BR102021025658A2 - KLOTHO MODULATION - Google Patents

Abstract

The present invention relates to a low voltage pulsed electrical stimulation device for modulating (e.g. upregulation or downregulation) the expression of klotho, a useful protein, by tissues. Methods for increasing expression of klotho in cells are also described.

Description

KLOTHO MODULATION Field of Invention

[0001] The present invention relates generally to the field of medical devices and associated treatments, and more specifically to the precise bioelectrical stimulation of an individual's tissue, possibly augmented with the administration of a composition comprising, among other things , stem cells and nutrients useful for stimulating and treating the subject, the subject's tissue(s), the subject's organ(s) and/or the subject's cells. More specifically, the present invention relates to a device, programmed bioelectric signal sequences and associated methods for the controlled expression of Klotho by means of precise bioelectric signal sequences.

Background of the Invention

[0002] Klotho protein is a hormone secreted by the kidney that is known to be membrane bound and secreted. In men, Klotho is associated with muscle regeneration, rejuvenation and neural protection. Klotho loss contributes to the aging features of human chronic kidney disease (“CKD”) and CKD progression. Its deficiency is also associated with degenerative processes and accelerated aging. See, for example, Lim, Kenneth et al. “α-Klotho Expression in Human Tissues”. The Journal of Clinical Endocrinology and Metabolism vol. 100, 10 (2015): E1308-18. doi: 10.1210 / jc.2015-1800; Thurston et al. “Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis”. Gastroenterology. April 2010; 138(4): 1384-94, 1394.e1-2. doi: 10.1053 / j.gastro.2009.12.002. Epub December 11, 2009. PMID: 20004202; PMCID: PMC3454518.

[0003] As found by S. Ranjit et al., “Since Klotho cannot cross the blood-brain barrier, it is speculated that there are two different groups of Klotho, one secreted from the kidney into the serum and the other secreted by the choroid plexus into the serum. the cerebrospinal fluid. Due to these reasons, the therapeutic use of Klotho to provide neuroprotection [to reduce neuroinflammation and oxidative damage] is limited.” S. Ranjit et al., "Potential neuroprotective role of astroglial exosomes against smoking-induced oxidative stress and HIV-1 replication in the central nervous system", Expert Opin Ther Targets. August 2018; 22(8): 703-714.

[0004] Ricardo Ferrari described that the improved regenerative response in aged muscle after two weeks of "Estim" electrical stimulation (i.e., a neuromuscular stimulator (Empi 300 PV, St Paul, MN, US)) was associated with a limitation, but it even increased Klotho expression (similar to that obtained with muscle contraction, eg exercise). Ricardo Ferrari, “The Effect of Electrical Stimulation on Aged Skeletal Muscle Regenerative Potential”, dscholarship.pitt.edu/28094/1/FerrariRJ_ETD_May_31_2016_PDF.pdf.

[0005] Ferrari also observed a direct relationship between Klotho expression and the percentage of senescent muscle precursor cells ("MPCs"). When Klotho was inhibited by siRNA in young MPCs and aged MPCs exposed to an Estim protocol, Ferrari observed a significantly increased percentage of senescent cells. Such findings suggest that Klotho is inversely associated with cell senescence and that Estim modulates Klotho expression in aged MPCs, and there is precedent to suggest that Klotho plays a role in inhibiting cell senescence. See also, Dalise et al., “Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats”, Sci. Rep. September 7, 2017; 7(1): 10830.

[0006] Using the skin and small intestine as models, others have demonstrated that Klotho increases the regenerative potential of stem cells and promotes tissue healing through an inhibition of Wnt signaling activation. Recent studies have demonstrated that Klotho is able to bind directly to Wnt ligands extracellularly, thus inhibiting the formation of renal fibrosis.

[0007] The findings suggest that Klotho is inversely associated with cell senescence and that Estim modulates Klotho expression in aged MPCs, and there is precedent to suggest that Klotho plays a role in inhibiting cell senescence. See also, Dalize et al., “Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats,” Sci. Rep. September 7, 2017; 7(1): 10830.

[0008] Schardong et al., " Intradialytic neuromuscular electrical stimulation reduces DNA damage in chronic kidney failure patients: a randomized controlled trial”, Biomarkers, 2018, 23: 5,495-501, DOI: 10.1080/1354750X.2018.1452049, described using electrical stimulation Generic Neuromuscular Therapy (NMES) to reduce DNA damage in hemodialysis patients Although this is a step in the right direction, the additional benefits of enhanced Klotho expression as described herein are still needed.

DESCRIPTION

[0009] A method of using a bioelectric stimulator programmed to produce one or more bioelectric signals for stimulating cellular tissue is described, wherein at least one of said bioelectric signal modulates Klotho expression by cellular tissue, the method comprising: applying the bioelectric stimulator to the subject's target tissue, and triggering the bioelectric stimulator to produce the programmed bioelectric signal.

[0010] Although any cellular tissue can be used, in such a method, the target tissue is typically selected from the group consisting of muscle, brain, kidney, pancreas, bone, tumor and nerve.

[0011] Typically, the cellular tissue is that of an individual who has been diagnosed as having a condition selected from the group consisting of cancer, a tumor, atrial fibrillation, aortic valve calcification, arterial calcification, tissue calcification, erectile dysfunction, fall of hair, and breast calcification.

[0012] The individual may be undergoing concomitant therapy (eg, radiation treatment or chemotherapy for cancer).

[0013] Typically, by applying the method, the amount of Klotho circulating in the subject's bloodstream is increased by at least 200% above normal.

[0014] In certain embodiments, Klotho expression is up-regulated in the individual. Typically, this is performed with a bioelectric stimulator programmed to deliver at least one bioelectric signal having a frequency of from 5 to 750 Hz, and more typically selected from the group consisting of 5 Hz, 10 Hz, 20 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz, 2,500 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz. Typically, the bioelectric signal is applied to the subject for anywhere from 5 minutes to 24 hours at one day.

[0015] In certain embodiments, Klotho expression is down-regulated. Typically, this is performed with a bioelectric stimulator programmed to deliver at least one bioelectric signal having a frequency selected from the group consisting of 25,000 Hz, 50,000 Hz, 750,000 Hz, and 1 MHz. Typically, the bioelectric signal is applied to the individual for from 5 minutes to 24 hours in a day.

[0016] in the present document A bioelectric stimulator particularly configured to activate the expression and/or release of Klotho in cellular tissue is described in the present document. In certain embodiments, the bioelectrical stimulator is further configured to activate expression and/or release of stromal cell-derived factor 1 ("SDF-1"), insulin-like growth factor 1 ("IGF-1"), insulin-like growth factor 1 ("IGF-1"), platelet-derived growth ("PDGF"), follistatin, tropoelastin, and any combination thereof.

[0017] Also described is a bioelectric stimulator that includes: an energy source (e.g., battery, capacitor, or other suitable source of electricity), and means for sending an electrical signal to a tissue of an individual (e.g., via lead(s) or cord). The bioelectric stimulator uses the electrical signal to precisely control tissue protein expression on demand.

[0018] In certain cases, the bioelectric stimulator is programmed to produce a bioelectric signal that stimulates target tissue to express and/or release Klotho polypeptide by the target tissue by using a bioelectric signal comprising a biphasic square pulse at 20 Hz (at, for example, 0.1 V (100mV)), and a 7.8 ms pulse duration per 24 hours of stimulation. In certain modalities, such as some related to heart care, the bioelectrical signal stimulation will cycle for a full 24 hours with, for example, 6 signals in a row, continuously.

[0019] In certain embodiments, the duration of stimulation of the bioelectrical signal for the subject to further regulate the expression of klotho stimulation is measured in minutes rather than in hours. For example, a typical stimulation time is from about 5 to 10 minutes twice a week only. In other modalities, stimulation times of up to 35 minutes to 1 hour are used.

[0020] The amount of Klotho expression increased by (or decreased by) the system described herein is greater than that seen with muscle stimulation or muscle contraction alone.

[0021] In certain cases, the bioelectric stimulator is further programmed to produce a bioelectric signal (to further regulate the expression of SDF-1) of 30 pulses per second with a voltage of 3.5 mV, and successively alternating currents of 700 at 1500 picoamps for one minute, and again with 700 to 1500 picoamps for one minute, further stimulated with a current of, say, 0.25 mA, pulse duration 40 pulses per second, pulse width 100 µs , and frequency of 100 Hz, each signal for 40 minutes to 8 hours a day.

[0022] In certain cases, the bioelectric stimulator is further programmed to produce (to further regulate PDGF expression) a bioelectric signal of 3 V/cm, 10 Hz at, for example, 2 µA (0.000002 amps), and pulse duration of 0.2 ms. In certain cases, the bioelectric stimulator is further programmed to produce (to further regulate PDGF expression) a bioelectric signal of 20 V/cm, 100 Hz at, for example, 0.25 µA (2.5e-7 amps) , and pulse duration of 40 pulses/s, width of 100 μs.

[0023] In certain cases, the bioelectric stimulator is further programmed to produce (to further regulate follistatin expression) a bioelectric signal of 10V at 50 Hz and 100 Hz, 0.25 mA for one (1) minute.

[0024] In certain cases, the bioelectric stimulator is further programmed to produce a bioelectric signal (to further regulate tropoelastin expression) of, for example, 0.06 V with 50 Hz alternating electric field and electric current at, for example, 1 mA for 15 minutes and 3 mA for 15 minutes.

