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

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.

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.

Force potentiation during eccentric contractions in rat skeletal muscle.

Postactivation potentiation refers to an acute enhancement of contractile properties following muscle activity. Previously, the effects of prior muscle activation on eccentric force at tetanic activation frequencies have only been sparsely reported. This paper aimed to study acute activity-induced effects on eccentric force of slow and fast-twitch muscles and characterize them in relation to postactivation potentiation. We elicited eccentric contractions in isolated rat extensor digitorum longus and soleus muscles by actively lengthening muscles at a constant velocity. We assessed contractile properties by measuring force over shortly interspaced, identical eccentric, and isometric contractions. We then analyzed stretch force, isometric peak force, rate of force development, and relaxation times. Finally, we compared the time courses for the development and cessation of changes in stretch force to known features of postactivation potentiation. In extensor digitorum longus, muscles stretch force consistently increased in a contraction-to-contraction manner by up to 49% [95% confidence interval (CI): 35-64%] whereas isometric peak force simultaneously showed minor declines (8%, 95% CI: 5-10%). The development and cessation of eccentric force potentiation coincided with the development of twitch potentiation and increases in rate of force development. In soleus muscles we found no consistent eccentric potentiation. Characterization of the increase in eccentric force revealed that force only increased in the very beginning of an active stretch. Eccentric force at tetanic activation frequencies potentiates substantially in extensor digitorum longus muscles over consecutive contractions with a time course coinciding with postactivation potentiation. Such eccentric potentiation may be important in sport performance.NEW & NOTEWORTHY Force during eccentric contractions can increase to a magnitude that may have profound consequences for our understanding of skeletal muscle locomotion. This increase in eccentric force occurs over consecutive, shortly interspaced, tetanic contractions in rat extensor digitorum longus muscles-not in rat soleus muscles-and coincides with well-known traits of postactivation potentiation. Eccentric force potentiation may significantly enhance muscle performance in activities involving stretch-shortening cycles.

Additional in-series compliance does not affect the length dependence of activation in rat medial gastrocnemius.

NEW FINDINGS: What is the central question of this study? The length dependence of activation (LDA) is typically explained by a length-dependent increase in calcium sensitivity, but recently calcium-independent mechanisms have been suggested: does active muscle shortening provided by a compliant in-series component impact the muscle length at which force output is maximized, thus contributing to LDA? What is the main finding and its importance? Using an in situ rat medial gastrocnemius set-up and varying the magnitude of muscle shortening via an artificial compliant series-elastic component, we were unable to observe any change in optimal length between conditions, contrary to some previous findings. More research is therefore required to explain these discrepancies. ABSTRACT: The force-length relationship dictates the amount of force a muscle can produce as a function of its length, during maximal isometric contractions. When activation is submaximal, it has been shown that the length at which force production is highest (the optimal length) is longer. This is typically explained by a length-dependent increase in Ca2+ sensitivity, known as the 'length dependence of activation'. Recent reports have implicated shortening against in-series compliance to be a potential factor in the observed optimal length (L0 ) of muscle, via the phenomenon of shortening-induced force depression (a phenomenon which describes the relative reduction in muscle force when a muscle is actively shortening to a given length compared to contracting isometrically at that same length). In the current study, rat medial gastrocnemius was stimulated with single and triple pulses (200 Hz) over a range of lengths, both with and without additional in-series compliance provided by a small piece of silicon tubing in series with the muscle, which allowed greater fascicle shortening upon activation. Fascicle length was measured using sonomicrometry crystals, and peak force (Fpeak ) and L0 were estimated by curve-fitting of the force-length data. The additional in-series compliance significantly reduced Fpeak by approximately 14% and 25% for the single and triple pulses, respectively (P = 0.003, P < 0.001), yet L0 remained unchanged (P = 0.405), suggesting that in our model, shortening against in-series compliance does not affect L0 . We offer potential explanations for the discrepancies seen and discuss whether the velocity of shortening may have a role in the length dependence of force.

Force-frequency relationship during fatiguing contractions of rat medial gastrocnemius muscle.

