Resolution of cisplatin-induced fatigue does not require endogenous interleukin-10 in male mice.

Based on previous results showing a pivotal role of endogenous interleukin-10 (IL-10) in the recovery from cisplatin-induced peripheral neuropathy, the present experiments were carried out to determine whether this cytokine plays any role in the recovery from cisplatin-induced fatigue in male mice. Fatigue was measured by decreased voluntary wheel running in mice trained to run in a wheel in response to cisplatin. Mice were treated with a monoclonal neutralizing antibody (IL-10na) administered intranasally during the recovery period to neutralize endogenous IL-10. In the first experiment, mice were treated with cisplatin (2.83 mg/kg/day) for five days and IL-10na (12 µg/day for three days) five days later. In the second experiment, they were treated with cisplatin (2.3 mg/kg/day for 5 days twice at a five-day interval) and IL10na (12 µg/day for three days) immediately after the last injection of cisplatin. In both experiments, cisplatin decreased body weight and reduced voluntary wheel running. However, IL-10na did not impair recovery from these effects. These results show that the recovery from the cisplatin-induced decrease in wheel running does not require endogenous IL-10 in contrast to the recovery from cisplatin-induced peripheral neuropathy.

Chronic restraint stress impairs voluntary wheel running but has no effect on food-motivated behavior in mice.

Chronic restraint stress is known to cause significant alterations of mitochondrial biology. However, its effects on effort-based behavior and the sensitivity of these effects to treatments that restore mitochondrial function have not been assessed. Based on the hypothesis that the behavioral consequences of this stressor should be more severe for an energy demanding activity than for an energy procuring activity, we compared the effects of chronic restraint stress on the performance of male mice trained to use a running wheel or to nose poke for a food reward in an operant conditioning cage. In accordance with our hypothesis, we observed that exposure of mice to 2-hour daily restraint sessions for 14 to 16 days during the light phase of the cycle reliably decreased voluntary wheel running but had no effect on working for food in a fixed ratio 10 schedule of food reinforcement or in a progressive ratio schedule of food reinforcement. This dissociation between the two types of behavioral activities could reflect an adaptive response to the constraint imposed by chronic restraint stress on mitochondria function and its negative consequences on energy metabolism. To determine whether it is the case, we administered mesenchymal stem cells intranasally to chronically restrained mice to repair the putative mitochondrial dysfunction induced by chronic restraint stress. This intervention had no effect on wheel running deficits. Assessment of mitochondrial gene expression in the brain of mice submitted to chronic restraint stress revealed an increase in the expression of genes involved in mitochondrial biology that showed habituation with repetition of daily sessions of restraint stress. These original findings can be interpreted to indicate that chronic restraint stress induces behavioral and mitochondrial adjustments that contribute to metabolic adaptation to this stressor and maintain metabolic flexibility.

HDAC6 inhibition reverses long-term doxorubicin-induced cognitive dysfunction by restoring microglia homeostasis and synaptic integrity.

Breast cancer is the most common female malignancy in both the developed and developing world. Doxorubicin is one of the most commonly used chemotherapies for breast cancer. Unfortunately, up to 60% of survivors report long-term chemotherapy-induced cognitive dysfunction (CICD) characterized by deficits in working memory, processing speed and executive function. Currently, no therapeutic standard for treating CICD exists. Here, we hypothesized that treatment with a blood-brain barrier permeable histone deacetylase 6 (HDAC6) inhibitor can successfully reverse long-term doxorubicin-induced cognitive dysfunction. Methods: The puzzle box test and novel object/place recognition test were used to assess cognitive function following a therapeutic doxorubicin dosing schedule in female mice. Mitochondrial function and morphology in neuronal synaptosomes were evaluated using the Seahorse XF24 extracellular flux analyzer and transmission electron microscopy, respectively. Hippocampal postsynaptic integrity was evaluated using immunofluorescence. Hippocampal microglia phenotype was determined using advanced imaging techniques and single-nucleus RNA sequencing. Results: A 14-day treatment with a blood-brain barrier permeable HDAC6 inhibitor successfully reversed long-term CICD in the domains of executive function, working and spatial memory. No significant changes in mitochondrial function or morphology in neuronal synaptosomes were detected. Long-term CICD was associated with a decreased expression of postsynaptic PSD95 in the hippocampus. These changes were associated with decreased microglial ramification and alterations in the microglia transcriptome that suggest a stage 1 disease-associated microglia (DAM) phenotype. HDAC6 inhibition completely reversed these doxorubicin-induced alterations, indicating a restoration of microglial homeostasis. Conclusion: Our results show that decreased postsynaptic integrity and a neurodegenerative microglia phenotype closely resembling stage 1 DAM microglia contribute to long-term CICD. Moreover, HDAC6 inhibition shows promise as an efficacious pharmaceutical intervention to alleviate CICD and improve quality of life of breast cancer survivors.