[0025] In certain cases, the bioelectric stimulator is also programmed to produce (for the expression of IGF-1) a bioelectric signal applied to the target tissue of, for example, 3 mV with an electrical frequency of 22 Hz, and current of 1 mA for 15 minutes and at, for example, 3 mA for 15 minutes.

[0026] In certain instances, a method of using the bioelectric stimulator to stimulate an individual's tissue includes connecting (directly or wirelessly) the bioelectric stimulator to the target tissue or cells of the individual. The target tissue can be selected from, for example, the group consisting of muscle, brain, kidney, pancreas, bone, tumor and nerve.

[0027] In certain cases, the individual is interested in bodybuilding.

[0028] In certain cases, the individual has been diagnosed as suffering from kidney failure, diabetes, bone degeneration, aging, cancer and/or immune system dysfunction.

[0029] In certain cases, the individual has been diagnosed with cancer, a tumor, atrial fibrillation, aortic valve calcification, arterial calcification, tissue calcification, and/or breast calcification.

[0030] In certain cases, the individual has age-related cognitive decline. In certain cases, the individual has cognitive decline resulting from a neurodegenerative disease. In certain cases, the individual has cognitive decline resulting from traumatic brain damage. In certain cases, the individual is receiving or has received radiation or chemotherapy treatment for cancer.

[0031] A preferred system includes: a bioelectric stimulator that controls/stimulates the release/production of Klotho by a target cell or tissue. The stimulator can be associated with (eg connected to) the organ or tissue to be treated with a stimulation infusion electrode (available from Nanoscribe of Eggenstein-Leopoldshafen, Germany) or wirelessly. In certain cases, the interface with the subject's tissue may be a soft conductive sheath.

[0032] The stimulator can be designed to externally deliver all wireless regeneration promoting signals to the subject's organ(s), tissue(s) and/or cells. In certain embodiments, a microinfusion pump can be included in the system to deliver other supporting substances in greater volume more quickly.

[0033] While not intended to be bound by theory, the described system uses precise bioelectrical signaling sequences that appear to communicate with DNA and cell membranes within the subject's stimulated tissues to cause the cells to produce high volumes of the Klotho protein. Potential indications include muscle regeneration and treatment, bodybuilding, kidney regeneration and treatment, brain regeneration and treatment, cognitive function and memory enhancement, skin regeneration and treatment, hair loss treatment and hair regeneration, wound healing, dysfunction erectile dysfunction, eye regeneration and treatment, anti-aging, multiple sclerosis, lung regeneration and treatment, COPD, liver regeneration and treatment, hearing regeneration and treatment, blood pressure control, polyp treatment, cyst treatment, fibroid treatment, fibrosis cystic cyst, heart failure and heart valve decalcification.

Brief Description of the Drawings

[0034] Figure 1 illustrates a bioelectric stimulator programmed for delivery to an individual connected to multiple soft conductive electrode pads.

[0035] Figure 2 illustrates a bioelectric stimulator programmed as described herein.

[0036] Figure 3 illustrates a soft conductive wrap for use with the system.

[0037] Figure 4 illustrates a programmed bioelectric stimulator illustrated next to a pen.

[0038] Figure 5 illustrates a picture of the signal (voltage and frequency) associated with follistatin at 10V/cm, 50 Hz, square wave.

[0039] Figure 6 illustrates a picture of the signal (voltage and frequency) associated with IGF-1: 3.0 mV, 22Hz, square wave.

[0040] Figure 7 illustrates a picture of a signal (voltage and frequency) associated with PDGF30%: 3V/cm (100 mV here), 10 Hz, pulse width 200 µs, square wave.

[0041] Figure 8 illustrates a picture of a signal (voltage and frequency) associated with PDGF230%: 20V/cm (7.0 V here), 100 Hz, pulse width 100 µs, square wave.

[0042] Figure 9 illustrates an image of a signal (voltage and frequency) associated with stem cell proliferation (or orientation): 15 mV, 70 Hz, square wave.

[0043] Figure 10 illustrates an image of a signal (voltage and frequency) associated with stem cell proliferation: 2.5-6.0 V (4V here), 20 Hz, pulse width 200-700 µs, square wave .

[0044] Figure 11 illustrates a picture of the signal (voltage and frequency) associated with SDF-1: 3.5 mV, 30 Hz, square wave.

[0045] Figure 12 illustrates a picture of the signal (voltage and frequency) associated with SDF-1 (2nd part): 0.25 mA (3.0 V shown here), 100 Hz, 100 µs pulse width, wave square.

[0046] Figure 13 illustrates a picture of the signal (voltage and frequency) associated with tropoelastin: 60 mV, 50 Hz, square wave.

[0047] Figure 14 is a table illustrating the results of Example I.

[0048] Figures 15 and 16 graphically illustrate the results of Example II.

[0049] Figures 17 - 20 graphically illustrate the results of Example II.

[0050] Figure 21 graphically illustrates klotho gene expression in C2C12 cells stimulated for 60 minutes according to Example III.

[0051] Figure 22 is a table showing gene expression of C2C12 cells normalized to GAPDH using RT-qPCR according to Example III.

Detailed Description

[0052] In certain embodiments, a bandage wrap is described that is applied to the affected region. A microstimulator can be conveniently located in the bandage wrap and is used to deliver specific bioelectrical signals to the affected tissue and nerves that regulate various protein expressions for stem cell orientation, stem cell proliferation, stem cell differentiation, blood vessel formation , blood circulation enhancement, muscle function repair and DNA repair.

[0053] Referring now to Figure 1, there is illustrated a stimulator for use in treating a human subject. The illustrated device is about the size of a pen (Figure 4) and is programmable.

[0054] Preferably, the system uses a bioelectrical stimulator programmed to control the expression and/or release of Klotho, SDF-1, IGF-1, PDGF, follistatin and tropoelastin. See, for example, US Patent 10,960,206 to Leonhardt et al. (Mar. 30, 2021) for “Bioelectric Stimulator”.

[0055] Klotho is as described above. Follistatin promotes muscle growth and counteracts myostatin. SDF-1 is generally for stem cell recruitment and maturation of blood vessels. IGF-1 is for DNA repair. PDGF is a second stem cell guidance factor and helps in tissue regeneration. Either protein expression signal works well on its own for organ regeneration, but they work best together. SDF-1 is a powerful regeneration protein, as is IGF-1.

[0056] The microvoltage signal generator can be produced using the same techniques for producing a standard cardiac pacemaker well known to a person skilled in the art. An exemplary microvoltage generator is available from Mettler of Anaheim, CA. The main difference is the special electrical stimulation signals needed to control, for example, the precise release of follistatin on demand (the signals of which are described later in this document). Major pacemaker manufacturers are Medtronic, Boston Scientific Guidant, Abbott St. Jude, BioTronik, and Sorin Biomedica.

[0057] The construction of electrical signal generators and pacemakers is known in the art and can be obtained from OEM suppliers, as well as the chargers and programmers that accompany them. The electrical signal generators are programmed to produce specific signals to drive specific protein expressions at precisely the right time for, for example, optimal organ treatment or regeneration.

[0058] The pacing infusion lead can be constructed or purchased from the same vendors who build standard cardiac pacemaker leads. Stimulation infusion electrodes can be purchased from several OEM suppliers. The pacing infusion electrode may, for example, be a standard currently used in heart failure pacing studies in combination with drug delivery.

[0059] A wide-area infusion and electrode patch can be constructed by cutting the conduction polymer to shape and forming the plastic into a flat bag with exit ports at strategic locations.

[0060] In certain embodiments, the bioelectric signal (or signals) is applied through a bioelectric suit. The body's bioelectric body suit provides bioelectric signals to increase circulation of Klotho, Follistatin and IGF-1 controlled bioelectric protein expressions to increase muscle building and recovery. VEGF, PDGF and other protein expressions to improve circulation and exercise recovery. Controlled bioelectric release of tropoelastin designed to improve skin and tendon elasticity. All components are typically contained within a comfortable stretch bodysuit that optimally fits the individual's body.

[0061] In such a suit, the electrodes are preferably located strategically around the body. For example, electrodes are placed above and below the kidneys and over skeletal muscle to help stimulate circulatory production of Klotho. Above and below the liver to help stimulate IGF-1 production. Electrical stimulation is divided into two (2) categories, starting with Leonhardt's patented (1) submuscular contraction bioelectric signal sequences designed to increase expressions of specific regenerative proteins such as klotho, follistatin, muscle LIM and IGF1 for regeneration and muscle building, IGF1, sonic hedgehog and LIM for nerve regeneration, SDF1 and PDGF for stem cell guidance, VEGF, eNOS, SDF1, PDGF, HIF1a, CXCL5 for improving blood circulation, tropoelastin and COL17A1 for improving muscle elasticity fabrics. Certain signaling sequences focus on reducing acute, systemic, and chronic inflammation, and others focus on pain relief. Other built-in electrical stimulation protocols send impulses to the deeper layers of the muscles, (2) generating a natural muscle contraction.

[0062] Preferably, all bioelectric signal and muscle contraction stimulation impulses are sent through 10+ pairs of electrodes that are flexible, interchangeable, antibacterial, and highly resistant to damage.

[0063] All bioelectric signal and muscle contraction stimulation impulses are sent through 10+ pairs of state-of-the-art electrodes that are flexible, interchangeable, antibacterial and highly resistant to damage.

