Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-ß-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-ß (TGF-ß)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-ß receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-ß signaling. Pharmacological inhibition of the downstream TGF-ß effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-ß1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-ß receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-ß-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-ß-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-ß signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.

The Type 2 Diabetes Factor Methylglyoxal Mediates Axon Initial Segment Shortening and Alters Neuronal Function at the Cellular and Network Levels.

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or location along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment. To determine the key type 2 diabetes-related factor that produces AIS shortening we modified levels of insulin, glucose, or the reactive glucose metabolite methylglyoxal in cultures of dissociated cortices from male and female mice and quantified AIS geometry using immunofluorescent imaging of the AIS proteins AnkyrinG and ßIV spectrin. Neither insulin nor glucose modification altered AIS length. Exposure to 100 but not 1 or 10 µm methylglyoxal for 24 h resulted in accumulation of the methylglyoxal-derived advanced glycation end-product hydroimidazolone and produced reversible AIS shortening without cell death. Methylglyoxal-evoked AIS shortening occurred in both excitatory and putative inhibitory neuron populations and in the presence of tetrodotoxin (TTX). In single-cell recordings resting membrane potential was depolarized at 0.5-3 h and returned to normal at 24 h. In multielectrode array (MEA) recordings methylglyoxal produced an immediate ∼300% increase in spiking and bursting rates that returned to normal within 2 min, followed by a ∼20% reduction of network activity at 0.5-3 h and restoration of activity to baseline levels at 24 h. AIS length was unchanged at 0.5-3 h despite the presence of depolarization and network activity reduction. Nevertheless, these results suggest that methylglyoxal could be a key mediator of AIS shortening and disruptor of neuronal function during type 2 diabetes.