Nasal administration of mesenchymal stem cells reverses chemotherapy-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most frequently reported adverse effects of cancer treatment. CIPN often persists long after treatment completion and has detrimental effects on patient's quality of life. There are no efficacious FDA-approved drugs for CIPN. We recently demonstrated that nasal administration of mesenchymal stem cells (MSC) reverses the cognitive deficits induced by cisplatin in mice. Here we show that nasal administration of MSC after cisplatin- or paclitaxel treatment- completely reverses signs of established CIPN, including mechanical allodynia, spontaneous pain, and loss of intraepidermal nerve fibers (IENF) in the paw. The resolution of CIPN is associated with normalization of the cisplatin-induced decrease in mitochondrial bioenergetics in DRG neurons. Nasally administered MSC enter rapidly the meninges of the brain, spinal cord and peripheral lymph nodes to promote IL-10 production by macrophages. MSC-mediated resolution of mechanical allodynia, recovery of IENFs and restoration of DRG mitochondrial function critically depends on IL-10 production. MSC from IL-10 knockout animals are not capable of reversing the symptoms of CIPN. Moreover, WT MSC do not reverse CIPN in mice lacking IL-10 receptors on peripheral sensory neurons. In conclusion, only two nasal administrations of MSC fully reverse CIPN and the associated mitochondrial abnormalities via an IL-10 dependent pathway. Since MSC are already applied clinically, we propose that nasal MSC treatment could become a powerful treatment for the large group of patients suffering from neurotoxicities of cancer treatment.

Mitochondrial transfer from mesenchymal stem cells to neural stem cells protects against the neurotoxic effects of cisplatin.

Mesenchymal stem cells (MSCs) transfer healthy mitochondria to damaged acceptor cells via actin-based intercellular structures. In this study, we tested the hypothesis that MSCs transfer mitochondria to neural stem cells (NSCs) to protect NSCs against the neurotoxic effects of cisplatin treatment. Our results show that MSCs donate mitochondria to NSCs damaged in vitro by cisplatin. Transfer of healthy MSC-derived mitochondria decreases cisplatin-induced NSC death. Moreover, mitochondrial transfer from MSCs to NSCs reverses the cisplatin-induced decrease in mitochondrial membrane potential. Blocking the formation of actin-based intercellular structures inhibited the transfer of mitochondria to NSCs and abrogated the positive effects of MSCs on NSC survival. Conversely, overexpression of the mitochondrial motor protein Rho-GTPase 1 (Miro1) in MSCs increased mitochondrial transfer and further improved survival of cisplatin-treated NSCs.In vivo, MSC administration prevented the loss of DCX+ neural progenitor cells in the subventricular zone and hippocampal dentate gyrus which occurs as a result of cisplatin treatment. We propose mitochondrial transfer as one of the mechanisms via which MSCs exert their therapeutic regenerative effects after cisplatin treatment.

Nasal administration of mesenchymal stem cells restores cisplatin-induced cognitive impairment and brain damage in mice.

Cognitive impairments are a common side effect of chemotherapy that often persists long after treatment completion. There are no FDA-approved interventions to treat these cognitive deficits also called 'chemobrain'. We hypothesized that nasal administration of mesenchymal stem cells (MSC) reverses chemobrain. To test this hypothesis, we used a mouse model of cognitive deficits induced by cisplatin that we recently developed. Mice were treated with two cycles of cisplatin followed by nasal administration of MSC. Cisplatin treatment induced deficits in the puzzle box, novel object/place recognition and Y-maze tests, indicating cognitive impairment. Nasal MSC treatment fully reversed these cognitive deficits in males and females. MSC also reversed the cisplatin-induced damage to cortical myelin. Resting state functional MRI and connectome analysis revealed a decrease in characteristic path length after cisplatin, while MSC treatment increased path length in cisplatin-treated mice. MSCs enter the brain but did not survive longer than 12-72 hrs, indicating that they do not replace damaged tissue. RNA-sequencing analysis identified mitochondrial oxidative phosphorylation as a top pathway activated by MSC administration to cisplatin-treated mice. Consistently, MSC treatment restored the cisplatin-induced mitochondrial dysfunction and structural abnormalities in brain synaptosomes. Nasal administration of MSC did not interfere with the peripheral anti-tumor effect of cisplatin. In conclusion, nasal administration of MSC may represent a powerful, non-invasive, and safe regenerative treatment for resolution of chemobrain.

The renal TRPV4 channel is essential for adaptation to increased dietary potassium.

