do not have the expertise and/or infrastructure required to design and carry out appropriate kidney phenotyping studies or studies investigating mouse models of kidney injury.

The Phenotyping and Pathophysiology Core has five distinct subcores that will offer and/or develop methodologies to define the pathogenesis of kidney disease and design therapeutic interventions.

The Phenotyping subcore will provide a battery of well-established invasive and non-invasive tests of kidney function.
The Injury Model subcore will provide investigators cost-effective testing of potential pharmaceutical or immunologic therapy strategies in appropriate mouse models of acute or progressive kidney injury. In addition, this subcore will provide murine models for screening of potential kidney side effects of drugs. This subcore provides a wide variety of experimental models of mouse kidney injury that offer consistent and reproducible results. Some of the mouse models available have been developed by the Center investigators.
The Metabolism and Bioenergetics subcore will offer direct measurements of kidney tissue partial oxygen consumption and will develop methodology for the measurements of whole kidney oxygen consumption based on kidney blood flow and measurements of arterial and renal venous partial oxygen. Furthermore, this subcore will provide expertise in metabolic flux analysis, glucose uptake, and quantification of various metabolites in kidney tissues using mass spectrometry methods.
The In Vivo Mouse Kidney Imaging subcore will develop and establish non-invasive imaging techniques for the assessments of mouse renal diseases. This subcore will help investigators to execute high throughput, quantitative, and non-invasive imaging studies in mouse models of renal disease.
The CRISPR/cas9 subcore will provide guidance for the design, construction, and generation of a CRISPR mouse

The Phenotyping and Pathophysiology Core provides a broad range of services that aid researchers interested in utilizing murine models for kidney-related research. The structure of this Core is unique in that it performs a variety of kidney functions, non-invasive quantitative kidney images analysis, and experimental platforms for the testing of drugs and interventions.

Most importantly, this Core performs all these services in-house and directly oversees these studies. We have been very successful in providing these services not only to the local scientific community interested in kidney research, but also serving as a national and international resource.

We not only provide innovative services, but also plan to continue to develop, test and implement new methodologies that can be utilized by both the local Biomedical Research Base, as well as interested national and international researchers, to advance our knowledge about the mechanisms of kidney disease, and to develop and test potential therapies.

Core Assays and Service

Phenotyping Subcore

Phenotyping subcore provides standardized methods to determine renal function in mice

Albuminuria: Urinary albumin and creatinine will be determined using a mouse specific ELISA kit (Albuwell M kit, EXOCELL, Philadelphia, Penn.).
Blood urea nitrogen (BUN): It will be measured using a Urea Assay Kit (BioAssay Systems).
Creatinine: Serum creatinine will be measured by HPLC
Glomerular filtration rate (GFR): GFR will be determined based on transcutaneously measured elimination kinetics of FITC-sinistrin. A device transcutaneously excites FITC-sinistrin at 480 nm and detects the emitted light through the skin at 520 nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of kidney function. The whole device fits on the back of freely moving rodents.
Plasma renin activity (PRA): PRA is evaluated in fresh or frozen plasma collected from conscious mice via the femoral vein between 9-11 AM. Plasma samples are incubated for 1 hour with excess exogenous renin substrate and then radioimmunoassay (Gammacoat, DiaSorin) is performed to measure the generation of angiotensin I
Plasma aldosterone: Plasma levels are determined using RIA Kits (COAT-A-COUNT, Siemens, Siemens Medical Solutions).
Urinary F2-isoprostanes: F2-isoprostanes are sensitive and specific biomarkers for oxidative stress will be determined by negative-ion gas-chromatography mass spectrometry.
Urinary osmolality: It will be measured using a microosmometer (μOsmette, Precision Systems, Natick, Mass.).
Blood and urine Na+, K+ levels: Blood and urine electolytes will be determined using a flame photometer (GMI, Ramsey, Minn.).
Hemodynamic measurements: Blood pressure will be measured in conscious mice:
- Tail cuff: Mice will be trained for three consecutive days before systolic blood pressure (SBP) is recorded on the following two days using a tail-cuff monitor (BP-2000 Blood Pressure Analysis System, Visitech Systems, Inc, Apex, N.C).
- Catheterization BP: Anesthetized mice undergo tracheostomy followed by insertion of a phycoerythrin 10 tubing into the right common carotid artery. The catheter is then tunneled under the skin, exteriorized, secured at the back of the neck, filled with heparinized saline, and sealed. The catheterized mice are singly housed, and BP is measured 24 and 48 hours later with a Blood Pressure Analyzer (Micro-Med, Lousville, Ky.). This service is provided in conjunction with the Vanderbilt MMPC.
- Radiotelemetry: A radiotelemetric catheter (PA-C10, Data Sciences International) is inserted into the left common carotid artery of anesthetized mice while a transmitter (TA11PA-C20, Data Sciences International, St. Paul, Minn.) is implanted s.c. After 10 days recovery, BP and HR are recorded. The data from the telemetric device are collected using the Dataquest ART system, version 4.0 (Data Sciences International) by way of an RPC-1 receiver placed under the mouse cage. The baseline measures are averaged for a period of one to seven days before subjecting the animal to the desired physiologic and/or pathologic maneuver. Blood pressure and heart rate data can be collected over a period of days to weeks as needed.
Sodium and water balance study with acute salt loading. Mice will be housed individually to acclimate in metabolic cages for three days on a normal salt diet with free access to water. Then, a single dose of one mEq of NaCl is given by gavage. Urine is collected every 12 hours for 72 hours for urine volume and Na and K excretion. Urinary Na and K levels will be determined using a flame photometer (GMI, Ramsey, Minn.).
Bone marrow transplantation (BMT): Briefly, the recipient mice are kept on acidified water containing neomycin and polymyxin B sulfate(21). One week later, recipient mice are lethally irradiated with nine Gy via a cesium γ source and then injected via tail vein with 5 x 106 bone marrow cells in 0.2 ml medium obtained from the tibia of donor mice. Five weeks later, DNA from whole blood is extracted to determine the degree of chimerism by PCR.
Tissue and cell sample preparation from kidney
- Kidney protein and mRNA isolation: Upon request, total kidney, kidney cortices, outer medullae and/or papillae can be dissected, immediately frozen in liquid N2 and stored at -80C. These samples can then be used for total protein and/or RNA extraction for Western blot and real time PCR.
- Glomerular isolation: Upon request, glomerular isolation can be performed using method described by Takemoto et al. Briefly, anesthetized mice are perfused through the ascending aorta with 8 × 107 Dynabeads (Dynal Biotech ASA, Oslo, Norway) in PBS. The kidneys are removed, minced, and digested with collagenase A (1 mg/ml) and deoxyribonuclease (100U/ml)) in Hank’s balanced salt solution (HBSS) at 37°C for 30 minutes. The digested kidneys are then pressed through a 100-μm cell strainer. The filtered cells are subsequently passed through a new cell strainer and the filtered cells are pelleted and re-suspended in HBSS. Finally, glomeruli that contain Dynabeads are harvested by a magnetic particle concentrator. The preparation consists of >90% glomeruli.

