We thank the authors and reviewers for their contributions to this collection. We also acknowledge funding from the NIH (DK106284 and DK123542) and the Oxalosis &#38; Hyperoxaluria Foundation &#8211; American Society of Nephrology KidneyCure Transition to Independence Grant.

<ol>
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The authors have nothing to disclose.

Introduction 
Research within the field of nephrology has significantly improved the well-being of individuals impacted by kidney disease. However, kidney disease is a still major healthcare problem that impacts more than 10% of the population and is responsible for increased healthcare costs and mortality worldwide1. Unfortunately, the precise mechanisms leading to kidney disease have not been fully elucidated. This JoVE Methods Collection highlights recent strategies to define kidney pathophysiology. Specifically, the research groups present a novel animal model and methods to evaluate or treat kidney disease using experimental models or clinical samples. The work presented in this collection is another series of excellent contributions to the field of nephrology and may provide new avenues to prevent, diagnose, or treat kidney disease in the future.

A translational animal model of acute kidney injury 
Acute kidney injury (AKI) is a common form of kidney disease2. Several experimental models have been used to study renal ischemia/reperfusion (I/R) injury3,4,5. However, translating these findings to improve the clinical setting has posed many challenges since there are significant differences amongst species6. Doulamis et al. developed a swine renal I/R model using a bilateral balloon catheter to address this issue7. Their model was highly reproducible, caused reduced urinary output and estimated glomerular filtration rate, and increased plasma creatinine and blood urea nitrogen levels in animals. Further, gross tissue and histological examination revealed infarction and hemorrhaging within the kidneys. This new model is clinically relevant and may aid in further understanding AKI in humans.
Strategies to examine cellular mechanisms involved in kidney disease 
Cytokines can contribute to kidney injury and repair8. Thus, it is important to identify the source and role of cytokines within the kidney. Taguchi et al. developed a technique to understand protein secretion using brefeldin A (BFA), a protein secretion inhibitor, in AKI and chronic kidney disease mouse models9. They injected BFA into the tail vein of mice and subsequently evaluated their kidneys using immunofluorescence staining. They determined that BFA inhibited protein secretion and caused cytokine buildup in certain cell types using cell-specific markers. This innovative protocol could be used to study protein secretion in several renal pathologies.
Another proposed mechanism thought to contribute to kidney disease is microRNAs (miRNA)10, which are known to degrade and inhibit mRNA transcription. It has been reported that there is a correlation between miRNA expression in tissue and serum in humans11. However, there is no method to purify and quantify both mouse kidneys and serum miRNA. Yanai et al. optimized a quantitative real-time polymerase chain reaction method to evaluate miRNA expression in the kidney and serum of mice with age-dependent renal impairment12. They also established a correlation between both miRNA levels. This protocol is high-throughput and could be applicable to many pathological conditions.
New imaging techniques to evaluate kidney disease 
Noninvasive imaging of the kidney can assess structural and functional defects. However, there are some limitations with the current methods. To circumvent this, Holmes et al. developed a simple, cost-effective 3D imaging technique based on robotic ultrasound (US) technology to quantify kidney, liver, and cardiac function13. Their protocol provided consistently high-quality images and could be used for studies where gross tissue, tumor sizes, or the benefits of therapeutics could be monitored. The greatest strength of this method is the high output of data. For example, three rats can be imaged at once, or 20&#8211;30 mice can be imaged per hour. This protocol is a quick alternative to traditional noninvasive imaging modalities.
Another research group in the collection used imaging to study unilateral ureteral obstruction (UUO), an injury model that induces surface glomeruli. Wagner et al. used intravital two-photon microscopy to monitor UUO in rats that develop surface glomeruli and those that do not develop surface glomeruli14. Two-photon microscopy provides an increased depth of penetration and examination of different regions of the kidney. The authors found that UUO induced inflammation and fibrosis, and also decreased red blood cell flow in rat kidneys. Quantifying blood flow, vasoconstriction, and dilatation in response to drugs and inflammation are a few benefits this method provides. Collectively, these studies provide new imaging strategies to study various tissues.
Estimating nanocrystalluria as a predictor of kidney disease 
It has been proposed that assessing urinary crystals could be useful to predict kidney stone risk15. We developed a protocol to quantify urinary nanocrystals (&#8804;1 &#181;m) from healthy adults before and after they consumed a dietary oxalate load known to induce urinary oxalate levels16. We isolated and analyzed calcium-containing urinary nanocrystals using a calcium fluorophore and nanoparticle tracking analysis (NTA). We determined that NTA can specifically detect calcium-containing nanocrystals in urine, and the results were consistent with our previous findings17. This protocol can be used to study urinary nanocrystals in stone formers or various fields of research where crystalopathies occur.
Therapeutic approaches to treat kidney disease 
Artificially synthesized miRNA mimics have been suggested as a possible treatment for kidney disease. However, serum RNAase can degrade exogenous miRNA mimics18. Yanai et al. produced non-viral vectors based on linear polymer, polyethylenimine nanoparticles (PEI-NPs), to safely deliver a miRNA mimic to the kidneys of mice18. They proved that the mimic was effective in overexpressing the targeted miRNA. The limitations of this method include the substantial amount of PEI-NPs required for larger animal models and the lack of specificity to the kidneys. However, based on the success of RNA vaccines, miRNA mimics could be an ideal therapeutic option for kidney disease.
Concluding remarks 
The comprehensive research presented here&#160;introduces a new animal model and techniques to study prevalent issues surrounding nephrology. It is important to note that some of these methods could apply to other areas outside of nephrology. The next logical steps will be to investigate how these methods could be tested in larger animal models and clinical trials. We hope that the sharing of these protocols will expand our knowledge about renal pathophysiology and birth new ideas to mitigate kidney disease in individuals.

novel translational approaches to study kidney disease

ARTICLES DISCUSSED:
Kumar, P., Bell, A., Mitchell, T., Estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis. Journal of Visualized Experiments. (168), e62192 (2021).
Doulamis, I. P. et al. A large animal model for acute kidney injury by temporary bilateral renal artery occlusion. Journal of Visualized Experiments. (168), e62230 (2021).
Taguchi, K., Sugahara, S., Elias, B. C., Brooks, C. R. Identification of the source of secreted proteins in the kidney by brefeldin A injection. Journal of Visualized Experiments. (177), e63178 (2021).
Yanai, K. et al. Quantitative real-time polymerase chain reaction evaluation of microRNA expression in kidney and serum of mice with age-dependent renal impairment. Journal of Visualized Experiments. (182), e63258 (2022).
Holmes, H. L. et al. Use of 3D robotic ultrasound for in vivo analysis of mouse kidneys. Journal of Visualized Experiments. (174), e62682 (2021).
Yanai, K., Kaneko, S., Ishii, H., Aomatsu, A., Morishita, Y. Delivery of exogenous artificially synthesized miRNA mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models. Journal of Visualized Experiments. (183), e63302 (2022).
Wagner, M. C., Sandoval, R. M., Campos-Bilderback, S. B., Molitoris, B. A. Using 2-photon microscopy to quantify the effects of chronic unilateral ureteral obstruction on glomerular processes. Journal of Visualized Experiments. (181), e63329 (2022).

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Novel Translational Approaches To Study Kidney Disease

ARTICLES DISCUSSED:
Kumar, P., Bell, A., Mitchell, T., Estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle ...

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