[0064] Microstimulators can be purchased or built in the same way cardiac pacemakers have been made since the 1960's. When used with a micro infusion pump, these pumps can be purchased or produced similarly to how they were produced for delivery of pharmaceuticals, insulin, and analgesics since the 1970s. See, also, US Patent 10,646,644 to Leonhardt et al. (May 12, 202)) for "Stimulator, Pump and Composition".

[0065] The programming computer can be a standard laptop. The programming wand common to wireless programming wands can be used to program cardiac pacemakers.

[0066] Both implantable wire cable-based and/or wireless non-invasive means ("electrode") can be used to deliver the bioelectric signals of promoting regeneration and healing to target organs.

[0067] A wireless single lumen infusion stimulation electrode or a wide range infusion driving patch can be used to deliver the regeneration signals and substances to the organ of interest to be treated or they can be used in combination .

[0068] A recharging wand for use herein is preferably similar to the pacemaker recharging wand developed by Alfred Mann in the early 1970's to recharge externally implantable pacemakers.

[0069] Bioelectric stimulation can be performed with the described microstimulator, which may have a stimulation infusion cable with, for example, a corkscrew cable placed / connected, for example, to the center of the tissue to be stimulated and/ or treated.

[0070] The microstimulator is activated and works through programmed signals to signal the release, for example, of Klotho. In such a method, when the electrical signal includes (within 15%): a biphasic square pulse at 20 Hz, 0.1 V (100 mV) and a pulse duration of 7.8 ms per 24 hours of stimulation (where the electrical signal is measured three (3) mm deep in the tissue), the expressed and/or released protein is Klotho.

[0071] In such a method, when the electrical signal includes (within 15%): 10V at 50 Hz and 100 Hz for about 12 hours each (1 minute duration) (wherein the electrical signal is as measured at three (3 mm deep within the tissue), the protein further expressed and/or released by the subject is follistatin.

[0072] In such a method, when the electrical signal includes (within 15%): 3 mV with a frequency of about 22 Hz, and current of about 1 mA for about fifteen (15) minutes and 3mA for about fifteen (15) minutes (5 minute duration) (where the electrical signal is as measured three (3) mm deep within the tissue), the protein further expressed and/or released by the subject is IGF-1.

[0073] For example, up-regulation of IGF-1, and SDF-1 has been achieved in cardiomyocytes using such signals. Up-regulation of SDF-1 was achieved in pig heart. It has been observed that signals to one cell tissue work with other cell tissues as well.

[0074] Also described is a method of activating a tissue to further produce stromal cell-derived factor 1 ("SDF-1"), the method including: stimulating the (e.g., human) tissue with an electrical signal, wherein the electrical signal includes (within 15%): 30 pulses per second with a voltage of about 3.5 mV, and successively alternating currents of about 700 to 1500 picoamps for about a minute, and again with 700 to 1500 picoamps for about one minute and stimulated with current of about 0.25 mA, pulse duration of about 40 pulses/sec, pulse width of about 100 μs, where the electrical signal is as measured at three (3 ) mm deep within the fabric. In such a method, the time period is typically at least 24 hours. In such a method, the field strength is typically at least 0.1 V/cm.

[0075] The following are preferential signals from the stimulator. For example, two PDGF expression control signals, one low voltage and one high voltage, are described. The test fabric is sheep heart tissue. The test cells are mesenchymal stem cells.

[0076] 30% PDGF increase > 3 V/cm, 10 Hz, 2 microamps (0.000002 amps) and pulse duration 0.2 ms.

[0077] 230% increase of PDGF > 20 V/cm 100 Hz, 0.25 mA (2.5e-7 amps) and pulse duration of 40 pulses/s, width of 100 μs.

[0078] PDGF signal: 20V for 1 minute, 20 mV for 10 minutes, current of 0.25 mA, pulse duration of 40 pulses/s, pulse width of 100 μs, and frequency of 100 Hz for 5 minutes in a row by 528 Hz for 3 minutes and 432 Hz for 3 minutes and 50 Hz for 3 minutes.

[0079] SDF-1 - Stem cell recruitment signal: 30 pulses per second with a voltage of 3.5 mV, and successively alternating currents of 700 to 1500 picoamps for one minute, and again with 700 to 1500 picoamps per one minute and stimulated with current of 0.25 mA, pulse duration of 40 pulses/s, pulse width of 100 μs, and frequency of 100 Hz - each signal for 40 minutes to 8 hours a day for 2 to 36 months as necessary for optimal results. Duration 7 minutes.

[0080] Stem cell proliferation signals: 15 mV and a current of 500 picoamps at 70 pulses per minute for 3 hours and 20 pulses per minute, a pulse amplitude of from 2.5 to 6 volts, and a width pulse from 0.2-0.7 milliseconds for 3 hours. Duration 3 minutes.

[0081] Follistatin production signal - (muscle growth): 10V at 50 Hz and 100 Hz 0.25 mA. Duration 1 minute.

[0082] IGF-1: 3mV with an electrical frequency of 22 Hz, and electrical current of 1 mA for 15 minutes and 3 mA for 15 minutes. Duration 5 minutes.

[0083] An example of bioelectric signal sequence in humans (after Klotho) is as follows.

[0084] SDF-1 (Stem Cell Guidance Signal) - 5 minutes

[0085] IGF-1 signal (DNA repair) - 3 minutes

[0086] Signal of follistatin (myostatin antagonist) at 1 volt (not 10 volts) - 3 minutes.

[0087] PDGF - 1 minute

[0088] One week after treatment, samples can be collected for morphometric evaluation by in-situ hybridization or RTPCR.

[0089] Among the accompanying figures are included images of the corresponding signals with the name, voltage and frequency of each signal written in each image. The signals must further be defined in terms of current and frequency, not voltage and frequency as shown. The voltage supplied to the cells will be different for each tissue type, but with current all signals can be kept constant, regardless of tissue type. The device must have a current-driven signal (instead of voltage-driven like most other devices).

[0090] Follistatin is a powerful myostatin antagonist. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. The system has precise bioelectrical signaling sequences that have demonstrated an ability to control follistatin protein release in target tissue on demand.

[0091] Activin A can be produced by a bioelectric signal of 6.0 mV, pulse width 100 μs, square wave, and/or 1.25 V at 5 to 10 Hz frequency. A bioelectrical signal to upregulate more TGF-β may also be helpful in this regard as well.

[0092] Up-regulation of expression of various proteins is described in US Patent Publication 201800649935 A1 (March 8, 2018), the contents of the entirety of which are incorporated herein by reference.

[0093] Figure 5 illustrates a picture of the signal (voltage and frequency) associated with follistatin at 10V/cm, 50 Hz, square wave. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. The system has precise bioelectrical signaling sequences that have demonstrated an ability to control follistatin protein release in target tissue on demand.

[0094] Figure 6 illustrates a picture of the signal (voltage and frequency) associated with IGF-1: 3.0 mV, 22Hz, square wave.

[0095] Figure 7 illustrates a picture of the signal (voltage and frequency) associated with PDGF30%: 3V/cm (100 mV here), 10 Hz, pulse width 200 µs, square wave. Figure 8 also illustrates a picture of the signal (voltage and frequency) associated with PDGF 230%: 20 V/cm (7.0 V here), 100 Hz, pulse width 100 µs, square wave.

[0096] Figure 9 illustrates a picture of the signal (voltage and frequency) associated with stem cell proliferation: 15 mV, 70 Hz, square wave.

[0097] Figure 10 illustrates a picture of the signal (voltage and frequency) associated with stem cell proliferation: 2.5 - 6.0 V (4V here), 20 Hz, pulse width 200 µs - 700 µs, square wave .

[0098] Figure 11 illustrates a picture of the signal (voltage and frequency) associated with SDF-1: 3.5 mV, 30 Hz, square wave.

[0099] Figure 12 illustrates a picture of the signal (voltage and frequency) associated with SDF-1 (2nd part): 0.25 mA (3.0 V shown here), 100 Hz, 100 µs pulse width, square wave.

[0100] Figure 13 illustrates an image of the signal (voltage and frequency) associated with tropoelastin: 60 mV, 50 Hz, square wave.

General Applications

[0101] In certain embodiments, a method of using the bioelectric stimulator described herein to stimulate tissue of an individual is described, the method comprising: connecting the bioelectric stimulator to the target tissue of the individual, and triggering the bioelectric stimulator to produce the signal programmed bioelectric. In such a method, the target tissue is typically selected from the group consisting of muscle, brain, kidney, pancreas, bone, tumor, liver, and/or nervous tissue. With such a method, the individual has typically been diagnosed as suffering from kidney failure, diabetes, bone degeneration, aging, cancer, and/or immune system dysfunction. In some embodiments, the individual has or is at risk of suffering from age-related cognitive decline, cognitive decline resulting from a neurodegenerative disease, and/or cognitive decline resulting from traumatic brain damage. In some embodiments, the individual is receiving or has received radiation or chemotherapy treatment for cancer.

[0102] In some embodiments, the individual has or is at risk of suffering from, for example, dementia, Alzheimer's disease, memory loss, inflammation, calcification, addiction, alcoholism, high blood pressure, aortic aneurysm recovery, lung regeneration, brain-immune interface difficulties, arthritis, vascular health, hypertension, vision recovery, tooth and gum regeneration, neurodegenerative disorders, hearing recovery, obesity, skin regeneration, aortic valve fibrosis, sun damage to skin, psoriasis , depression and anxiety, aging, diabetic neuropathy, brain-gut health neurogenesis difficulties, pancreas recovery and diabetes, age-related inflammation, ALS and/or Parkinson's disease. In some embodiments, the individual is seeking life extension (klotho-supplemented mice live 30% longer). In some embodiments, the individual is looking to build muscle and/or aid recovery from muscle injuries. In some embodiments, the individual is seeking to improve cognition. In some embodiments, the individual is looking to improve the immune system.

