The role of Klotho in mineral metabolism and inflammation

Accumulated data suggest that a disrupted FGF23-Klotho axis is a major contributor to the development of chronic kidney disease (CKD). This axis might have a broader role in the regulation of inflammation, yet the underlying mechanisms remain largely elusive. To understand the biological actions of FGF23, it is important to elucidate the function of the receptors that mediate the FGF23 signaling. Klotho, a type I membrane-bound protein directly interacts with the FGF-receptor 1c and functions as a specific FGF23-receptor. FGF23-induced activation of the FGF receptor/Klotho complex exerts a reduction in re-absorption of phosphate as well as in the down-regulation in the synthesis of vitamin-D in renal tubules. In addition to its potential role in phosphate metabolism, Klotho might have a wider role in the regulation of inflammation; i.e., it has been shown that its production is substantially reduced in different inflammatory diseases such as acute kidney injury (AKI), CKD, inflammatory bowel disease (IBD), and rheumatoid arthritis (RA). The overall aim of the present research project is to elucidate the pathogenic mechanisms of the FGF23-Klotho axis in the development of CKD and hematopoietic stem cell transplantation (HSCT)-associated AKI. In order to understand the basic mechanisms underlying these conditions, we generated distal tubule-specific Klotho (Ksp-KL-/-) using cre-lox recombination technology. These mice were hyperphosphatemic with elevated serum FGF23 levels, indicating that distal tubular Klotho affects phosphate reabsorption in the proximal tubules. In contrast to Ksp-KL-/- mice, β -KL-/- mice (systemic Klotho knockout mice) exhibited the phenotype of existing Klotho null mice confirming that the Klotho gene functions as anti-aging gene. The mechanism of this proposed distal-to-proximal tubular signaling remains to be explored (Study I). To further elucidate the role of renal Klotho in the development of aging phenotype, we generated mice with Klotho deleted throughout the nephron (Six2-KL-/-). Six2-KL-/- mice were infertile, kyphotic, growth retarded and had a decreased life span, closely resembling the phenotype seen in systemic Klotho knockout mice. Further, the serum and urine biochemistries, low serum Klotho levels, as well as profound histological abnormalities, were indistinguishable from systemic Klotho knockout mice, unravelling the kidney as the principal contributor to circulating Klotho and as the mediator of Klotho anti-ageing traits (Study II). In order to prevail the significant controversies regarding the presence or absence of Klotho in the vascular system and whether the vascular tissue is directly responsive to FGF23 endocrine action, we analyzed the expression of the FGF23 co-receptor Klotho in mouse arteries and generated a novel mouse model, harboring a vascular smooth muscle cell-specific deletion of Klotho (Sm22-KL-/-). Arterial Klotho transcripts were detected at very low levels, whereas the corresponding protein levels were undetectable. FGF23-Klotho signaling was collectively absent in the mouse arteries, and the vascular phenotype was unaffected by FGF23 treatment. Thus, our data do not support direct, Klotho-dependent FGF23 vasculotoxicity although confirmative studies in humans are warranted (Study III). Lastly, to better define the role FGF23-Klotho axis in the regulation of inflammation, we utilized chemotherapy-based conditioning, (Bu)-cyclophosphamide (Cy) murine model for acute graft versus host disease (aGVHD). FGF23-Klotho axis was dysregulated in the aGVHD-induced kidney injury (AKI). Renal Klotho was ablated in aGVHD mice. Further, serum biochemistry was also indistinguishable in the LPS-induced AKI model. Thus, our findings demonstrate that FGF23-Klotho signalling is substantially disturbed in aGVHD which might contribute to the development AKI (Study IV). Taken together, the studies included in the present thesis substantially contribute to the understanding of the role FGF23-Klotho axis and its mechanism of action in several disease conditions. Urgent studies to identify current and novel therapeutic interventions that restitute the deranged FGF23-Klotho axis in CKD and the inflammatory disease, aGVHD-associated AKI and identify individuals who will benefit the most from such treatment are warranted

Renal Klotho in mineral metabolism

Calcium and phosphorous are critical elements in a number of physiological processes, including maintenance of bone structure, cell signaling and energy metabolism. Their endocrine regulation is tightly controlled through a number of feedback mechanisms involving parathyroid hormone (PTH), vitamin D and the recently discovered fibroblast growth factor 23 (FGF23). The kidney is a key organ in maintaining normal serum levels of calcium and inorganic phosphorous, and disturbances in mineral metabolism are commonly observed in patients with chronic kidney disease (CKD). Klotho is a membrane-bound protein expressed in the renal tubules that acts as a co-receptor for FGF23. In addition, Klotho can be shedded from the cell surface to extra-cellular compartments and function as a hormone with effects on mineral metabolism independent of FGF23. During the progression of CKD the expression of Klotho rapidly declines, and accumulating evidence point to lack of Klotho as a pathogenic factor driving clinical complications in CKD. The main focus of this thesis has been to elucidate the role of renal Klotho in mineral metabolism and on systemic effects. In Study I we generated distal tubule-specific Klotho knockout mice (Ksp-KL-/-) by employing cre-lox recombination. Ksp-KL-/- mice were hyperphosphatemic with elevated serum Fgf23 levels, indicating that distal tubular Klotho affects phosphate reabsorption in the proximal tubules. The exact mechanism of this proposed distal-toproximal tubular signaling remains unknown. In Study II we generated mice with Klotho deleted throughout the nephron (Six2-KL-/- ). Six2-KL-/- mice were infertile, kyphotic, growth retarded and had a decreased life span, closely resembling the phenotype seen in systemic Klotho knockout mice. Also the serum and urine biochemistries, low serum Klotho levels as well as profound histological abnormalities were indistinguishable from systemic Klotho knockout mice, unraveling the kidney as the principle contributor to circulating Klotho and mediator of Klotho anti-ageing traits. Taken together, the studies presented in this licentiate thesis substantially contribute to the understanding of renal Klotho function

Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes.

BACKGROUND: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies. RESULTS: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation compared with cells from normal-weight subjects. In contrast, trimethylation at lysine 4 was 40% higher in adipocytes from overweight diabetic subjects compared with normal-weight and overweight non-diabetic subjects. There was no association between level of modification and age of subjects. CONCLUSIONS: The findings define genome-wide molecular modifications of histones in adipocytes that are directly associated with overweight and diabetes, and thus suggest a molecular basis for existing epidemiological evidence of epigenetic inheritance

RESEARCH Open Access

Global differences in specific histone H3 methylation are associated with overweight and type 2 diabete

Open Access

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trial

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer : a retrospective study including patients from the randomised Stockholm tamoxifen trials.

INTRODUCTION: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. METHODS: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. RESULTS: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. CONCLUSION: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer : a retrospective study including patients from the randomised Stockholm tamoxifen trials.

INTRODUCTION: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. METHODS: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. RESULTS: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. CONCLUSION: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer