Muscle proprioceptive afferents provide feedback critical for successful execution of motor tasks via specialized mechanoreceptors housed within skeletal muscles: muscle spindles, supplied by group Ia and group II afferents, and Golgi tendon organs, supplied by group Ib afferents. The morphology of these proprioceptors and their associated afferents has been studied extensively in the cat soleus, and to a lesser degree, in the rat; however, quantitative analyses of proprioceptive innervation in the mouse soleus are comparatively limited. The present study employed genetically-encoded fluorescent reporting systems to label and analyze muscle spindles, Golgi tendon organs, and the proprioceptive sensory neuron subpopulations supplying them within the intact mouse soleus muscle using high magnification confocal microscopy. Total proprioceptive receptors numbered 11.3 ± 0.4 and 5.2 ± 0.2 for muscle spindles and Golgi tendon organs, respectively, and these receptor counts varied independently (n = 27 muscles). Analogous to findings in the rat, muscle spindles analyzed were most frequently supplied by two proprioceptive afferents, and in the majority of instances, both were classified as primary endings using established morphological criteria. Secondary endings were most frequently observed when spindle associated afferents totaled three or more. The mean diameter of primary and secondary afferent axons differed significantly, but the distributions overlap more than previously observed in cat and rat studies.

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We investigated the calcium dynamics of dorsal root ganglion (DRG) neurons using transgenic mice to target expression of the genetically encoded calcium indicator (GECI), GCaMP6s, to a subset of neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low-threshold mechanoreceptors. This study provides the first analysis of GECI calcium transient parameters from large-diameter DRG neurons. Our approach generated calcium transients of consistent shape and time-course, with quantifiable characteristics. Four parameters of calcium transients were determined to vary independently from each other and thus are likely influenced by different calcium-regulating mechanisms: peak amplitude, rise time (RT), decay time, and recovery time. Pooled analysis of 188 neurons demonstrated unimodal distributions, providing evidence that PV+ DRG neurons regulate calcium similarly as a population despite their differences in size, electrical properties, and functional sensitivities. Calcium transients increased in size with elevated extracellular calcium, longer trains of action potentials, and higher stimulation frequencies. RT and decay time increased with the addition of the selective sarco/endoplasmic reticulum calcium ATPases (SERCA) blocker, thapsigargin (TG), while peak amplitude and recovery time remained the same. When elevating bath pH to 8.8 to block plasma-membrane calcium ATPases (PMCA), all measured parameters significantly increased. These results illustrate that GECI calcium transients provide sufficient resolution to detect changes in electrical activity and intracellular calcium concentration, as well as discern information about the activity of specific subclasses of calcium regulatory mechanisms.

Background Zinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated. Hypothesis Since nuclear factor‐kB (NFκB) mediates ZnD‐induced detrimental effects, we tested the hypothesis that NFκB drives ZnD‐induced NCC upregulation and subsequently renal Na+ retention and hypertension. Experimental Design To determine the role of NFκB in ZnD‐induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor ‐ caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFκB mediates ZnD‐induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′, N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT‐PCR, Western blot and immunofluorescence, respectively. Results In mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFκB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN‐induced NCC upregulation was prevented by CAPE treatment. Conclusions Together, these results demonstrate that 1) NFκB is a novel NCC regulator and 2) NFκB mediates ZnD‐induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFκB activation thereby stimulating NCC‐mediated Na+ retention and promoting hypertension. Significance This study identifies NFκB as a possible pharmacological target to mitigate hypertension. Support or Funding Information American Heart Association ‐ Scientist Development Grant #16SDG27080009 National Institutes of Health ‐ R21 Exploratory/Development Grant #R21DK119879

Background Zn2+ deficiency (ZnD) is a worldwide problem. In the United States, 14% of Americans are Zn2+ deficient, which represents 1 out of 7 people. In ZnD populations, the prevalence of hypertension is higher. In our recent studies, we demonstrated that ZnD induces hypertension by promoting renal Na+ reabsorption by the sodium chloride co‐transporter (NCC). However, the exact molecular mechanisms involved in NCC upregulation were undefined. Nuclear factor‐κB (NFκB) is a transcription factor found to play a role in ZnD‐mediated detrimental effects throughout the body. Hypothesis As such, we hypothesized that ZnD drives renal NFκB activation. Experimental Design To examine the effects of ZnD on renal NFκB activation, adult, male C57Bl/6 mice were fed a ZnA‐ or ZnD‐diet for 6 weeks. NFκB expression and nuclear translocation were examined by immunohistochemistry. To confirm the role of Zn2+ in NFκB regulation, mouse distal convoluted tubular (mDCT) cells were treated with the Zn2+ chelator ‐ N,N,Nʹ,Nʹ‐tetrakis(2‐pyridylmethyl)ethane‐1,2‐diamine (TPEN) or vehicle ± Zn2+ supplementation. Results In ZnD mice, NFκB protein expression and nuclear localization were increased compared to ZnA mice. Consistently, in mDCT cells, TPEN‐induced ZnD stimulated NFκB expression and nuclear translocation. However, Zn2+ supplementation reversed TPEN‐induced NFκB upregulation. Conclusion These results indicate that 1) NFκB is a Zn2+‐regulated nuclear transcription factor, and 2) ZnD drives renal NFkB activation. Significance NFκB represents a potential mediator that drives ZnD‐induced NCC upregulation and consequently renal Na+ reabsorption and hypertension.