[0103] For example, applying a bioelectric signal to up-regulate klotho expression and a bioelectric signal to up-regulate follistatin expression to an individual contributes to the individual's liver health and/or liver regeneration. For patients with more severe liver injury, the treatment regimen may further include repeated delivery of a composition via a micropump comprising: adipose tissue-derived stem cells and stromal fraction; selected growth factors such as Klotho, IGF1 and SDF1; Platelet Rich Fibrin (PRF); regenerative fluid of amniotic origin; exosomes; micro RNA gel; oxygenated nanoparticles; nutrient hydrogel; and alkaloids.

[0104] Applying a bioelectric signal to further regulate klotho expression to an individual contributes to healing of an injury in the individual. Yamauchi et al. “Wound healing delays in α-Klothodeficient mice that have skin appearance similar to that in aged humans – Study of delayed wound healing mechanism” Biochemical and Biophysical Research Communications Volume 473, Item 4, May 13, 2016, Pages 845-852.

[0105] For the treatment of psoriasis, a bioelectric signal treatment regimen for klotho (for example, about 10 minutes), COL17A1 (for about 10 minutes), IGF-1 (for about 10 minutes), and for Sonic Hedgehog can be used for approximately 35 minutes treatment three times a week for four weeks. See, Zhang, Beibei et al. “Klotho Protein Protects Human Keratinocytes from UVB-Induced Damage Possibly by Reducing Expression and Nuclear Translocation of NF-κB”, Medical Science Monitor: International Medical Journal of Experimental and Clinical Research vol. 24 8583-8591. November 27, 2018, doi: 10.12659 / MSM.910687; Lim et al. “Klotho: A Major Shareholder in Vascular Aging Enterprises” Int. J. Mol. Sci. 2019, 20(18), 4637: doi.org/10.3390/ijms20184637; Liu, N., Matsumura et al. “Stem cell competition orchestrates skin homeostasis and aging”. Nature 568, 344–350 (2019); doi.org/10.1038/s41586-019-1085-7; Savastano et al. “Insulin-like Growth Factor-1, Psoriasis, and Inflammation: A Threesome?” European Journal of Inflammation Volume: 9 item: 3, page(s): 277-283 (2011); Sadagurski, Marianna et al. “Insulin-like growth factor 1 receptor signaling regulates skin development and inhibits skin keratinocyte differentiation”, Molecular and Cellular Biology vol. 26.7 (2006): 2675-87. doi:10.1128/MCB.26.7.2675-2687.2006; U.S. Patent Application Publication 20210228870 A1 by Leonhardt et al. (July 29, 2021) for “COL17A1 Modulation”; U.S. Patent Application Publication 20200324106 A1 by Leonhardt et al. (October 15, 2020) for “Bioelectric Stimulation for Sonic Hedgehog Expression”; Papaioannou et al. “Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation” J Clin Invest. 2019; 129(8):3153-3170: doi.org/10.1172/JCI125170; Caradu et al. “Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice”. Cardiovasc Res. April 1, 2018; 114(5):759-770. doi: 10.1093/cvr/cvy017. PMID: 29365079.

[0106] The application of a bioelectric signal to further regulate the expression of klotho to an individual suffering from Parkinson's may also contribute to the individual's cure. Compare, Leon et al. "Peripheral Elevation of a Klotho Fragment Enhances Brain Function and Resilience in Young, Aging, and α-Synuclein Transgenic Mice" Cell Reports Volume 20, Item 6, August 6, 2017, pages 1360-1371.

[0107] For the treatment of the individual's pancreas, application of a signal that up-regulates Klotho expression may be sufficient. Compare, Prud'homme et al. “The anti-aging protein Klotho is induced by GABA therapy and exerts protective and stimulatory effects on pancreatic beta cells”. Biochem Biophys Res Commun. December 2, 2017; 493(4): 1542-1547. doi: 10.1016 / j.bbrc.2017.10.029. Epub October 6, 2017. PMID: 28993191; Sharma et al. “Insulin demand regulates β cell number via the unfolded protein response”. Journal of Clinical Investigation, 2015; DOI: 10.1172 / JCI79264; Chera et al. “Diabetes recovery by age-dependent conversion of pancreatic δ cells to insulin producers”, Nature, 2014; DOI: 10.1038 / nature13633. See also Guyot et al. “Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers”. Nat Biotechnol 37, 1446–1451 (2019): doi.org/10.1038/s41587-019-0295-8 and Wang, Na et al. “Secreted klotho from exosomes alleviates inflammation and apoptosis in acute pancreatitis”. American Journal of Translational Research vol. 11, 6 3375-3383. June 15, 2019. In certain cases, however, this bioelectrical signal regimen is used with a bioelectrical signal that further regulates stem cell guidance factors and a rechargeable micro infusion pump that can be recharged with a mixed composition of stem cells (derived adipose tissue) and supporting factors such as selected growth factors including SDF1, Klotho, PDGF and selected BMPs, stromal fraction, amniotic fluid, PRF, microRNA gel, selected alkaloids, nutrient hydrogel, oxygenated nanoparticles and pancreatic matrix.

[0108] For the treatment of an individual's brain, a bioelectric signal for Klotho expression can be combined with other treatments and modalities, such as exercises and those described in US patent 10,646,644 incorporated by Leonhardt et al. (May 12, 2020)) for “Stimulator, Pump &Composition”; US Patent 10,960,206 to Leonhardt et al. (March 30, 2021) for “Bioelectric Stimulator”; US Patent 11,110,274 to Leonhardt (September 7, 2021) for "System and Method for Treating Inflammation"; Tang-Schomer MD. “3D axon growth by exogenous electrical stimulus and soluble factors”. Brain Res. Jan 01, 2018; 1678: 288-296. doi:10.1016 / j.brainres.2017.10.032. Epub 2017, October 31st. PMID: 29097106; Malyshevskaya et al. “Role of Electrical Activity in Horizontal Axon Growth in the Developing Cortex: A Time-Lapse Study Using Optogenetic Stimulation” PLOS ONE (2013): doi.org/10.1371/journal.pone.0082954; Pai et al. “Endogenous Gradients of Resting Potential Instructively Pattern Embryonic Neural Tissue via Notch Signaling and Regulation of Proliferation”. Journal of Neuroscience, 2015; 35(10): 4366 DOI: 10.1523 / JNEUROSCI.1877- 14.2015; Rhee et al. "Neural factors stem cells secrete facilitating brain regeneration upon constitutive Raf-Erk activation" Sci Rep 6, 32025 (2016): doi.org/10.1038/srep32025; Diaco et al. “Amniotic fluid-derived stem cells as an effective cell source for transplantation therapy in stroke”. Brain Circ 2015; 1: 119-24; Zhou et al. Front. “Advance of Stem Cell Treatment for Traumatic Brain Injury”. Front. Cell. Neurosci., (August 13, 2019): doi.org/10.3389/fncel.2019.00301; Geribaldi-Doldán et al. Protein Kinase C: Targets to Regenerate Brain Injuries?” Front. Cell Dev. Biol., March 20, 2019): doi.org/10.3389/fcell.2019.00039; Ghuman et al. “Biodegradation of ECM hydrogel promotes endogenous brain tissue restoration in a rat model of stroke”. Acta Biomater. October 15, 2018; 80: 66-84. doi: 10.1016 / j.actbio.2018.09.020. Epub 2018, September 16th. PMID: 30232030; PMCID: PMC6217851; Nih et al. “Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke”. Nature Mater 17, 642–651 (2018): doi.org/10.1038/s41563-018-0083-8; Sood et al. “Fetal Brain Extracellular Matrix Boosts Neuronal Network Formation in 3D Bioengineered Model of Cortical Brain Tissue” ACS Biomater. Sci. Eng. 2016, 2, 1, 131-140; Morales-García et al. “The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro”. Sci Rep 7, 5309 (2017): doi.org/10.1038/s41598-017-05407-9; Sieg, F.. “Mini-review of neural regeneration peptides in brain development”, Journal of Stem Cell Research & Therapeutics 1 (2016): DOI: 10.15406 / JSRT.2016.01.00025 Corpus ID: 14566389; Jayaraj et al. “Neuroinflammation: friend and foe for ischemic stroke”. J Neuroinflammation 16, 142 (2019): doi.org/10.1186/s12974-019-1516-2; Zhao and Willing “Enhancing endogenous capacity to repair a strokedamaged brain: An evolving field for stroke research” Progress in Neurobiology Volumes 163-164, April-May 2018, Pages 5-26; Cheng, Xi et al. “The Role of SDF-1 / CXCR4 / CXCR7 in Neuronal Regeneration after Cerebral Ischemia”, Frontiers in Neuroscience vol. 11 590. October 24, 2017, doi: 10.3389 / fnins.2017.00590; Deng et al. “Effects of SDF-1 / CXCR4 on the Repair of Traumatic Brain Injury in Rats by Mediating Bone Marrow Derived Mesenchymal Stem Cells” Cell Mol Neurobiol. March 2018; 38(2): 467-477. doi: 10.1007 / s10571-017-0490-4. Epub 2017, May 8. Errata in: Cell Mol Neurobiol. 2021 April; 41(3): 617-618. PMID: 28484859; Li et al. “GDF10 is a signal for axonal sprouting and functional recovery after stroke” Nat Neurosci 2015; Epub 2015, October 15; Mir et al. "IGF-1 mediated Neurogenesis Involves a Novel RIT1/Akt/Sox2 Cascade". Sci Rep 7, 3283 (2017): doi.org/10.1038/s41598-017-03641-9; Bourdillon et al. Front. “Electromagnetic Brain Stimulation in Patients with Disorders of Consciousness”. Front. Neurosci., (March 18, 2019): doi.org/10.3389/fnins.2019.00223; and/or Qi et al. "Enhancement of neural stem cell survival, proliferation and differentiation by IGF-1 delivery in graphene oxide-incorporated PLGA electrospun nanofibrous mats" RSC Adv., 2019,9, 8315-8325.

