Name: using 2-photon microscopy to quantify the effects of chronic unilateral ureteral obstruction on glomerular processes
Uploaded: 2022-03-04T12:30:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes at JoVE.com

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.

<ol>
	<li>Dunn, K. W., Molitoris, B. A., Dagher, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Indiana+O'Brien+center+for+advanced+renal+microscopic+analysis.">The Indiana O'Brien center for advanced renal microscopic analysis.</a> American Journal of Physiology-Renal Physiology. 320 (5), 671-682 (2021).</li><li>Dunn, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+of+the+kidney+of+living+animals+using+multicolor+two-photon+microscopy.">Functional studies of the kidney of living animals using multicolor two-photon microscopy.</a> American Journal of Physiology-Cell Physiology. 283 (3), 905-916 (2002).</li><li>Eisenbach, G., Liew, J., Boylan, J., Manz, N., Muir, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+angiotensin+on+the+filtration+of+protein+in+the+rat+kidney:+a+micropuncture+study.">Effect of angiotensin on the filtration of protein in the rat kidney: a micropuncture study.</a> Kidney International. 8 (2), 80-87 (1975).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+multiphoton+microscopy+as+a+tool+for+studying+renal+physiology+and+pathophysiology.">Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology.</a> Methods. 128, 20-32 (2017).</li><li>Sandoval, R. M., Molitoris, B. A., Palygin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+imaging+and+microscopy+for+dynamic+processes+in+rats.">Fluorescent imaging and microscopy for dynamic processes in rats.</a> Methods in Molecular Biology. 2018, 151-175 (2019).</li><li>Huber, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+functional+podocin-nephrin+complex:+mutations+in+the+NPHS2+gene+disrupt+nephrin+targeting+to+lipid+raft+microdomains.">Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains.</a> Human Molecular Genetics. 12 (24), 3397-3405 (2003).</li><li>Kawachi, H., Koike, H., Kurihara, H., Sakai, T., Shimizu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+rat+homologue+of+podocin:+expression+in+proteinuric+states+and+in+developing+glomeruli.">Cloning of rat homologue of podocin: expression in proteinuric states and in developing glomeruli.</a> Journal of the American Society of Nephrology JASN. 14 (1), 46-56 (2003).</li><li>Roselli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+glomerular+filtration+defect+and+severe+renal+disease+in+podocin-deficient+mice.">Early glomerular filtration defect and severe renal disease in podocin-deficient mice.</a> Molecular and Cellular Biology. 24 (2), 550-560 (2004).</li><li>Schie&#223;l, I., Bardehle, S., Castrop, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superficial+nephrons+in+BALB/c+and+C57BL/6+mice+facilitate+in+vivo+multiphoton+microscopy+of+the+kidney.">Superficial nephrons in BALB/c and C57BL/6 mice facilitate in vivo multiphoton microscopy of the kidney.</a> PloS One. 8 (1), 52499 (2013).</li><li>Chevalier, R., Forbes, M., Thornhill, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureteral+obstruction+as+a+model+of+renal+interstitial+fibrosis+and+obstructive+nephropathy.">Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.</a> Kidney International. 75 (11), 1145-1152 (2009).</li><li>Forbes, M., Thornhill, B., Chevalier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximal+tubular+injury+and+rapid+formation+of+atubular+glomeruli+in+mice+with+unilateral+ureteral+obstruction:+a+new+look+at+an+old+model.">Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model.</a> American Journal of Physiology. Renal physiology. 301 (1), 110-117 (2011).</li><li>Yang, H. -. C., Zuo, Y., Fogo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+chronic+kidney+disease.">Models of chronic kidney disease.</a> Drug Discovery Today. Disease Models. 7 (1-2), 13-19 (2010).</li><li>Hackl, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+fate+of+glomerular+epithelial+cells+in+vivo+using+serial+multiphoton+imaging+in+new+mouse+models+with+fluorescent+lineage+tags.">Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.</a> Nature Medicine. 19 (12), 1661-1666 (2013).</li><li>Kitching, A., Kuligowski, M., Hickey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+recruitment+to+glomeruli+in+mice+using+intravital+microscopy.">In vivo imaging of leukocyte recruitment to glomeruli in mice using intravital microscopy.</a> Methods in Molecular Biology. 466, 109-117 (2009).</li><li>Savin, V. J., Terreros, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filtration+in+single+isolated+mammalian+glomeruli.">Filtration in single isolated mammalian glomeruli.</a> Kidney International. 20 (2), 188-197 (1981).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nature Protocols. 1 (2), 581-585 (2006).</li><li>El Karoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+stress+drives+proteinuria-induced+kidney+lesions+via+Lipocalin+2.">Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2.</a> Nature Communications. 7, 10330 (2016).