Advances in Renal Cell Imaging

Abstract

A great variety of cell imaging technologies are used routinely every day for the investigation of kidney cell types in applications ranging from basic science research to drug development and pharmacology, clinical nephrology, and pathology. Quantitative visualization of the identity, density, and fate of both resident and nonresident cells in the kidney, and imaging-based analysis of their altered function, (patho)biology, metabolism, and signaling in disease conditions, can help to better define pathomechanism-based disease subgroups, identify critical cells and structures that play a role in the pathogenesis, critically needed biomarkers of disease progression, and cell and molecular pathways as targets for novel therapies. Overall, renal cell imaging has great potential for improving the precision of diagnostic and treatment paradigms for individual acute kidney injury or chronic kidney disease patients or patient populations. This review highlights and provides examples for some of the recently developed renal cell optical imaging approaches, mainly intravital multiphoton fluorescence microscopy, and the new knowledge they provide for our better understanding of renal pathologies.

Keywords: Genetic cell fate tracking; calcium signaling; cell metabolism; ischemia-reperfusion injury; multiplex imaging fibrosis.

Figure 1. Intravital MPM imaging of renal cell types and lineages in the mouse kidney cortex based on genetic cell identification and fate tracking

A–C: Individual cells of the renin lineage are labeled in Ren1d-Confetti mouse kidneys in one of four colors (membrane-targeted CFP, nuclear GFP, cytosolic YFP or cytosolic RFP), and identify the classical vascular site of intra-renal renin synthesis in the juxtaglomerular apparatus around the terminal afferent arteriole (AA), but not the efferent arteriole (EA, panel A). Cells of the Bowman’s capsule around glomeruli (G, panels A–B) and the tubular site of renin production in two connecting tubule segments (CNT1-2) merging into the common cortical collecting duct (CCD, panel C) are also labeled. B: Nanotubes (arrows) are visible interconnecting the cells of the parietal and visceral Bowman’s capsule over the open filtration space. D: Proximal tubule segments (green) are labeled in γGT-mTmG mice. Note the fragmented rather than continuous epithelial cell labeling (arrows). E: Interstitial pericytes (green) are labeled in Coll1a1-GFP mice, and are visible around the proximal tubule (PT) peritubular capillaries. F: Lymphatic endothelial cells (green) are labeled in Prox1-GFP mouse kidney. Note the branching lymphatic vessels (arrows) in a mouse kidney 21 days after unilateral ureter obstruction (UUO). In all images plasma was labeled red using Albumin-Alexa594. Bar is 50 μm.

Figure 2. Quantitative intravital MPM imaging of the changes in renal cortical CD44+ cell density after ischemia-reperfusion injury (IRI)

A: Control, non-ischemic images were obtained at baseline (Pre-IRI) after labeling endogenous CD44+ cells by iv injection of Alexa488-conjugated anti-CD44 antibody (green). Plasma was labeled red using Albumin-Alexa594. Note the intense autofluorescence in proximal (PT) but not in distal tubule (DT) segments owing to the high density of NADH-rich mitochondria, and the low number of circulating CD44+ cells (arrows, magnified in insets). Bar is 20 μm. B–C: MPM images of the mouse renal cortex 1 (B) and 2 (C) days after IRI. Note the high number of CD44+ cells homing in peritubular capillaries surrounding PT segments. D: Statistical summary of the density of CD44+ cells in the renal cortex before and after IRI. CD44+ cell numbers were counted per microscope field. *p<0.05, n=4 each.

Figure 3. Intravital MPM imaging of cell metabolism in the living mouse kidney

A–D: Serial MPM imaging of the changes in mitochondrial membrane potential in the same glomerulus and surrounding tubule segments before (A, control) and after iv injected MitoTracker-Red (red)(B, Pre-IRI), and 10 min after ischemia-reperfusion injury (C, Post-IRI). Plasma was labeled with FITC-conjugated albumin (green). G: glomerulus, PT: proximal tubule. D: Statistical summary of the changes in MitoTracker-Red fluorescence intensity in the PT in response to IRI. *p<0.05, n=10 each. E: The highest intensity of MitoTracker-Red fluorescence was observed in cells around glomerular capillaries (podocytes, arrows), and in the distal tubule (DT). F: Intravital MPM imaging of mitochondrial reactive oxygen species (ROS) generation using iv injected MitoSox-Red (red) in STZ+L-NAME-treated diabetic and hypertensive mice. High intensity of MitoSox-Red fluorescence was observed in the distal tubule and cortical collecting duct (CCD) in addition to proximal tubules (PT). Scale bars are 20 μm.

Figure 4. Intravital MPM imaging of injury-induced proximal tubule cell calcium signaling in Ksp-GCaMP3 mice

A–C: Select proximal tubule segments (PT, green) are labeled by the Ksp-driven Cre/lox-mediated expression of the genetically encoded calcium indicator GCaMP3. Note the fragmented rather than continuous epithelial cell labeling. The site of laser-induced single PT cell injury is indicated by “X”, and the resulting elevations in cell [Ca²⁺] that propagated to downstream tubule segments within 30 s are shown by arrows on panels B–C. Plasma was labeled red using Albumin-Alexa594, which also appears endocytosed in select early PT segments. Bar is 50 μm. D: Representative recordings of GCaMP3 fluorescence intensity at an unaffected upstream site (ROI1, green) and downstream from the injury site (ROI2, red).

Figure 5. Multiplex imaging analysis of the pathological changes in cell functions and tissue architecture in fixed human kidney sections

A–C: Quantitative imaging of the level of tissue fibrosis by the ratio of second harmonic generation (SHG, cyan) and tissue autofluorescence (green) in fixed nephrectomy samples of 50–60 years old age-matched female (A) and male (B) patients. Note the moderate, homogenous SHG signal around the peritubular interstitium in female (A) versus the intense, focally accumulated SHG signal in male (B, arrow) kidney. C: Example of potential quantitative imaging analysis of kidney fibrosis showing lower levels in female kidney tissues. D–E: Representative examples of four-channel multiplex analysis of cell and tissue variables in human renal pathologies. Measuring tissue fibrosis by SHG (cyan) and tissue autofluorescence (green) signals, cell proliferation (Ki67 immunofluorescence, arrows, red in D) or podocyte density (p57 immunofluorescence, arrows, red in E), and cell nuclei (DAPI, dark blue).