[0109] The described bioelectric signals can be used to treat depression, anxiety and/or other stress-related disorders in an individual. See, for example, Prather et al. “Longevity factor klotho and chronic psychological stress”. Translational Psychiatry, 2015; 5(6):e585 DOI:10.1038/tp.2015.81, in which klotho levels were lower in stressed and depressed women. See also Paroni et al. “Klotho Gene and Selective Serotonin Reuptake Inhibitors: Response to Treatment in Late-Life Major Depressive Disorder”. Mole Neurobiol. March 2017; 54(2): 1340-1351. doi: 10.1007 / s12035-016-9711-y. Epub 2016, February 3rd. PMID: 26843110; Zhang WG et al. “Association of Klotho and interleukin 6 gene polymorphisms with aging in Han Chinese population”, J Nutr Health Aging. December 2014; 18(10): 900-4. doi: 10.1007 / s12603-014-0470-z. PMID: 25470806; Xia, Weiwei et al. “Klotho Contributes to Pravastatin Effect on Suppressing IL-6 Production in Endothelial Cells”. Mediators of Inflammation vol. 2016 (2016): 2193210. doi: 10.1155 / 2016/2193210; Zhu et al. “Klotho controls the brain–immune system interface in the choroid plexus” PNAS November 27, 2018 115 (48) E11388- E11396; first published November 9, 2018; and Hoyer et al. “Electroconvulsive therapy enhances the anti-aging hormone Klotho in the cerebrospinal fluid of geriatric patients with major depression”, Eur Neuropsychopharmacol. March 2018; 28(3): 428-435. doi: 10.1016 / j.euroneuro.2017.12.012. Epub 2017 Dec. PMID: 29274997. Compare, US Patent Application Publication 20190290541 by Greiner et al. (September 26, 2019) for "Implantable Electroacupuncture System and Method for Treating Depression and Similar Mental Conditions". Several mechanisms are believed to be at work, such as up-regulation of klotho suppresses IL6 and TNF, which are released with stress and cause depression, and/or klotho increases the release of serotonin, which promotes brain health.

[0110] For cancer, klotho fights chronic inflammation created by chronic stress that can lead to cancerous tumors. Xuan, Nguyen Thi and Nong Van Hai. “Changes in expression of klotho affect physiological processes, diseases, and cancer”. Iranian Journal of Basic Medical Sciences vol. 21.1 (2018): 3-8; Xie et al. “Klotho Acts as a Tumor Suppressor in Cancers”, July 2013 Pathology & Oncology Research 19 (4) DOI: 10.1007 / s12253-013-9663-8; Sachdeva et al. “Klotho and the Treatment of Human Malignancies” Cancers 2020, 12, 1665; doi: 10.3390 / cancers12061665; Sun, Huidong et al. “Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating wnt/β-catenin signaling pathway”. World Journal of Surgical Oncology vol. 13 307. October 24, 2015, doi: 10.1186 / s12957-015-0717-0.

[0111] For the treatment of rheumatoid arthritis, a bioelectric signal for klotho may be sufficient. Witkowski et al. “Klotho — a Common Link in Physiological and Rheumatoid Arthritis-Related Aging of Human CD4 + Lymphocytes” J Immunol (2007), 178(2): 771-777; DOI: doi.org/10.4049/jimmunol.178.2.771; Martinez-Redondo et al. “αKLOTHO and sTGFβR2 treatment counteract the osteoarthritic phenotype developed in a rat model”. Protein Cell 11, 219-226 (2020): doi.org/10.1007/s13238-019-00685-7.

[0112] Klothos may be involved in inflammation and exert antifibrogenic effects. Some of these pathways can be altered in alcoholism or liver cirrhosis. Martín-González et al. “Soluble α-Klotho in Liver Cirrhosis and Alcoholism, Alcohol and Alcoholism”, Volume 54, Issue 3, May 2019, pages 204–208.

Kidney Treatment and Disease Prevention:

[0113] In certain embodiments, a method of treating a subject's kidney is described, the method comprising: bioelectric stimulation of the subject's muscle(s) for 30 minutes with a bioelectric stimulator programmed to produce a bioelectric signal that stimulates the muscle(s) to enhance expression and/or release of Klotho polypeptide, wherein the bioelectrical signal comprises a biphasic pulse at (within 15%) 20 Hz, 0.1 V, and at 7.8 ms of pulse duration. Typically, said bioelectrical stimulation takes place twice a week. Preferably, the muscle at least includes a quadratus lumborum of the subject.

[0114] Preferably in such a method of treating an individual's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of 30 pulses per second with a voltage of (within 15%) 3.5 mV, square wave from 700 to 1500 picoamps.

[0115] Preferably in such a method of treating an individual's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 0.25 mA, pulse duration of 40 pulses per second , pulse width 100 μs, and frequency 100 Hz, each signal.

[0116] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 100 mV, 50 Hz, square wave.

[0117] Preferably in such a method of treating an individual's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 3 mV with electrical frequency of 22 Hz, and current of 1 mA and 3 mA.

[0118] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 10V/cm, 50 Hz, square wave.

[0119] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produce a bioelectric signal of (within 15%): 3.5 V stimulation in 10 second bursts, one (1) burst every 30 seconds at a frequency of about 50 Hz (duration 5 minutes) (wherein an electrical signal is measured three (3) mm deep in the tissue).

[0120] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal that includes (within 15%): alternating high frequency and medium frequency signals, pulses symmetric, biphasic, trapezoidal, with 400 µs pulse duration and 1.5/1-s acceleration/deceleration duration, respectively (where an electrical signal is as measured three (3) mm deep in the tissue).

[0121] Preferably in such a method of treating an individual's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 0.06 V with alternating electric field of 50 Hz and current 1 mA electrical.

[0122] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 6 mV at 150 Hz Monophasic square wave pulse sw 0, 1 ms.

[0123] Preferably in such a method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal (eGF) of (within 15%) 10 V/cm, 500 Hz, width of pulse 180 µs, square wave.

[0124] In such a method of treating an individual's kidney, the bioelectric stimulator can advantageously be further programmed to produce a bioelectric signal of (within 15%) 3.0 mV, 2 Hz, square wave, frequency alternating with the 3 mA current. In one such method of treating a subject's kidney, the bioelectric stimulator can be further programmed to produce and produces a bioelectric signal of (within 15%) 15 Hz, one (1) EM Gauss field, consisting of 5 millisecond bursts with 5 microsecond pulses followed by a pulse lasting 200 μs at 30 Hz and a current amplitude of 140 mA.

[0125] Such methods can be combined with other therapies, such as those described in Missoum et al. “Recent Updates on Mesenchymal Stem Cell Based Therapy for Acute Renal Failure” Curr Urol 2019; 13: 189–199; DOI: 10.1159 / 000499272.

Heart and Heart Valve Treatment and Health

[0126] In certain embodiments, a bioelectrical signal is applied to a subject in need thereof to further regulate Klotho expression, which is used to treat or prevent cardiac disease, such as aortic valve fibrosis and/or cardiac calcification. See, for example, Chen et al. “Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPKα-mediated activation of RUNX2”, Aging Cell vol. 15.5 (2016): 853-60. doi:10.1111/acel.12494, Martín-Núñez et al. “Implications of Klotho in vascular health and disease” World J Cardiol. Dec 26, 2014; 6(12): 1262-1269, and Bre LP, McCarthy R, Wang W. Prevention of bioprosthetic heart valve calcification: strategies and outcomes. Curr Med Chem. 2014; 21(22):2553-64. doi: 10.2174/0929867321666131212151216. PMID: 24358975; and The et al. “Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells” Cells 2020, 9, 385; doi:10.3390/cells9020385.

[0127] After cleaning the heart valve(s), a combination of bioelectrical and biological signal sequences are applied to the valve(s), particularly bioelectrical signals for SDF1 (for stem cell guidance ), follistatin and klotho for leaflet regeneration. Patients are typically treated for about 20 minutes to 1 hour a month with electrodes applied to the subject's thigh muscle to release Klotho and/or other beneficial proteins, which help to prevent calcification of valves and arteries going forward.

[0128] Recalcification can be prevented by monthly bioelectric stimulation (at home) with a bioelectric signal to regulate for more than 30 minutes Klotho, for example, the subject's thigh muscles with non-invasive gel electrodes, in order to regulate for plus the expression and release of beneficial proteins, such as klotho, by muscles.

[0129] The valves can be decalcified and regenerated with the aid of an external non-invasive chest / back wireless transmission of lithotripsy signals and regeneration signals with a temporal interference method of crossing two frequencies to create an energy envelope, for example For example, 2000 Hz and 2030 Hz create - at the crossover point - a 30 Hz energy envelope at the heart valve site.