Injury Model Subcore

The Injury Model subcore provides models of both acute and chronic kidney injury.

Models of type I and type II diabetic nephropathy

Type II diabetic nephropathy: The eNOS-/-;db/db mice have significant decreases in GFR and increases in albuminuria, arteriolar hyalinosis, increased GBM thickness, mesangial expansion, mesangiolysis and focal segmental and early nodular glomerulosclerosis. The eNOS-/-;db/db mice become obese by three to four weeks of age. By 20-24 weeks, these mice present significant renal histologic damage, offering the advantage that significant pathologic abnormalities can be detected within a relatively short time of period.
Streptozotocin-induced Type I diabetic nephropathy: To mitigate non-specific cytotoxicity, we use the approved DCC protocol of multiple low-dose streptozotocin injections (50 mg/kg/day for five consecutive day), which induces diabetes mellitus by causing repetitive low-grade ß-cell damage without significant extra-pancreatic toxicity.

Models of progressive non-diabetic renal injury

5/6 nephrectomy
Angiotensin II induced nephropathy: This model of glomerulosclerosis and tubulointerstitial injury is induced in unilaterally nephrectomized mice with angiotensin II infusion over a prolonged period of time. Depending on the susceptibility of the mouse strain and the dose of angiotensin II, infusion for eight weeks produces moderate to severe renal lesions, mainly consisting of glomerulosclerosis, tubular atrophy and/or dilation with little microcyst formation, mild to moderate interstitial fibrosis and a multifocal mononuclear cell infiltration.
Unilateral ureteral obstruction (UUO): This is a model of tubulointerstitial injury induced by performing a unilateral ureteral obstruction. Significant tubulointerstitial matrix accumulation is evident within five to seven days, and there is progressive destruction of tubular architecture.
Aristolochic acid-induced nephropathy: This is a model of acute kidney injury followed by chronic sequelae, which is induced by a single injection of aristolochic acid (4.7 mg/kg body weight). Mice initially develop acute tubular injury and after four weeks they still show evidence of substantial and widespread tubular atrophy and tubulointerstitial fibrosis.
Overexpression of human heparin-binding EGF in kidney proximal tubules. We have developed a mouse line that overexpresses the EGFR ligand, human heparin-binding EGF (hHB-EGF), in the proximal tubule epithelial cells. Homozygous hHB-EGF transgenic mice develop early (three to four weeks of age), spontaneous, and progressive tubulointerstitial fibrosis

Models of acute kidney injury (AKI)

Ischemia-reperfusion: We routinely offer unilateral clamping and contralateral nephrectomy in order to reduce variability of results but bilateral clamping is also available.
Toxic nephropathy: The Core offers a variety of toxin-induced AKI models, including mercuric chloride, folic acid, adenine, and aristolochic acid as well as a novel transgenic model of selective proximal tubule injury that we have recently developed. This model has selective expression of the diphtheria toxin (DT) receptor in the proximal tubule. Injection of different concentration of DT induces graded and reproducible apoptotic injury selectively in the proximal tubule, thus offering a robust model of AKI.

Kidney Oxygen Metabolism and Bioenergetics Subcore

To assist investigators in the screening and characterization of murine kidney disease model with potential defects in O2 and energy metabolism.

The In Vivo Mouse Kidney Imaging Subcore

To enable investigators to execute high quality, high throughput, and quantitative kidney imaging (MRI, SPECT/PET, CT, ultrasound, optical, etc.) for the non-invasive characterization of mouse kidney disease.

The CRISPR/cas9 Subcore

To provide guidance to design and construction of a CRISPR mouse, help with the preparation of reagents, oversee the generation of the mouse, and provide guidance and/or technical help for identifying positive pups and selecting the line.