Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners

Renal Interstitial Platelet-Derived Growth Factor Receptor-β Cells Support Proximal Tubular Regeneration

Background The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-beta (PDGFR beta) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined. Methods In mice with fluorescent protein expression in renal tubular cells and PDGFR beta-positive interstitial cells, weablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration. Results Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFR beta-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFR beta-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFR beta-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFR beta impeded PDGFR beta-positive cell migration to the tubular injury site and compromised the recovery of tubular function. Conclusions Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFR beta-mediated communication between the renal interstitium and the tubular system

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron

Abstract Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology

Essential role and therapeutic targeting of the glomerular endothelial glycocalyx in lupus nephritis

Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN

Supporting information.

This supporting information includes figures S1 to S6 and their figure legends as well as the legends to the supporting movies S1 to S3. (PDF)</p

Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice

Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (μm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine

Sequence of the single stranded DNA donor (ssDNA donor).

(PDF)</p

Clta-eGFP in primary MEFs.

(A) Western blot of three Clta-reporter MEF and two WT MEF cell lines. Clta-eGFP fusion protein is detected using anti-eGFP and anti-Lc antibodies. On each lane 20 μg of total protein were loaded. (B) Co-IP of CHC and Clta-eGFP fusion protein in MEFs. (C) qPCR of Clta and Cltb in MEFs. No significant changes were observed in Clta-reporter and WT MEFs. WT: n = 4; Clta-reporter n = 6 (students t-test). (D) Cy3-Tf uptake in Clta-reporter and WT MEFs: no significant changes in the uptake of Cy3-Tf were detected between WT and Clta-reporter MEFs at 0 min and 30 min (students t-test). WT: n = 6; Clta-reporter n = 9; Scale bar: 50 μm.</p

Tissue expression of Clta-eGFP fusion protein.

(A) Western Blot Analysis of small intestine, brain, kidney and liver lysates show the expression of the Clta-eGFP fusion protein. Additional band in small intestine and kidney arises possibly through tissue specifc splicing or exon-skipping through insertion of eGFP. Partial degradation may not be excluded. On each lane 25 μg of total protein were loaded. *: unspecific cross reactivity of the eGFP antibody. (B) Immunofluorescence of dye separated WT and Clta-reporter cryo sections of kidney and small intestine using confocal microscopy (left, scale bar 30 μm) and 2-PM (right, scale bar 15 μm). The related WT emission spectra in the green spectral range recorded with confocal microscopy, indicating an autofluoresence maxima at 550 nm can be found in S3B Fig in S1 Appendix. (C) Confocal and associated STED images of small intestine of WT and Clta-reporter cryo sections revealed a strong apical occurrence. Scale bar: 5 μm and 1 μm for the cut-outs (1–4). Clta-eGFP appears in clusters of 74 nm in size or smaller. (D) Confocal and associated STED images of kidney of WT and Clta-reporter cryo sections. A strong apical localization of the Clta-eGFP fusion protein was detected. Scale bar: 5 μm and 1 μm for the cutouts (1–2). Darker regions in the STED image are shown by a saturated color table with overglow (OGL) in blue.</p

TIRFM of Clta-eGFP in the cell shown in Fig 5A–5C.

(AVI)</p