[0130] This treatment can be combined with non-invasive wearable device of tuned harmonic resonance vibrational energy, as well as to prevent plaque, calcification, blood formation in heart valves, after such procedures for preventive purposes. See, for example, US Patent Application Publication 20190125932A1 to Leonhardt & Donofrio, May 2, 2019) for "Preventing blood clot formation, calcification and/or plaque formation on blood contact surface(s)". The basically focused transmission of Mozart-like music to the heart valve vibrates them harmonically just enough to prevent plaque formation, but below the threshold for hemolysis.

[0131] The electrical surface charge of heart valves and surface protein expressions can be modified to ward off the early stages of plaque formation with bioelectrical signaling non-invasively for prevention purposes.

[0132] In certain modalities, the entire process of preventing decalcification and calcification can be completely non-invasive with temporal interference, focused electromagnetic waves or focused ultrasound (for example, at 40 kHz) and peripheral bioelectrical stimulation, such as stimulating the thigh muscle of the subject to release Klotho, which prevents calcification. See, for example, Chen, Tianlei et al. “The Role and Mechanism of α-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells”, BioMed Research International vol. 2015 (2015): 194362. doi: 10.1155 / 2015/194362; Leibrock et al. “You have access NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency” Journal of the American Society of Nephrology, October 2015, 26 (10) 2423-2433; Nakamura et al. Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice. PLoS One. August 3, 2017; 12 (8): e0181009. doi:10.1371/journal.pone.0181009. PMID: 28771600; PMCID: PMC5542469; Lang et al. “Therapeutic Interference With Vascular Calcification — Lessons From KlothoHypomorphic Mice and Beyond” Front. Endocrinol. (May 2018): doi.org/10.3389/fendo.2018.00207.

[0133] Focused electromagnetic waves are described in Ayden et al. “Focusing of electromagnetic waves by a left-handed metamaterial flat lens” Optics Express (October 31, 2005) 13 (22): 8753-8759; Kinney BM, Lozanova P. “High intensity focused electromagnetic therapy evaluated by magnetic resonance imaging: Safety and efficacy study of a dual tissue effect based non-invasive abdominal body shaping”. Lasers Surg Med. January 2019; 51(1): 40-46. doi:10.1002 / lsm.23024. Epub 2018 October 10th. PMID: 30302767; PMCID: PMC6585690.

[0134] After completing a third cleaning, for example with citric acid, the valves are treated to prevent recalcification, which can be done before and after the regeneration procedure. O'Neill, W Charles and Koba A Lomashvili. “Recent progress in the treatment of vascular calcification”, Kidney International vol. 78.12 (2010): 1232-9. doi:10.1038/ki.2010.334; Chen, Nai-Ching et al. "The Strategy to Prevent and Regress the Vascular Calcification in Dialysis Patients", BioMed Research International, vol. 2017, Article ID 9035193, 11 pages, 2017; Lei, Yang et al. “Efficacy of reversal of aortic calcification by chelating agents”, Calcified Tissue International Vol. 93.5 (2013): 426-35. doi:10.1007/s00223-013-9780-0; US Patent 4,976,733 to Giradot (Dec. 11, 1990) for "Prevention of prosthesis calcification"; Lei, Yang “Mechanisms And Reversal Of Elastin Specific Medial Arterial Calcification”, (2014). All Dissertations. 1307; tigerprints.clemson.edu/all_dissertations/1307/; Yarbrough et al. “Specific binding and mineralization of calcified surfaces by small peptides”, Calcified Tissue International Vol. 86.1 (2010): 58-66. doi:10.1007/s00223-009-9312-0; Yan Cai et al. “Intermedin inhibits vascular calcification by increasing the level of matrix γ-carboxyglutamic acid protein”, Cardiovascular Research, Volume 85, Item 4, March 1, 2010, Pages 864–873, doi.org/10.1093/cvr/cvp366; Back et al. “Endogenous Calcification Inhibitors in the Prevention of Vascular Calcification: A Consensus Statement From the COST Action EuroSoftCalcNet” Front. Cardiovasc. Med., 918 January 2019): doi.org/10.3389/fcvm.2018.00196.

Treatment and Prevention of Erectile Dysfunction

[0135] In certain embodiments, the bioelectrical stimulation therapy described herein is used as part of a system and/or method to treat and/or prevent erectile dysfunction in an individual. In this case, smooth cavernosal muscle regeneration by bioelectrical stimulation aims at the spontaneous return of erectility. Stief et al. “Functional electromyostimulation of the corpus cavernosum penis — preliminary results of a novel therapeutic option for erectile dysfunction”, World J. Urol. (1995) 13: 243-247. This is in contrast to oral therapy, injection and/or the use of a prior art vacuum pump when the patient is treatment dependent. van Kampen et al., "Treatment of Erectile Dysfunction by Perineal Exercise, Electromyographic Biofeedback, and Electrical Stimulation", Phys. The R. 2003; 83(6): 536-543.

[0136] Furthermore, klotho levels are associated with libido. Dote-Montero et al. “Predictors of Sexual Desire and Sexual Function in Sedentary Middle-Aged Adults: The Role of Lean Mass Index and SKlotho Plasma Levels. The FIT-AGEING Study”, J Sex Med. 2020 Apr; 17(4):665-677. doi: 10.1016/j.jsxm.2020.01.016. Epub February 20, 2020. PMID: 32089483.

[0137] In certain embodiments, the bioelectric stimulator is configured to up-regulate Klotho expression. Preferably, the upregulation is at least 200% above normal. Even preferably, Klotho expression is up-regulated such that the amount of Klotho circulating in the subject's blood stream is increased by 200%.

[0138] In certain modalities, the treatment regimen includes applying bioelectrical signal(s) to the individual for as little as 15 minutes every other day for just 4 weeks to see results.

[0139] Secretor Klotho results in the reduction of TNFα and IFNγ, which may have anti-inflammatory properties. Klotho can interact with Wnt, which results in the inhibition of Wnt pathway activity, thus inhibiting the aging process. Torbus-Paluszczak et al., “Klotho protein in neurodegenerative disorder,” Neurol. Sci. 39, 1677–1682 (2018): doi.org/10.1007/s10072-018-3496-x.

[0140] The bioelectric stimulator is further programmed to upregulate more IGF-1 to enhance nerve regeneration and neuromuscular recovery. Apel, P.J., Ma, J., Callahan, M., Northam, C.N., Alton, T.B., Sonntag, W.E., & Li, Z. (2010), Effect of locally delivered IGF-1 on nerve regeneration during aging: an experimental study in rats, Muscle & nerve, 41(3), 335–341: doi.org/10.1002/mus.21485.

[0141] The bioelectric stimulator is further programmed to up-regulate follistatin, which promotes muscle regeneration and recovery. Follistatin is able to effect accelerated muscle restoration, not only by potentiating the regenerative effects of myostatin inhibition, but potentially through modulation of inflammation. Yaden et al., "Follistatin: a novelapeutic for muscle regeneration," Journal of Pharmacology and Experimental Therapeutics March 13, 2014, jpet.113.211169; DOI: doi.org/10.1124/jpet.113.211169.

[0142] In most such methods, bioelectrical stimulation can be wirelessly administered to the subject's muscle(s).

[0143] In such a method, bioelectrical stimulation may be administered to the muscle(s) via a gel tape electrode applied to the subject's skin adjacent to the muscle(s). In one such method, the gel strip electrode can be applied to at least one thigh of the subject.

[0144] Such a method may be useful to an individual who has been diagnosed with kidney failure, diabetes, aging, and/or cancer.

Relationship Between Components:

[0145] The microvoltage signal generator is connected to the stimulation infusion electrode, for example, with a corkscrew tip, deep vein stimulation electrode (Medtronic) (for example, for bioelectrical stimulation of the brain), or bandage of conductive or curative polymer to the tissue or organ to be treated. An external signal programmer can be used to program the microvoltage signal generator with the appropriate signals for treatment, including the Klotho output signal. The device's battery can be recharged with an external battery charging wand.

[0146] The essential elements are the microvoltage signal generator and the means for sending the signal to the target tissue.

[0147] The signal generator can be external or internal. Signal transmission can be wireless, via liquid and/or via wires.

[0148] The contact interface with the tissue can be, for example, a dressing or bandage or it can be via electrodes or cables. FDA approved gel strip electrodes (Mettler) can be used for application to the skin. Electroacupuncture needles can be used to ensure that signals positively reach target tissues under the skin.

[0149] In certain embodiments, an individual's organs and/or tissues are first scanned or analyzed with a device to determine what their needs may be before treatment begins. Scanning / analysis may be, for example, generating mechanical vibrations in position adjacent to the location to be analyzed as described, for example, in US 2003/0220556 A1 by Porat et al. (the contents of which are incorporated herein by reference) and/or measuring the transmembrane voltage potential of a cell (see, for example, Chernet & Levin, “Transmembrane voltage potential is an essential cellular parameter for the detection and control of tumor development in a Xenopus model”, Dis. Models & Mech. 6, pp. 595-607 (2013); doi: 10.1242 / dmm.010835, the contents of which are also incorporated herein by reference. See also Brooks et al ., “Bioelectric impedance predicts total body water, blood pressure, and heart rate during hemodialysis in children and adolescents,” J. Ren. Nutr., 18(3): 304-311 (May 2008); doi: 10.1053 / j .jrn .2007.11.008, the contents of which are incorporated herein by reference, describing the use of bioelectrical impedance to assess blood pressure variability, systolic blood pressure, etc.