Background : Zinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown. Hypothesis : Since nuclear factor‐κB (NFκB) is a key transcription factor implicated in ZnD‐mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension. Experimental Design : Adult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFκB in ZnD‐induced hypertension, mice were switched from a ZnA‐diet to a ZnD‐diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFκB inhibitor, for 1 week. In mice, NCC expression (qRT‐PCR) and blood pressure (tail‐cuff) were measured. To further investigate the role of NFκB in NCC expression (qRT‐PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE. Results : Compared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFκB nuclear localization. In mDCT cells, TPEN‐induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFκB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFκB inhibition by CAPE prevented TPEN‐induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice. Summary : These results demonstrate that 1) renal NFκB is a Zn2+‐regulated transcription factor, 2) NFκB is involved in NCC regulation and 3) NFκB mediates ZnD‐induced blood pressure increases. Significance : Taken together, these data indicate that ZnD stimulates NFκB activation thereby driving NCC upregulation and hypertension.

Background : Zinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated. Hypothesis : Since nuclear factor‐kB (NFkB) mediates ZnD‐induced detrimental effects, we tested the hypothesis that NFκB drives ZnD‐induced NCC upregulation and subsequently Na+ retention and hypertension. Experimental Design : To determine the role of NFκB in ZnD‐induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor ‐ caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFkB mediates ZnD‐induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′,N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT‐PCR, Western blot and immunofluorescence, respectively. Results : In mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFkB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN‐induced NCC upregulation was prevented by CAPE treatment. Conclusions : Together, these results demonstrate that 1) NFkB is a novel NCC regulator and 2) NFkB mediates ZnD‐induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFkB activation thereby stimulating NCC‐mediated Na+ retention and promoting hypertension. Significance : This study identifies NFkB as a possible pharmacological target to mitigate hypertension.

Background Zn2+ deficiency (ZnD) is a worldwide problem. In the United States, 14% of Americans are Zn2+ deficient, which represents 1 out of 7 people. In ZnD populations, the prevalence of hypertension is higher. In our recent studies, we demonstrated that ZnD induces hypertension by promoting renal Na+ reabsorption by the sodium chloride co‐transporter (NCC). However, the exact molecular mechanisms involved in NCC upregulation were undefined. Nuclear factor‐κB (NFκB) is a transcription factor found to play a role in ZnD‐mediated detrimental effects throughout the body. Hypothesis As such, we hypothesized that ZnD drives renal NFκB activation. Experimental Design To examine the effects of ZnD on renal NFκB activation, adult, male C57Bl/6 mice were fed a ZnA‐ or ZnD‐diet for 6 weeks. NFκB expression and nuclear translocation were examined by immunohistochemistry. To confirm the role of Zn2+ in NFκB regulation, mouse distal convoluted tubular (mDCT) cells were treated with the Zn2+ chelator ‐ N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethane‐1,2‐diamine (TPEN) or vehicle ± Zn2+ supplementation. Results In ZnD mice, NFκB protein expression and nuclear localization were increased compared to ZnA mice. Consistently, in mDCT cells, TPEN‐induced ZnD stimulated NFκB expression and nuclear translocation. However, Zn2+ supplementation reversed TPEN‐induced NFκB upregulation. Conclusion These results indicate that 1) NFκB is a Zn2+‐regulated nuclear transcription factor, and 2) ZnD drives renal NFkB activation. Significance NFκB represents a potential mediator that drives ZnD‐induced NCC upregulation and consequently renal Na+ reabsorption and hypertension. Support or Funding Information American Heart Association ‐ Scientist Development Grant #16SDG27080009 National Institutes of Health ‐ R21 Exploratory/Development Grant #R21DK119879