FluoBolt™ α-KLOTHO Protein Immunoassay

MATERIAL SUPPLIED WITH THE KIT

• 1 x 96 well- Anti-human Klotho antibody, pre-coated MEF-microtiter plate
• 1 x 25 ml- Wash buffer concentrate 20x, natural cap
• 1 x 5 ml- Anti-human Klotho antibody, labeled with either: FITC, Cy3, Cy5 or AlexaFluor®680
• 6 x 0.25 ml- Human Klotho protein Standards 1-6, (400, 200, 100, 50, 25, 0 pmol/l),white caps, lyophilized
• 2 x 0.25 ml- Control A and B, yellow cap, lyophilized (for concentrations see label)
• 1 x 10 ml- Sample diluent, natural cap, ready to use

 ADDITIONAL MATERIAL SUPPLIED WITH THE KIT

• 2 self-adhesive plastic films
• QC data sheet
• Protocol sheet
• Instruction for use manual
• 2 desiccant bags for plate storage

MATERIAL AND EQUIPMENT REQUIRED BUT NOT SUPPLIED

• Precision pipettes calibrated to deliver 10 μl, 20 μl, 50 μl, 200 μl, 500 μl, and disposable tips
• Distilled or deionized water
• Plate washer, multichannel pipette, or manifold dispenser for washing
• Refrigerator with 4°C (2-8°C)
• Fluorescence microplate reader

Read more about the MEF technology here diapharma.com/fluobolt/

Figure 1- Example of typical klotho protein calibration curve

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit lot at production date.

Fluorescence intensity obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life.

However, this does not affect validity of results as long as the supplied kit controls read according to specifications (target ranges, see labels).

Figure 2- α-KLOTHO protein and does not cross-react with β-KLOTHO proein

Figure 3-  FGF-23 does not interfere with α-KLOTHO detection

Intra assay:
4 samples of known concentrations were tested 3 times within 1 assay run
CVs ranged from 1 – 11.0%

Inter assay:
4 samples of known concentrations were tested in duplicates within 3 different assay runs
CVs ranged from 6 – 10%

Spike/Recovery:
The recovery of KLOTHO in serum was evaluated by adding known amounts of human recombinant KLOTHO to 4 different human serum samples. Mean recovery was 82% (69 101%).
Mean recovery in Citrate and Heparin Plasma was each 75% (n=4) and 88% in EDTA Plasma (n=4).

Cross-Reactivity:
Human α-KLOTHO protein shares between 84-100% aa sequence identity with primates, mice, rats, hamster, and pig protein (shown in the chart below). Cross-reactivity of this assay with species other than human has not been tested with FluoBolt™ α-KLOTHO.

Basic Structure and Biology

The name for the klotho (KL) family of proteins was derived from Greek mythology.  Clotho was the daughter of Zeus and Themis and one of the three fates.  Clotho was the fate that spun the thread of Life, while the other Fates would each draw out or cut this thread.  There are three subfamilies of Klotho: α-Klotho, β-Klotho, and γ-Klotho.  Α-Klotho and β-Klotho are essential components of the endocrine fibroblast growth factor (FGF) receptor complexes.  They are required for the high-affinity binding of FGF-19, FGF-21, and FGF-23 to their cognate FGF receptors (FGFRs).  When the subfamily is not specified, the word “Klotho” generally means the α-klotho protein subfamily.

Figure 1     Klotho protein can exist in a membrane-bound or soluble state. The membrane bound Klotho is mainly expressed on cells of the kidney and acts locally on kidney biology via signaling interactions with FGF-23. Soluble Klotho protein can be produced by protease cleavage sites within the membrane bound isoform, or a proposed directly secreted isoform. As a soluble form, Klotho can enter the blood stream and act distally, similar to a hormone or cytokine.

Membrane-bound Klotho protein is a single transmembrane protein with a short (10 amino acid) intracellular domain that is not yet credited for any signal transduction (shown in Figure 1). The extracellular part of Klotho protein contains two homologous domains, termed KL1 and KL2.  Between the membrane and KL2 and in between KL1 and KL2 are cleavage sites for ADAM10 and ADAM17.  Cleavage produces soluble Klotho proteins that either contains independent KL1 domain, KL2 domain, or both.  The form containing both KL1 and KL2 is 130 kDa in size and appears that the predominant form of soluble Klotho protein.  In fact, neither secondary cleavage product has been detected in human serum so far, only in in vitro systems. In addition to cleaved membrane Klotho protein, an alternatively spliced Klotho mRNA transcript is hypothesized to code for a secreted Klotho protein, comprised of the KL1 domain with a 10 amino acid tail.  The soluble forms of human klotho protein are detected in blood, urine and cerebrospinal fluid.

