Background and Hypothesis
Klotho is a recently discovered anti-aging gene. The purpose of this study was to investigate if klotho gene transfer attenuates superoxide production and oxidative stress in rat aorta smooth muscle (RASM) cells.
Methods and Results
RASM cells were transfected with AAV plasmids carrying mouse klotho full-length cDNA (mKL) or LacZ as a control. Klotho gene transfer increased klotho expression in RASM cells. Notably, klotho gene expression decreased Nox2 NADPH oxidase protein expression but did not affect Nox2 mRNA expression, suggesting that the inhibition may occur at the post-transcriptional level. Klotho gene transfer decreased intracellular superoxide production and oxidative stress in RASM cells. Klotho gene expression also significantly attenuated the angiotensin II (AngII)-induced superoxide production, oxidative damage, and apoptosis. Interestingly, klotho gene delivery dose-dependently increased the intracellular cAMP level and PKA activity in RASM cells. Rp-cAMP, a competitive inhibitor of cAMP, abolished the klotho-induced increase in PKA activity, indicating that klotho activated PKA via cAMP. Notably, inhibition of cAMP-dependent PKA activity by RP-cAMP abolished klotho-induced inhibition of Nox2 protein expression, suggesting an important role of the cAMP-dependent PKA in this process.
The present finding revealed a previously unidentified role of klotho in regulating Nox2 protein expression in RASM cells. Klotho not only downregulated Nox2 protein expression and intracellular superoxide production but also attenuated AngII-induced superoxide production, oxidative damage, and apoptosis. The klotho-induced suppression of Nox2 protein expression may be mediated by the cAMP PKA pathway.
klotho; Nox2; NADPH oxidase; superoxide; cAMP; PKA; smooth muscle cell
The klotho gene is expressed in a limited number of tissues, most notably in distal convoluted tubules in the kidney and choroid plexus in the brain. A previous study suggested that Klotho increases resistance to oxidative stress. However, changes of Klotho expression in high glucose-induced oxidative stress remain unclear. In the present study, we used streptozotocin-induced diabetic rats (STZ rats) to examine the effects of insulin, phloridzin or antioxidant, tiron on diabetic nephropathy. Both insulin and phloridzin reversed the lower Klotho expression levels in kidneys of STZ rats by the correction of hyperglycemia. Also, renal functions were improved by these treatments. In addition to the improvement of renal functions, the decrease of Klotho expression in kidney of STZ rats was also reversed by tiron without changing blood glucose levels. The reduction of oxidative stress induced by high glucose can be considered for this action of tiron. This view was further confirmed in vitro using high glucose-exposed Madin-Darby canine kidney (MDCK) epithelial cells. Thus, we suggest that decrease of oxidative stress is not only responsible for the improvement of renal function but also for the recovery of Klotho expression in kidney of STZ rats.
Klotho is a single-pass transmembrane protein that exerts its biological functions through multiple modes. Membrane-bound Klotho acts as coreceptor for the major phosphatonin fibroblast growth factor-23 (FGF23), while soluble Klotho functions as an endocrine substance. In addition to in the distal nephron where it is abundantly expressed, Klotho is present in the proximal tubule lumen where it inhibits renal Pi excretion by modulating Na-coupled Pi transporters via enzymatic glycan modification of the transporter proteins – an effect completely independent of its role as the FGF23 coreceptor. Acute kidney injury (AKI) and chronic kidney disease (CKD) are states of systemic Klotho deficiency, making Klotho a very sensitive biomarker of impaired renal function. In addition to its role as a marker, Klotho also plays pathogenic roles in renal disease. Klotho deficiency exacerbates decreases in, while Klotho repletion or excess preserves, glomerular filtration rate in both AKI and CKD. Soft tissue calcification, and especially vascular calcification, is a dire complication in CKD, associated with high mortality. Klotho protects against soft tissue calcification via at least 3 mechanisms: phosphaturia, preservation of renal function and a direct effect on vascular smooth muscle cells by inhibiting phosphate uptake and dedifferentiation. In summary, Klotho is a critical molecule in a wide variety of renal diseases and bears great potential as a diagnostic and prognostic biomarker as well as for therapeutic replacement therapy.