</li><li>VA, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+measurements+of+gene+signatures+in+different+analytes+using+the+Nanostring+nCounter+Assay+System.">Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.</a> BMC Research Notes. 2, 80 (2009).</li><li>Springer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+transcriptome+and+bioinformatics+approach+to+unilateral+ureteral+obstructive+uropathy+in+the+fetal+sheep+model.">A combined transcriptome and bioinformatics approach to unilateral ureteral obstructive uropathy in the fetal sheep model.</a> The Journal of Urology. 187 (2), 751-756 (2012).</li><li>Braun, F., Becker, J., Brinkkoetter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+or+let+die:+Is+there+any+cell+death+in+podocytes.">Live or let die: Is there any cell death in podocytes.</a> Seminars in Nephrology. 36 (3), 208-219 (2016).</li><li>Kim, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+angiopoietin-1+in+kidney+disease.">The role of angiopoietin-1 in kidney disease.</a> Electrolyte &amp; Blood Pressure E &amp; BP. 6 (1), 22-26 (2008).</li><li>Liu, F., Zhuang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+receptor+tyrosine+kinase+signaling+in+renal+fibrosis.">Role of receptor tyrosine kinase signaling in renal fibrosis.</a> International Journal of Molecular Sciences. 17 (5), 972 (2016).</li><li>Martini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+biology+identifies+shared+transcriptional+networks+in+CKD.">Integrative biology identifies shared transcriptional networks in CKD.</a> Journal of the American Society of Nephrology: JASN. 25 (11), 2559-2572 (2014).</li><li>M&#252;hlberger, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+bioinformatics+analysis+of+proteins+associated+with+the+cardiorenal+syndrome.">Integrative bioinformatics analysis of proteins associated with the cardiorenal syndrome.</a> International Journal of Nephrology. 2011, 809378 (2010).</li><li>Satirapoj, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin:+novel+tissue+and+urinary+biomarker+of+progressive+renal+injury+induces+a+coordinated+mesenchymal+phenotype+in+tubular+cells.">Periostin: novel tissue and urinary biomarker of progressive renal injury induces a coordinated mesenchymal phenotype in tubular cells.</a> Nephrology, Dialysis, Transplantation. 27 (7), 2702-2711 (2012).</li><li>Fengxin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+contributes+to+renal+fibrosis+by+promoting+tubular+epithelial+cell+cycle+arrest+at+G2/M.">Numb contributes to renal fibrosis by promoting tubular epithelial cell cycle arrest at G2/M.</a> Oncotarget. 7 (18), 25604-25619 (2016).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+glomerular+permeability+of+fluorescent+macromolecules+using+2-photon+microscopy+in+Munich+Wistar+rats.">Quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in Munich Wistar rats.</a> Journal of Visualized Experiments: JoVE. (74), e50052 (2013).</li><li>Russo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+tubular+uptake+explains+albuminuria+in+early+diabetic+nephropathy.">Impaired tubular uptake explains albuminuria in early diabetic nephropathy.</a> Journal of the American Society of Nephrology: JASN. 20 (3), 489-494 (2009).</li><li>Russo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+kidney+filters+nephrotic+levels+of+albumin+retrieved+by+proximal+tubule+cells:+retrieval+is+disrupted+in+nephrotic+states.">The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.</a> Kidney International. 71 (6), 504-513 (2007).</li><li>Sandoval, R. M., Wang, E., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+bottom+and+using+it:+Offsets+and+sensitivity+in+the+detection+of+low+intensity+values+in+vivo+with+2-photon+microscopy.">Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy.</a> Intravital. 2 (1), 23674 (2014).</li><li>Kuligowski, M. P., Kitching, A. R., Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+recruitment+to+the+inflamed+glomerulus:+a+critical+role+for+platelet-derived+P-selectin+in+the+absence+of+rolling.">Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling.</a> Journal of Immunology. 176 (11), 6991-6999 (2006).</li><li>Devi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nature Medicine. 19 (1), 107-112 (2013).</li><li>Finsterbusch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patrolling+monocytes+promote+intravascular+neutrophil+activation+and+glomerular+injury+in+the+acutely+inflamed+glomerulus.">Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (35), 5172-5181 (2016).</li><li>Shroff, U. N., Gyarmati, G., Izuhara, A., Deepak, S., Peti-Peterdi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+view+of+macula+densa+cell+protein+synthesis.">A new view of macula densa cell protein synthesis.</a> American Journal of Physiology. Renal Physiology. 321 (6), 689-704 (2021).</li></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.

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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.

Watch this Scientific Journal Video about Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes at JoVE.com

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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