To maintain potassium homeostasis, kidneys exert flow-dependent potassium secretion to facilitate kaliuresis in response to elevated dietary potassium intake. This process involves stimulation of calcium-activated large conductance maxi-K (BK) channels in the distal nephron, namely the connecting tubule and the collecting duct. Recent evidence suggests that the TRPV4 channel is a critical determinant of flow-dependent intracellular calcium elevations in these segments of the renal tubule. Here, we demonstrate that elevated dietary potassium intake (five percent potassium) increases renal TRPV4 mRNA and protein levels in an aldosterone-dependent manner and causes redistribution of the channel to the apical plasma membrane in native collecting duct cells. This, in turn, leads to augmented TRPV4-mediated flow-dependent calcium ion responses in freshly isolated split-opened collecting ducts from mice fed the high potassium diet. Genetic TRPV4 ablation greatly diminished BK channel activity in collecting duct cells pointing to a reduced capacity to excrete potassium. Consistently, elevated potassium intake induced hyperkalemia in TRPV4 knockout mice due to deficient renal potassium excretion. Thus, regulation of TRPV4 activity in the distal nephron by dietary potassium is an indispensable component of whole body potassium balance.

Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

Control of ENaC-mediated sodium reabsorption in the distal nephron by Bradykinin.

Kinins, such as Bradykinin (BK), are peptide hormones of the kallikrein-kinin system. Apart from being a vasodilator, BK also increases urinary sodium excretion to reduce systemic blood pressure. It is becoming appreciated that BK modulates function of the epithelial Na(+) channel in the distal part of the renal nephron to affect tubular sodium reabsorption. In this chapter, we outline the molecular details, as well as discuss the physiological relevance of this regulation for the whole organism sodium homeostasis and setting chronic blood pressure.

IGF-1 and insulin exert opposite actions on ClC-K2 activity in the cortical collecting ducts.

Despite similar stimulatory actions on the epithelial sodium channel (ENaC)-mediated sodium reabsorption in the distal tubule, insulin promotes kaliuresis, whereas insulin-like growth factor-1 (IGF-1) causes a reduction in urinary potassium levels. The factors contributing to this phenomenon remain elusive. Electrogenic distal nephron ENaC-mediated Na(+) transport establishes driving force for Cl(-) reabsorption and K(+) secretion. Using patch-clamp electrophysiology, we document that a Cl(-) channel is highly abundant on the basolateral plasma membrane of intercalated cells in freshly isolated mouse cortical collecting duct (CCD) cells. The channel has characteristics attributable to the ClC-K2: slow gating kinetics, conductance ∼10 pS, voltage independence, Cl(-)>NO3 (-) anion selectivity, and inhibition/activation by low/high pH, respectively. IGF-1 (100 and 500 nM) acutely stimulates ClC-K2 activity in a reversible manner. Inhibition of PI3-kinase (PI3-K) with LY294002 (20 µM) abrogates activation of ClC-K2 by IGF-1. Interestingly, insulin (100 nM) reversibly decreases ClC-K2 activity in CCD cells. This inhibitory action is independent of PI3-K and is mediated by stimulation of a mitogen-activated protein kinase-dependent cascade. We propose that IGF-1, by stimulating ClC-K2 channels, promotes net Na(+) and Cl(-) reabsorption, thus reducing driving force for potassium secretion by the CCD. In contrast, inhibition of ClC-K2 by insulin favors coupling of Na(+) reabsorption with K(+) secretion at the apical membrane contributing to kaliuresis.

Direct inhibition of basolateral Kir4.1/5.1 and Kir4.1 channels in the cortical collecting duct by dopamine.

It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about the molecular targets of dopamine actions on water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine, which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K(+) channels in the distal renal tubule are critical for K(+) recycling and controlling basolateral membrane potential to establish the driving force for Na(+) reabsorption. Here, we demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCD. Using patch-clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40-pS and scarce 20-pS single channel conductances, most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased the open probability of both channels, with a relatively greater action on the Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. PKC blockade abolished the inhibition of basolateral K(+) channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential, as directly monitored using current-clamp mode in isolated CCDs. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of the basolateral membrane and a decreased driving force for Na(+) reabsorption in the distal renal tubule.

Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C.

We have recently documented that the Ca(2+)-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca(2+) responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca(2+)]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca(2+)]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca(2+)]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca(2+)]i responses and greatly increased basal [Ca(2+)]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.

TRPV4 dysfunction promotes renal cystogenesis in autosomal recessive polycystic kidney disease.

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca(2+)-permeable TRPV4 channel underlies flow-dependent Ca(2+) signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca(2+)]i, and loss of flow-mediated [Ca(2+)]i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca(2+) signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca(2+) signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.