Acute kidney injury; Chronic kidney disease; Klotho; Phosphaturia; Vascular calcification
Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone mainly produced by bone that acts in the kidney through FGF receptors and Klotho. Here we investigated whether the kidney was an additional source of FGF23 during renal disease using a model of type 2 diabetic nephropathy. Renal expression of FGF23 and Klotho was assessed in Zucker diabetic fatty (ZDF) and control lean rats at 2, 4, 6, 8 months of age. To evaluate whether the renoprotective effect of angiotensin converting enzyme (ACE) inhibitor in this model was associated with changes in FGF23 and Klotho, ZDF rats received ramipril from 4, when proteinuric, to 8 months of age. FGF23 mRNA was not detectable in the kidney of lean rats, nor of ZDF rats at 2 months of age. FGF23 became measurable in the kidney of diabetic rats at 4 months and significantly increased thereafter. FGF23 protein localized in proximal and distal tubules. Renal Klotho mRNA and protein decreased during time in ZDF rats. As renal disease progressed, serum phosphate levels increased in parallel with decline of fractional phosphorus excretion. Ramipril limited proteinuria and renal injury, attenuated renal FGF23 upregulation and ameliorated Klotho expression. Ramipril normalized serum phosphate levels and tended to increase fractional phosphorus excretion. These data indicate that during progressive renal disease the kidney is a site of FGF23 production which is limited by ACE inhibition. Interfering pharmacologically with the delicate balance of FGF23 and phosphorus in diabetes may have implications in clinics.
We have previously shown that myelin abnormalities and loss characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. While the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. In the present study, we found significant effects of Klotho on oligodendrocyte functions including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein Western analysis indicated Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPCs maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knockout mice and their control littermates revealed that knockout mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knockout mice. Strikingly, at the ultrastructural level, Klotho knockout mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. In order to decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Taken together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes.
The aim of this study was to investigate whether the effects of aging on oxidative stress markers and expression of major oxidant and antioxidant enzymes associate with impairment of renal function and increases in blood pressure. To explore this, we determined age-associated changes in lipid peroxidation (urinary malondialdehyde), plasma and urinary hydrogen peroxide (H2O2) levels, as well as renal H2O2 production, and the expression of oxidant and antioxidant enzymes in young (13 weeks) and old (52 weeks) male Wistar Kyoto (WKY) rats. Urinary lipid peroxidation levels and H2O2 production by the renal cortex and medulla of old rats were higher than their young counterparts. This was accompanied by overexpression of NADPH oxidase components Nox4 and p22phox in the renal cortex of old rats. Similarly, expression of superoxide dismutase (SOD) isoforms 2 and 3 and catalase were increased in the renal cortex from old rats. Renal function parameters (creatinine clearance and fractional excretion of sodium), diastolic blood pressure and heart rate were not affected by aging, although slight increases in systolic blood pressure were observed during this 52-week period. It is concluded that overexpression of renal Nox4 and p22phox and the increases in renal H2O2 levels in aged WKY does not associate with renal functional impairment or marked increases in blood pressure. It is hypothesized that lack of oxidative stress-associated effects in aged WKY rats may result from increases in antioxidant defenses that counteract the damaging effects of H2O2.
aging; oxidative stress; kidney; hydrogen peroxide; NADPH oxidase; antioxidant enzymes; Wistar Kyoto rat
Klotho is highly expressed in the kidney and a soluble form of Klotho functions as an endocrine substance that exerts multiple actions including the modulation of renal solute transport and the protection of the kidney from a variety of insults in experimental models. At present, the Klotho database is still largely preclinical, but the anticipated forthcoming impact on clinical nephrology can be immense. This manuscript puts these potentials into perspective for the clinician. There is renal and systemic Klotho deficiency in both acute kidney injury (AKI) and chronic kidney disease (CKD). Klotho plummets very early and severely in AKI and represents a pathogenic factor that exacerbates acute kidney damage. In CKD, Klotho deficiency exerts a significant impact on progression of renal disease and extra renal complications. In AKI, soluble Klotho levels in plasma and/or urine may serve as an early biomarker for kidney parenchymal injury. Restoration by exogenous supplementation or stimulation of endogenous Klotho may prevent and/or ameliorate kidney injury and mitigate CKD development. In CKD, Klotho levels may be an indicator of early disease and predict the rate of progression, and presence and severity of soft tissue calcification. The correction of Klotho deficiency may delay progression and forestall development of extra renal complications in CKD. Rarely does one find a molecule with such broad potential applications in nephrology. Klotho can possibly emerge on the horizon as a candidate for an unprecedented sole biomarker and intervention. Nephrologists should monitor the progress of the preclinical studies and the imminently emerging human database.
acute kidney injury; biomarker; cardiac hypertrophy; chronic kidney disease; FGF23; Klotho; prognosis; secondary hyperparathyroidism
Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice.