[0150] As used herein, "scanning" means measuring the bioelectrical electrical activity of organs, sometimes by placing a bion coil reader and transmitter on the organ, and directing this information to a computer. The computer stores the bioelectrical reading measurements of diseased and healthy organs and performs a comparative examination by classifying the organ into one category or another, much like a doctor using information to make a diagnosis.

[0151] Currently, the best approach to whole body and individual organ scanning is to use a combination of: a. 3D Body Scannint, b. Quantum magnetic resonance scanning, c. Biofeedback scan, d. Bioelectric scanning, e.g. Bion implant scan, f. Nervous system scan and g. Readout of light-activated cell reaction.

[0152] Readers such as the Ina’Chi reader, Quantum Magnetic Resonance Analyzer (QMRA), 3D Quantum Health Analyzer Scan Whole Body Organ Integrity 2, BodyScan® scanner, and “BIONic Muscle Spindle” are also helpful.

[0153] For example, the individual is positioned for analysis with a device, preferably with a non-invasive testing device to assess, for example, the autonomic nervous system, organ function(s) and risk factors associated with heart disease , diabetes, and stroke. The non-invasive testing device can analyze data, for example, the galvanic response of the subject's skin, skin color, oximeter, blood pressure and body composition analyzer to determine the hardening and thickening of the subject's arteries, the subject's heart health , exercise capacity, thyroid function, neurotransmitter balance, and numerous other health markers. See also Fatemi et al., "Imaging elastic properties of biologie tissue vibration," Proceedings of the IEEE, 91(10): 1503-1519 (October 2003).

[0154] In an alternative embodiment, the analysis conducted by the device comprises (or even includes) the detection of tiny energy fields around the human body, for example, with a "SQUID magnetometer" (SQUID is an acronym for "Superconducting Quantum Interference Device)", capable of detecting biomagnetic fields associated with physiological activities in the individual's body. A quantum resonant magnetic analyzer analyzes these fields. The magnetic frequency and energy of an individual's organ(s) and/or tissue(s) is collected by properly positioning the sensor relative to the portion of the organ(s) and/or tissue(s) of the individual to be analyzed, and after amplification of the signal by the instrument, the data are compared with the standard quantum resonant spectrum of diseases, nutrition and other indicators / markers to determine if the sample waveforms are irregular using a Fourier approach .

[0155] The treatment may include, for example, moving magnets or altering magnetic fields (pulsed electromagnetic fields) over the tissue and/or organ, for example, to reduce inflammation or treat pain or induce tissue growth in the individual.

[0156] The present invention is further described with the help of the following illustrative Examples.

EXAMPLES EXAMPLE I - Control of Expression and/or Release of Klotho

[0157] Twelve samples of gingival cells were stimulated with a biphasic square pulse at 20 Hz, 0.1 V (100 mV) and a pulse of 7.8 ms lasting 24 hours of stimulation. The cells were gingival fibroblasts from a 28-year-old Caucasian male (internet at atcc.org/en/Products/All/CRL-2014.aspx), which were passaged less than 8 times. RT-PCR was used to measure outcomes before and after the described bioelectrical stimulation. Results: Klotho expression up to an average of 248% (n = 5) and as high as 465% in skeletal muscle cells (see Figure 14).

EXAMPLE II – Control of Expression and/or Release of Klotho

[0158] Samples of cultured cells (eg, urinary smooth muscle cells) were stimulated for 30 minutes using a biphasic square waveform at 50% work (1V at 5 Hz at 1 MHz). Bioelectrical stimulation was applied to in vitro cultured cells using a commercially available constant voltage waveform generator RIGOL LXI 1022Z (Beaverton, OR, US) through a 6-well stimulation plate interface (IONOPTIX, Westwood, MA , USA).

[0159] RT-qPCR was used to measure klotho expression at varying frequencies. Triplicate qPCR measurements with standard deviations greater than 1 were removed.

[0160] Figures 15 and 16 graphically illustrate the results of the experiment. Data in graphs are Mean ± SE.

[0161] The main effect of frequency was tested with an ANOVA (with frequency as a continuous and categorical factor), Polynomial Model and General Additive Model.

[0162] A repeated one-sample t-test was performed to test whether modulation due to treatment (2ˆ-delta-delta Ct) was significant and was corrected using the method of Benjamini & Hochberg (1995).

[0163] Conclusions: There was a main effect of frequency for klotho for all methods used: ANOVA with numeric frequency (p < 0.001) and factor (p = 0.048), polynomial model (p < 0.001) and a general additive model (p = 0.008).

[0164] After adjustments (22 tests), the one-sample T Test showed that the klotho expression (log modulation due to treatment) was elevated above controls (0 Hz) for the following frequencies: 5 Hz, 25 Hz at 750 Hz, and 2500 Hz.

EXAMPLE III – Control of Expression and/or Release of Klotho

[0165] Samples of cultured cells (eg, osteoblasts) were stimulated for 30 minutes using a biphasic square waveform at 50% work (1V at 5 Hz at 1 MHz). Bioelectrical stimulation was applied to in vitro cultured cells using a commercially available constant voltage waveform generator RIGOL LXI 1022Z (Beaverton, OR, US) through a 6-well stimulation plate interface (IONOPTIX, Westwood, MA , USA).

[0166] Figures 17 - 20 graphically illustrate the results of the experiment. Data in graphs are Mean ± SE.

[0167] Triplicate qPCR measurements with standard deviations greater than 1 were removed.

[0168] The normality of the tests was assessed using scatterplots.

[0169] The main effects of frequency and volts were tested with a polynomial model (8º for frequency and 1º for volts) and a generalized additive model using cubic B-ribs (df = 11).

[0170] A one-sample t-test was performed to test whether log modulation due to treatment (2'-delta-delta Ct) was significant.

[0171] Repeated T-tests were corrected using the Benjamini & Hochberg (1995) method.

[0172] The frequency for the 8-fit polynomial was the best fit of the polynomial models tested for Klotho gene expression, adjusted Rˆ2 = 0.58, p < 0.001. The General Additive Model fits the data, but not better than the polynomial model, adjusted Rˆ2 = 0.36, p < 0.001. However, the General Additive Model provided the best explanation of the underlying biological theory for KLOTHO gene expression from electrical stimuli.

[0173] Strain was not related to klotho gene expression.

[0174] After adjustments (29 tests), the T-test of a sample showed that klotho expression was reduced for stimuli at 25,000 Hz, 50,000 Hz and 750,000 Hz. Expression was increased for stimuli at 100,000 Hz and 500,000 Hz. Lastly, frequencies at and above 1,000,000 Hz had mixed results. A cutoff score at 100,000 Hz was identified between frequencies that decreased (below 100,000 Hz) and increased (at or above 100,000 Hz) klotho expression (Sensitivity: 0.86; Specificity: 0.48; AUC 60%) .

EXAMPLE IV - Control of Expression and/or Release of Klotho

[0175] The purpose of this study was to analyze Klotho gene expression in C2C12 cells using RT-qPCR. Electrical signals: C2C12 cells were stimulated for 60 minutes, continuous cycle of 300 µs using a MettlerTM stimulator at various frequencies and currents ranging from 0.5 mA, 2 mA and 5 mA.

[0176] Methods: Bioelectric stimulation was applied to cells in vitro using a commercially available Mettler stimulator through a 6-well stimulation plate interface (IONOPTIX, Westwood, MA, USA). To induce uniform electric fields in all stimulation chambers, 1.3 mL of DMEM solution was added to each well before application of the BES signal.

[0177] Cells were seeded onto plates and grown to 8)% to 100% confluency. Once confluent, the cells were stimulated using an electrode array, which was inverted and introduced into the 6-well plate where the cells were cultured. Each cavity received uniform stimulation through a pair of carbon electrodes positioned on opposite sides.

[0178] Gene expression was determined by mRNA extraction according to the manufacturer's instructions. RNA quality was determined using a spectrophotometer and was reverse transcribed using a cDNA conversion kit. cDNA and TaqMan Master Mix were used.

[0179] Figure 21 graphically represents klotho gene expression in C2C12 cells stimulated for 60 minutes. Figure 22 is a table showing gene expression of C2C12 cells normalized to GAPDH using RT-qPCR.

[0180] The effects of C2C12 stimulated for 60 minutes at various frequencies and currents using a Mettler stimulator. After adjustments, a one-sample T-Test showed that klotho increased under the following conditions: 10 Hz at 2 mA, 20 Hz at 0.5 mA and 5 mA, and 50 Hz at 5 mA (which increased klotho 6088 times). ANOVA revealed that current influenced the magnitude of the klotho increase, probably meaning that 0.5 mA had smaller increases in klotho than 2 mA and 5 mA.

EXAMPLE V – Treatment of the Kidney

[0181] A bioelectric stimulator is programmed to regulate Klotho expression (20 Hz for 30 minutes, 0.1 V (100 mV) and a pulse duration of 7.8 ms minutes of application twice a week) for the treatment of a renal patient. The bioelectric signal is applied to the patient's thigh. Both the patient's muscles and the patient's kidneys have improved function.

