Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy (EM) of rat, rabbit, and human kidney. An elaborate network of major and minor cell processes here named maculapodia were found at the cell base, projecting towards other MD cells and the glomerular vascular pole. The extent of maculapodia showed up-regulation by low dietary salt intake and female gender. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. EM of rat, rabbit, and human kidneys, and three-dimensional (3D) volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and the afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus (JGA) between MD cells and between MD and other target cells.
Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo due to technical limitations. We aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days-weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked due to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti + cells were found in the renal cortex. The heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared to the brain, pancreas, liver and spleen. Serial MPM of Cdh5-Confetti mice in vivo is a powerful new technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.
Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that leads to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis and interstitial fibrosis by 5 months of age with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing, and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.
Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.
Background and Aims Preliminary preclinical and emerging clinical evidence indicates strong antiproteinuric actions of dual endothelin type A (ETA) and angiotensin II type 1 (AT1) receptor antagonism with sparsentan. These nephroprotective effects have been more pronounced in different experimental and clinical settings compared to current standard of care using an AT1 receptor blocker (ARB). Considering the broad spectrum of renal actions of endothelin (ET) and angiotensin II (Ang II), inhibition of both pathways using sparsentan is postulated to target multiple renal cell types via a variety of renoprotective mechanisms. The aim of this study was to determine glomerular action of sparsentan as compared to the ARB losartan (Los) by direct visualization of effects on renal hemodynamics and tissue remodeling in the intact living kidney. Method Intravital multiphoton microscopy (MPM) of the glomerular vasculature and filtration barrier structure and function was performed in genetically engineered mice combined with traditional urinalysis and histology-based phenotyping. Glomerular hemodynamic parameters (afferent and efferent arteriole (AA and EA) diameters and single nephron glomerular filtration rate (SNGFR)) and podocyte calcium entry, as a measure of cell injury, were quantitatively visualized in the FSGS model Pod-GCaMP5/Tomato TRPC6 transgenic mice (1.5 years of age), in which TRPC6 is overexpressed together with the calcium reporter GCaMP5 in podocytes. Single cell identification and fate tracking of cells of the renin lineage (CoRL) was performed over time using a second physiologic control mouse model, Ren1d-Confetti mice that feature a multicolor CFP/GFP/YFP/FP reporter. Three groups of mice in each model received treatment with either vehicle (CTRL), the ARB losartan (Los; 10 mg/kg/day), or sparsentan (120 mg/kg/day) for 6 weeks (FSGS model) or 2 weeks (control physiology model). Results Both Los and sparsentan treatment attenuated the acute ET + Ang II-induced elevation of podocyte calcium by ∼80%, and the development of albuminuria, and glomerulosclerosis and tissue fibrosis in the FSGS model. Notably, sparsentan prevented the ET + Ang II increases in podocyte calcium more than Los and was significantly more effective in dilating both AA and EA (Fig. 1A, B), increasing SNGFR (Fig. 1C), increasing capillary blood flow (2-fold; p<0.0001 vs. CTRL), and decreasing albuminuria (20%; p<0.05 vs. CTRL). Sparsentan also preserved p57+ podocyte number by 50% compared to Los (p<0.0001 vs. Los). Similarly, pretreatment with sparsentan was more effective in preventing glomerular arteriolar vasoconstriction induced by acute ET + Ang II iv injection compared to Los (p<0.05 vs. Los). Following a 2-week treatment in control healthy Ren1d-Confetti mice, sparsentan resulted in a more robust increase compared to Los in the number of Confetti+ cells, clones, and individual cells per clone in the glomeruli and AA (Fig. 1D-F). Renal tubule segments also showed active cellular remodeling in response to sparsentan. Conclusion Serial MPM imaging directly visualized several mechanisms underlying beneficial antiproteinuric and structural effects of sparsentan in both FSGS and in the normal mouse kidney and differences between dual ETA/AT1 receptor antagonism of sparsentan and a mono-selective ARB. The sparsentan-induced glomerular hemodynamic pattern was driven by both AA and EA dilation resulting in an increase in capillary blood flow. Compared to Los, sparsentan was more effective in attenuating ET/Ang II-induced podocyte injury and in activation of resident progenitor cells and tissue remodeling. These findings suggest multiple layers of renal protective actions by dual ETA and AT1 receptor antagonism.
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