FGF23; phosphate; PTH
Diabetes is associated with a high incidence of macrovascular disease (MVD), including peripheral and coronary artery disease. Circulating soluble-Klotho (sKlotho) is produced in the kidney and is a putative anti-aging and vasculoprotective hormone. Reduced Klotho levels may therefore increase cardiovascular risk in diabetes. We investigated if sKlotho levels are decreased in type 2 diabetes and associate with MVD in the absence of diabetic nephropathy, and whether hyperglycemia affects renal Klotho production in vitro and in vivo.
sKlotho levels were determined with ELISA in diabetic and non-diabetic patients with and without MVD, and healthy control subjects. Human renal tubular epithelial cells (TECs) were isolated and exposed to high glucose levels (15 and 30 mM) in vitro and Klotho levels were measured with qPCR and quantitative immunofluorescence. Klotho mRNA expression was quantified in kidneys obtained from long term (3 and 8 months) diabetic Ins2Akita mice and normoglycemic control mice.
No significant differences in sKlotho levels were observed between diabetic patients with and without MVD (527 (433–704) pg/mL, n = 35), non-diabetic MVD patients (517 (349–571) pg/mL, n = 27), and healthy control subjects (435 (346–663) pg/mL, n = 15). High glucose (15 and 30 mM) did not alter Klotho expression in TECs. Long-term hyperglycemia in diabetic Ins2Akita mice (characterized by increased HbA1c levels [12.9 ± 0.3% (3 months) and 11.3 ± 2.0% (8 months)], p < 0.05 vs. non-diabetic mice) did not affect renal Klotho mRNA expression.
These data indicate that sKlotho levels are not affected in type 2 diabetes patients with and without MVD. Furthermore, hyperglycemia per se does not affect renal Klotho production. As type 2 diabetes does not alter sKlotho levels, sKlotho does not seem to play a major role in the pathogenesis of MVD in type 2 diabetes.
Atherosclerosis; Coronary artery disease; Klotho; Macrovascular disease; Peripheral artery disease; Type 2 diabetes
The expression of the renoprotective antiaging gene Klotho is decreased in uremia. Recent studies suggest that Klotho may be a tumor suppressor, and its expression may be repressed by DNA hypermethylation in cancer cells. Here we investigated the effects and possible mechanisms by which Klotho expression is regulated during uremia in uninephrectomized B-6 mice given the uremic toxins indoxyl sulfate or p-cresyl sulfate. Cultured human renal tubular HK2 cells treated with these toxins were used as an in vitro model. Injections of indoxyl sulfate or p-cresyl sulfate increased their serum concentrations, kidney fibrosis, CpG hypermethylation of the Klotho gene, and decreased Klotho expression in renal tubules of these mice. The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased. Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2′-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro. Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.
DNA methylation; DNA methyltransferase; Klotho; uremic toxin
Klotho is a recently discovered antiaging gene. The objective of this study was to test the hypothesis that klotho gene delivery attenuates the progression of spontaneous hypertension and renal damage in spontaneous hypertensive rats (SHRs). An adeno-associated virus (AAV) carrying mouse klotho full-length cDNA (AAV.mKL) was constructed for in vivo expression of klotho. Four groups of male SHRs and 1 group of sex- and age-matched Wistar-Kyoto rats (5 rats per group) were used. Blood pressure was measured twice in all of the animals before gene delivery. Four groups of SHRs received an IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The Wistar-Kyoto group received PBS and served as a control. AAV.mKL stopped the further increase in blood pressure in SHRs, whereas blood pressures continued to increase in other SHR groups. One single dose of AAV.mKL prevented the progression of spontaneous hypertension for at least 12 weeks (length of the study). Klotho expression and production were suppressed in SHRs, which were reverted by AAV.mKL. AAV.mKL increased plasma interleukin 10 levels but decreased Nox2 expression, NADPH oxidase activity, and superoxide production in kidneys and aortas in SHRs. AAV.mKL abolished renal tubular atrophy and dilation, tubular deposition of proteinaceous material, glomerular collapse, and collagen deposition seen in SHRs, indicating that klotho gene delivery attenuated renal damage. Therefore, the suppressed klotho expression may play a role in the progression of spontaneous hypertension and renal damage in SHRs. AAV delivery of klotho may offer a new approach for the long-term control of hypertension and for renoprotection.
klotho; Nox2; blood pressure; adeno-associated virus; renoprotection
Background. The Klotho gene plays a role in suppressing ageing-related disorders. It is suggested that activation of renin–angiotensin system (RAS) or oxidative stress suppresses Klotho in the kidney. This study evaluated the association between Klotho expression and RAS in cyclosporine (CsA)-induced renal injury.