The force-frequency relationship presents the amount of force a muscle can produce as a function of the frequency of activation. During repetitive muscular contractions, fatigue and potentiation may both impact the resultant contractile response. However, both the apparent fatigue observed, and the potential for activity-dependent potentiation can be affected by the frequency of activation. Thus, we wanted to explore the effects that repetitive stimulation had on the force-frequency relationship. The force-frequency relationship of the rat medial gastrocnemius muscle was investigated during consecutive bouts of increasing fatigue with 20 to 100 Hz stimulation. Force was measured prior to the fatiguing protocol, during each of three levels of fatigue, and after 30 min of recovery. Force at each frequency was quantified relative to the pre-fatigued 100 Hz contractions, as well as the percentage reduction of force from the pre-fatigued level at a given frequency. We observed less reduction in force at low frequencies compared to high frequencies, suggesting an interplay of fatigue and potentiation, in which potentiation can "protect" against fatigue in a frequency-dependent manner. The exact mechanism of fatigue is unknown, however the substantial reduction of force at high frequency suggests a role for reduced force per cross-bridge.

Fatiguing stimulation increases curvature of the force-velocity relationship in isolated fast-twitch and slow-twitch rat muscles.

In skeletal muscles, the ability to generate power is reduced during fatigue. In isolated muscles, maximal power can be calculated from the force-velocity relationship. This relationship is well described by the Hill equation, which contains three parameters: (1) maximal isometric force, (2) maximum contraction velocity and (3) curvature. Here, we investigated the hypothesis that a fatigue-induced loss of power is associated with changes in curvature of the force-velocity curve in slow-twitch muscles but not in fast-twitch muscles during the development of fatigue. Isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles were incubated in Krebs-Ringer solution at 30°C and stimulated electrically at 60 Hz (soleus) and 150 Hz (EDL) to perform a series of concentric contractions to fatigue. Force-velocity data were fitted to the Hill equation, and curvature was determined as the ratio of the curve parameters a/F0 (inversely related to curvature). At the end of the fatiguing protocol, maximal power decreased by 58±5% in the soleus and 69±4% in the EDL compared with initial values in non-fatigued muscles. At the end of the fatiguing sequence, curvature increased as judged from the decrease in a/F0 by 81±20% in the soleus and by 31±12% in the EDL. However, during the initial phases of fatiguing stimulation, we observed a small decrease in curvature in the EDL, but not in the soleus, which may be a result of post-activation potentiation. In conclusion, fatigue-induced loss of power is strongly associated with an increased curvature of the force-velocity relationship, particularly in slow-twitch muscles.

Activation of mTORC1 signalling in rat skeletal muscle is independent of the EC-coupling sequence but dependent on tension per se in a dose-response relationship.

AIM: mTORC1 is regarded as an important key regulator of protein synthesis and hypertrophy following mechanical stimuli in skeletal muscle. However, as excitation and tension development is tightly coupled in most experimental models, very little and largely indirect evidence exist for such a mechanosensitive pathway. Here, we sought to examine whether activation of mTORC1 signalling is dependent on tension per se in rat skeletal muscle. METHODS: To examine the mechanosensitivity of mTORC1, rat EDL muscles were exposed to either excitation-induced eccentric contractions (ECC), passive stretching (PAS) with identical peak tension (Tpeak ) and Tension-Time-Integral (TTI), or ECC with addition of inhibitors of the myosin ATPases (IMA ). To further explore the relationship between tension and mTORC1 signalling, rat EDL muscles were subjected to PAS of different magnitudes of Tpeak while standardizing TTI and vice versa. RESULTS: PAS and ECC with equal Tpeak and TTI produced similar responses in mTORC1 signalling despite different modes of tension development. When active tension during ECC was nearly abolished by addition of IMA , mTORC1 signalling was reduced to a level comparable to non-stimulated controls. In addition, when muscles were exposed to PAS of varying levels of Tpeak with standardized TTI, activation of mTORC1 signalling displayed a positive relationship with peak tension. CONCLUSIONS: The current study directly links tension per se to activation of mTORC1 signalling, which is independent of an active EC-coupling sequence. Moreover, activation of mTORC1 signalling displays a positive dose-response relationship with peak tension.