EXAMPLE VI – Additional Kidney Treatment

[0182] In addition to being programmed to up-regulate Klotho expression, a preferred bioelectric stimulator for treating an individual's kidney(s) is programmed to produce bioelectric signals in the following order: 1. SDF-1 (see , eg Figures 11 and 12; a stem cell orientation factor - recruits a person's own stem cells); 2. VEGF (see, for example, Figure 7; for new blood vessel growth); 3. IGF-1 (see, for example, Figure 6; for DNA repair at the nucleus level); 4. Follistatin (see, for example, Figure 5; for muscle and tissue regeneration); 5. RANKL (see, for example, Figure 8; for demineralization and tissue loosening when needed); 6. Hepatocyte growth factor (see, for example, Figure 9; tissue regeneration); 7. eNOS (to dilate blood vessels to increase flow - for example, using a bioelectric signal includes (within 15%): alternating high frequency (HF) and medium frequency (MF) signals, symmetrical, biphasic, trapezoidal pulses , with 400 -μs pulse duration and 1.5 / 1-s acceleration / deceleration duration, respectively). 8. Tropoelastin (see, for example, Figure 13; it increases the elasticity of any tissues, such as skin, arteries, aorta, heart and promotes wound healing); 9. Activin A + B (see, for example, Figure 10); and 10. EGF (for regeneration, using eg 10 V/cm (5 V here), 500 Hz, 180 µs pulse width, square wave). These bioelectric signals are subsequently applied to Klotho's bioelectric signals.

EXAMPLE VII – Treatment of the Kidney

[0183] In addition to being programmed to further regulate Klotho expression, a preferred bioelectrical stimulator for treating an individual's kidney(s) is programmed to produce PDGF bioelectrical signals. A bioelectrical signal for platelet-derived growth factor (“PDGF”) is (within 15%) 3 V/cm, 10 Hz, 2 µA, and pulse duration of 0.2 ms. Another bioelectrical signal for PDGF is (within 15%) 20 V/cm, 100 Hz, 0.25 mA (2.5e-7 amps), and pulse duration 40 pulses/sec, width 100 µsec. Another bioelectrical signal for PDGF is (within 15%) 20 V for 1 minute, 20 mV for 10 minutes, current 0.25 mA, pulse duration 40 pulses/s, pulse width 100 μs, and frequency of 100 Hz for 5 minutes followed by 528 Hz for 3 minutes and 432 Hz for 3 minutes and 50 Hz for 3 minutes. These bioelectrical signals are applied to the individual subsequent to Klotho's bioelectrical signals.

EXAMPLE VIII – Erectile Dysfunction

[0184] A bioelectric stimulator is first programmed to up-regulate Klotho expression (20 Hz for 30 minutes (for example, 15 minutes twice a day), 0.1 V (100 mV), and at 7.8 ms minutes of pulse duration with twice-weekly application). The bioelectric stimulator is further programmed to produce an appropriate bioelectric signal to further regulate IGF-1 expression (e.g., 3 mV with electrical frequency of 22 Hz, and current of 1 mA for 15 minutes and 3 mA for 15 minutes. ) The bioelectric stimulator is further programmed to further regulate follistatin expression (e.g., a 10V bioelectric signal at 50 Hz and 100 Hz, 0.25 mA for one (1) minute.)

[0185] This regimen is applied to a male subject in the groin area to prevent and/or treat erectile dysfunction.

EXAMPLE IX – Loss of hair

[0186] A bioelectric stimulator is programmed to produce bioelectric signals that further regulate the expression of HGF, IGF-1, PDGF and Klotho (see, for example, US Patent 10,960,206 incorporated to Leonhardt et al. (Mar. 30, 2021 ) to “Bioelectric Stimulator” for examples of bioelectric signals). A bioelectric signal for hepatocyte growth factor (“HGF”) is (within 15%) 3.5 V stimulation in 10 second bursts, 1 burst every 30 seconds at a frequency of about 50 Hz (duration 5 minutes ) (where the electrical signal is as measured three (3) mm deep within the tissue). A bioelectrical signal for insulin-like growth factor 1 (IGF1) is (within 15%) 3 mV with a frequency of about 22 Hz, and current of about 1 mA, followed by 3 mA. A bioelectrical signal for platelet-derived growth factor (“PDGF”) is (within 15%) 3 V/cm, 10 Hz, 2 µA, and pulse duration of 0.2 ms. Another bioelectrical signal for PDGF is (within 15%) 20 V/cm, 100 Hz, 0.25 mA (2.5e-7 amps), and pulse duration 40 pulses/sec, width 100 µsec. Another bioelectrical signal for PDGF is (within 15%) 20 V for 1 minute, 20 mV for 10 minutes, current 0.25 mA, pulse duration 40 pulses/s, pulse width 100 μs, and frequency of 100 Hz for 5 minutes followed by 528 Hz for 3 minutes and 432 Hz for 3 minutes and 50 Hz for 3 minutes. A bioelectrical signal for Klotho is a biphasic pulse at (within 15%) 20 Hz, 0.1 V, and 7.8 ms pulse duration.

[0187] The bioelectric stimulator is connected to an individual's scalp (eg via electrodes) where the individual suffers from hair loss and/or baldness. A bioelectric signal regimen is applied to the subject's scalp. Increased hair growth is experienced by the individual.

[0188] LED light signaling sequences can also be applied to the subject's scalp. See, for example, Joo, Hong Jin et al. "Various Wavelengths of Light-Emitting Diode Light Regulate the Proliferation of Human Dermal Papilla Cells and Hair Follicles via Wnt/β-Catenin and the Extracellular Signal-Regulated Kinase Pathways”, Annals of Dermatology vol. 29, 6 (2017): 747- 754. doi:10.5021/ad.2017.29.6.747, and Kim et al. “Wnt/β-catenin and ERK pathway activation: A possible mechanism of photobiomodulation therapy with light-emitting diodes that regulate the proliferation of human outer root sheath cells” . Lasers Surg Med. 2017 Dec;49(10):940-947. doi: 10.1002/lsm.22736. Epub 2017 Sep 25. PMID: 28944964.

[0189] The subject's treatment can also be supplemented with the administration of Polygonum multiflorum Radix (PMR). See, for example, Li, Yunfei et al. “Hair Growth Promotion Activity and Its Mechanism of Polygonum multiflorum”, Evidence-Based Complementary and Alternative Medicine: eCAM vol. 2015 (2015): 517901. doi:10.1155/2015/517901.

[0190] The individual may also be administered with adipose-derived stem cells. Fukuoka et al. “Hair Regeneration Therapy: Application of Adipose-Derived Stem Cells”. Current Stem Cell Research & Therapy vol. 12.7 (2017): 531-534. doi:10.2174/1574888X12666170522114307.

EXAMPLE X – Diabetic Neuropathy

[0191] A 20 Hz bioelectric signal was applied to 10 patients (5 treated and 5 controls) included in a clinical study. As the means and SD show, an overexpression of Klotho protein (168%) is observed in the treated group over 4 and 8 weeks. Creatinine values so far did not change between groups.

[0192] References: Lee et al. “Klotho ameliorates diabetic nephropathy via LKB1-AMPK-PGC1α-mediated renal mitochondrial protection” Biochemical and Biophysical Research Communications Volume 534, January 1, 2021, Pages 1040-1046; and Hu, Ming Chang et al. “Renal and extrarenal actions of Klotho”, Seminars In Nephrology vol. 33.2 (2013): 118-29. doi:10.1016/j.semnp.hrol.2012.12.013.

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Claims (8)

Bioelectric stimulator programmed to produce at least one bioelectric signal having a frequency selected from the group consisting of 5 Hz, 10 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz, 2500 Hz , 25,000 Hz, 50,000 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz,
characterized in that said bioelectric signal(s) modulates Klotho expression in mammalian cell tissue stimulated with said bioelectric signal. Bioelectric stimulator, according to claim 1, characterized in that the bioelectric signal is selected from the group consisting of 5 Hz, 10 Hz, 75 Hz, 250 Hz, 500 Hz, 750 Hz, 2,500 Hz, 100,000 Hz , 500,000 Hz, and 1 MHz and klotho expression is up-regulated. Bioelectric stimulator according to claim 1, characterized in that the bioelectric stimulator is programmed with at least one bioelectric signal having a frequency selected from the group consisting of 25,000 Hz, 50,000 Hz, 750,000 Hz, and 1 MHz and klotho expression is down-regulated. A method of using a bioelectric stimulator programmed to produce one or more bioelectric signals for stimulating cellular tissue, wherein at least one such bioelectric signal modulates Klotho expression by cellular tissue, characterized in that it comprises:
applying the bioelectric stimulator to the subject's target tissue, and
trigger the bioelectric stimulator to produce the programmed bioelectric signal,
wherein the bioelectric stimulator is programmed with at least one bioelectric signal having a frequency selected from the group consisting of 5 Hz, 10 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz , 2,500 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz, and
wherein the bioelectrical signal is applied to the individual for from 5 minutes to 24 hours in a day. Method according to claim 4, characterized in that the bioelectric stimulator is programmed with at least one bioelectric signal having a frequency selected from the group consisting of 25,000 Hz, 50,000 Hz, 750,000 Hz, and 1 MHz and the expression is down-regulated. Method according to claim 4, characterized in that the bioelectric signal is selected from the group consisting of 5 Hz, 10 Hz, 75 Hz, 250 Hz, 500 Hz, 750 Hz, 2,500 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz and klotho expression is up-regulated. Method, according to claim 6, characterized by the fact that the amount of Klotho circulating in the individual's bloodstream is increased by at least 200% above normal. Use of a bioelectric stimulator programmed to produce at least one bioelectric signal having a frequency selected from the group consisting of 5 Hz, 10 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz , 2,500 Hz, 25,000 Hz, 50,000 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz,
characterized in that said use is for the preparation of a device for modulating Klotho expression in mammalian cell tissue

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