Methods. Chronic CsA nephropathy was induced by administering CsA (30 mg/kg) to mice on a low-salt diet (LSD) for 4 weeks. A normal-salt diet (NSD) was used as the control. Reverse transcription–polymerase chain reaction, western blot and immunohistochemistry were performed for Klotho and intrarenal RAS activity was measured using immunohistochemistry for angiotensinogen and renin. Oxidative stress was measured with urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG).
Results. CsA treatment decreased Klotho mRNA and protein in mouse kidney in a dose-dependent and time-dependent manner, but a concurrent treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Klotho expression with histological improvement. This finding was more marked in the LSD than the NSD. Klotho expression was correlated with angiotensinogen and renin expression, tubulointerstitial fibrosis score and urinary 8-OHdG excretion.
Conclusions. Angiotensin II may play a pivotal role in regulating Klotho expression in CsA-induced renal injury. AT1 receptor blocker may inhibit the ageing process by decreasing oxidative stress caused by CsA.
cyclosporine; kidney transplantation; nephrotoxicity; renin–angiotensin system; senescence
Klotho is a single-pass transmembrane protein that is highly expressed in the kidney and is known to act as a coreceptor for fibroblast growth factor 23. The extracellular domain can be produced independently or shed from membrane-bound Klotho and functions as an endocrine substance with multiple functions including antioxidation, modulation of ion transport, suppression of fibrosis, and preservation of stem cells. Emerging evidence has revealed that Klotho deficiency is an early event in acute kidney injury (AKI), and a pathogenic factor that exacerbates acute kidney damage and contributes to long-term consequences. Restoration by exogenous supplementation or stimulation of endogenous Klotho might prevent and ameliorate injury, promote recovery, and suppress fibrosis to mitigate development of chronic kidney disease. Although data are still emerging, in this Perspectives article we discuss why this renal-derived protein is a highly promising candidate as both an early biomarker and therapeutic agent for AKI.
Klotho knockout mice (klotho-/-) have increased renal expression of sodium/phosphate co-transporters (NaPi2a), associated with severe hyperphosphatemia. Such serum biochemical changes in klotho-/-mice lead to extensive soft tissue anomalies and vascular calcification. To determine the significance of increased renal expression of the NaPi2a protein and concomitant hyperphosphatemia and vascular calcification in klotho-/-mice, we generated klotho and NaPi2a double knockout (klotho-/-/NaPi2a-/-) mice.
Methods and Results
Genetic inactivation of NaPi2a activity from klotho-/-mice reversed the severe hyperphosphatemia to mild hypophosphatemia or normophosphatemia. Importantly, despite significantly higher serum calcium and 1,25-dihydroxyvitamin D levels in klotho-/-/NaPi2a-/- mice, the vascular and soft tissue calcifications were reduced,. Extensive soft tissue anomalies and cardiovascular calcification were consistently noted in klotho-/-mice by 6 weeks of age; however, these vascular and soft tissue abnormalities were absent even in 12-week-old double knockout mice. Klotho-/-/NaPi2a-/- mice also regained body weight and did not develop the generalized tissue atrophy often noted in klotho-/-single knockout mice.
Our in vivo genetic manipulation studies have provided compelling evidence for a pathologic role of increased NaPi2a activities in regulating abnormal mineral ion metabolism and soft tissue anomalies in klotho-/- mice. Notably, our results suggest that serum phosphate levels are the important in vivo determinant of calcification, and that lowering serum phosphate levels can reduce or eliminate soft tissue and vascular calcification, even in presence of extremely high serum calcium and 1,25-dihydroxyvitamin D levels. These in vivo observations have significant clinical importance and therapeutic implications for chronic kidney disease patients with cardiovascular calcification.
Klotho; Vitamin-D; NaPi2a; Calcification
Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL−/−). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL−/− and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.
The majority of neurodegenerative diseases have an important age component, and thus, understanding the molecular changes that occur during normal aging of the brain is of utmost relevance. In search for the basis of the age-related cognitive decline found in humans, monkeys and rodents, we study the rhesus monkey. Surprisingly, there is no loss of neurons in aged monkey brains. However, we reported white matter and myelin abnormalities in aged monkeys, similar to those observed in Alzheimer’s disease and multiple sclerosis patients. In a microarray analysis comparing young and old monkey white matter, we discovered that Klotho is downregulated in the aged brain. We then asked whether there is a connection between the age-related cognitive decline, myelin abnormalities and Klotho downregulation. If such a connection is found, compounds that upregulate Klotho expression could become of therapeutic interest for the treatment of multiple sclerosis, and perhaps even Alzheimer’s disease.