Human klotho protein is often described as the fountain of youth protein and seems to translate across species in studies of function and biomarker usefulness.  When Klotho was first discovered, Klotho deficiencies in a mouse model produced a phenotype that was similar to human aging.  These mice died prematurely, but also showed decreased muscle strength, endurance, cognitive ability, and osteoporosis.  Although the vast majority of research has been based on lack of Klotho in mouse models, overexpression of Klotho extends lifespan by 20–30% compared to wild type mice and is protective for kidney disease, cardiac and vascular disease, lung damage, neurodegenerative disease, and diabetes.  Exogenous Klotho protein in Klotho-deficient mice or even aged animals improves the aging phenotype.  In humans, variations in the Klotho gene are associated with both life extension and increased cognition in human populations.  In addition, soluble Klotho protein has been shown to be a circulating factor detectable in serum that declines with age.

The functions of Klotho are mainly linked to FGF-23 induced signaling and related to their role in mineral homeostasis.  The Klotho gene is under the transcriptional control of PPARy and is most predominantly expressed in the epithelial cells of the renal tubules in the kidney, but is also detected in the brain and to a lesser extent a variety of other tissues. Most publications referencing klotho to date utilize it as a kidney disease biomarker.

Klotho exists in at least two forms including a membrane form and a soluble form. The two forms of Klotho have distinct functions in the regulation of various physiological and pathophysiological processes.  Membrane Klotho forms a complex with the fibroblast growth factor 23 (FGF-23) and FGF receptors to regulate renal phosphate excretion and calcium reabsorption. As mentioned above, the soluble form can be produced as a result of a protease cleavage of the extracellular portion of the membrane bound Klotho or a proposed alternative spliced explicitly secreted form of Klotho.  In either case, soluble Klotho can be released into circulation and act as a hormone on distal tissues and organs.

Klotho as a Biomarker in Research

Klotho is linked to a variety of diseased states associated with aging, so it is a likely target to be examined when searching for sensitive and specific biomarkers that can be measured in the blood or urine.

• Kidney Disease Biomarker
  Throughout the past 20 years, evidence from clinical and basic science studies has showed that Klotho is significantly correlated with the development and progression of Chronic Kidney Disease (CKD) and its complications. The expression level of Klotho protein decreases in human and animal subjects with CKD as the severity of kidney disease increases.

In a rat model of Acute Kidney Injury (AKI) a rapid decrease in plasma and urine Klotho protein is detectable as early as at 3 h after injury. Changes in Klotho protein levels were detected earlier than serum creatinine (24 h) and NGAL (5 h). This report also suggests that Klotho protein levels in the urine of AKI subjects are lower than healthy subjects, but more work focused on timing and specificity need to be established.

• Brain Disease Biomarker
  Klotho is also highly expressed in the brain and animal studies suggest it has a role with neuro dysfunction or disease.  Among these studies, mice deficient in Klotho have a reduced number of synapses and cognitive deficits, while overexpressing klotho enhanced learning, memory and was neuroprotective.  Overexpression of Klotho in a mouse model of Alzheimer’s disease can improve cognitive function and prevent cell loss in the brain.  In a microarray analysis comparing young and old monkeys, Klotho is shown to be downregulated in the aged brain.  These animal studies suggest that Klotho expression level correlates with brain health and increase interest in Klotho protein as a human biomarker.

Consistent with animal reports, humans with higher plasma Klotho protein showed both higher cognitive assessment scores and less decline at follow up exams.  Clinical research studies show that Klotho protein levels have potential as a biomarker of multiple sclerosis severity, psychological dysfunction from chronic stress to bipolar disorder, epilepsy, and Alzheimer’s disease. Together, lower brain Klotho correlates with neurological disease while higher brain Klotho with neurological health.

• Bone Disease Biomarker
  The kidneys play an important role in maintaining healthy bone mass and structure by balancing phosphorus and calcium levels in the blood. Chronic kidney disease causes mineral and bone disorder (CKD-MBD) because the kidneys do not properly balance the mineral levels in the body. Klotho is mainly expressed in the kidney and is vital to the regulation of phosphate and calcium.  Decreased Klotho levels are associated with decreased bone mineral density of bones in mice.   More studies with human bone metabolism need to be done to investigate Klotho’s role as a protein biomarker for bone diseases.
• Cardiovascular Disease Biomarker
  Vascular calcification (VC) appears early in the course of CKD but becomes much more prevalent as kidney function deteriorates, creating a strong risk of cardiovascular mortality and morbidity in subjects with CKD. Klotho deficient mice demonstrate arteriosclerosis. In humans, Klotho gene variants have been found to be protective or detrimental for the development of cardiovascular and cerebrovascular disease.  Also, higher Klotho protein levels are reportedly associated with lower likelihood of cardiovascular disease and higher Klotho protein protects against endothelial dysfunction.
• Cancer Biomarker
  Klotho seems to have tumor suppressor activity via by inhibiting insulin/IGF-1 signaling and induction of cancer cell apoptosis. Klotho has been linked as a tumor suppressor in studies of breast cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, and ovarian cancer.  In xenograft studies of breast cancer, injections of Klotho protein decreased tumor size.    Structure-function studies pin-point this activity to the KL1 part of Klotho protein and is independent of FGF-23 signaling.  Additionally, exogenous Klotho reduced lung cancer metastasis and increased overall survival.  Klotho is a proposed biomarker for human hepatocellular carcinoma (HCC) and Klotho-positive tumors correlated with higher survival.
• Diabetes Biomarker
  In humans and mice, Klotho is significantly decreased in pancreatic islets of subjects with Type 2 diabetes versus those of healthy donors. Diabetic mice treated with Klotho decreased blood glucose levels increased insulin levels.  Klotho seems to protect β-cells by decreasing apoptosis and protect against diabetes.
• Fibrosis Biomarker
  Wnt signaling plays a role in stem cell proliferation and maintenance and is also a contributor to fibrosis in a variety of organs and disease states. Another substantial contributor to fibrosis is TGFβ-1 signaling. Studies support that circulating Klotho directly binds multiple soluble Wnt molecules, including at least Wnt1, Wnt3, Wnt4 and Wnt5a, and resulted in the suppression of Wnt biological activity. Klotho also blocks TGFβ-1 signaling by binding to the TGF-β type-II receptor, blocking TGF-β1 receptor interactions, thereby inhibiting TGF-β1 signaling.  Tissues and organs from klotho-deficient animals showed evidence of increased Wnt and TGFβ-1 signaling.   As an antagonist of fibrotic signaling, Klotho is predicted to be protective against fibrosis.

Klotho Protein Detection- Clinical Research Hurdles

Commercial products for detecting soluble Klotho protein in biological samples has been riddled with obstacles that have hampered Klotho studies.  While commercially available antibodies against human Klotho are available, several reports claim them to be non-specific and cross-react with other proteins. Many of the commercially available antibodies are not well characterized and it is unclear whether commercial assays are detecting the 130 kDa and/or 65 kDa circulating isoforms.  It is reported that novel anti-Klotho antibodies exist, but are currently not commercially available. Some use an immunoprecipitation-immunblot (IP-IB) assay in fresh urine samples, but the technique is labor intensive and difficult to scale up.

In addition to the antibody issues, generation of recombinant Klotho is difficult and the unpredictable quality of commercially produced recombinant proteins may affect the reproducibility of ELISA products.  For example, when one study compared a time-resolved fluorescence immunoassay (TRF) to an ELISA, no correlation was found between the assays and the levels of serum Klotho protein differed with by a factor 1000.  Furthermore, a related technical challenge is that soluble Klotho protein appears to be unstable in biological samples.  Standardization of techniques to prevent degradation and optimization of stable sample storage (fresh vs frozen) would eliminate conflicting data for use as a kidney disease biomarker, but no consensus protocol has yet been determined.

While improvements in sample preparation still need to be addressed, Fianostics is adding to the toolbox for Klotho researchers with FluoBolt™ alpha-Klotho.  The hope is to increase the options available to researchers so that they can produce accurate results and reliable conclusions concerning Klotho as a kidney disease biomarker.