The molecular interaction of fibroblast growth factor 23 (FGF23) and klotho is essential for physiologic regulation of phosphate balance. In the absence of klotho, the FGF23 protein cannot exert its physiologic functions, as demonstrated by in vivo mouse genetic studies. Bioactive FGF23 protein loses its phosphate lowering effects in genetically modified mice with no klotho activity. The FGF23–klotho system not only affects phosphate homeostasis but can also influence parathyroid hormone (PTH) and vitamin D activities. Dysregulation of the FGF23–klotho system is noted in a number of human acquired and genetic diseases, including chronic kidney disease. Vitamin D is a strong inducer of both FGF23 and klotho expression, while FGF23 can suppress the renal expression of 1α(OH)ase to reduce 1,25(OH)2D activity. An understanding of the complex interactions of phosphate, vitamin D and PTH with the FGF23–klotho system has paved the way to explore the therapeutic benefits of modulating the FGF23–klotho system in diseases associated with abnormal mineral ion balance. The patent (WO2009095372) under discussion proposes using fusion polypeptides to manipulate the FGF23–klotho system.
Background & Aims
Klotho deficiency in hypomorphic KL mice leads to premature senescence and phenotype consistent with impaired mineral homeostasis. Klotho has anti-inflammatory properties protecting from NO-induced endothelial dysfunction, reduces the expression of endothelial adhesion molecules, and may contribute to T-cell dysfunction. Since defective Ca2+/Pi homeostasis leading to osteopenia/osteoporosis is frequently associated with human IBD, we investigated the changes in Klotho gene expression as a consequence of experimental colitis.
We utilized three murine IBD models: TNBS colitis, microflora-induced colitis in gnotobiotic IL-10−/− mice, and adoptive CD4+CD45RBhigh T-cell transfer colitis. These studies were followed by in vitro approaches using renal epithelial cells (mpkDCT4 and mIMCD3), and the cloned murine KL gene promoter.
Renal expression of Klotho mRNA and protein was significantly inhibited in all three models of human IBD. This degree of inhibition was correlated with the severity of colitis, and was reversed by neutralizing anti-TNF antibodies. In vitro, TNF resulted in a significant inhibition of KL expression and was further potentiated by IFN-γ. TNF/IFN-γ combination resulted in increased iNOS expression and significantly elevated the concentration of NO in medium. The effect of IFN-γ could be reproduced by cell exposure to SNAP (NO donor), and reversed by iNOS inhibitor, L-NIL. The cytokine effects were transcriptionally mediated since Klotho mRNA stability remained unaffected, while reporter constructs with the mKL gene promoter displayed significant downregulation in transiently transfected renal epithelial cells.
These novel findings could help explain several extraintestinal complications including abnormalities in bone homeostasis in patients with chronic colitis.
kidney; mineral homeostasis; distal convoluted tubules; bone metabolism
The molecular mechanism linking aldosterone and endothelin-1 in the development of diabetic nephropathy has not been completely elucidated. Here, we provide evidence showing that streptozotocin-induced diabetic rats have significantly increased aldosterone and endothelin-1 in the kidney tissue and markedly decreased expression of Dot1a and Af9. Blocking aldosterone with spironolactone significantly reduced proteinuria, glomerulosclerosis, tubulointerstitial injury and endothelin-1 expression, and significantly increased Dot1a and Af9 expression. Increasing Dot1a and Af9 expression by spironolactone or by stable transfection led to impaired endothelin-1 expression in NRK-52 cells. In contrast, downregulation of Dot1a and Af9 by aldosterone in NRK-52E cells caused upregulation of endothelin-1. Genetic inactivation of Dot1l, which encodes Dot1a, in Aqp2-expressing principal cells of mouse kidney impaired association of Dot1a and H3 dimethyl K79 with the specific subregions of endothelin-1 promoter, and upregulates endothelin-1 mRNA and protein expression. Our data suggest that Dot1a and Af9 repress endothelin-1 in vitro and in vivo. Excessive aldosterone induces kidney injury, in part possibly by downregulating Dot1a and Af9, and thus relieving Dot1a-Af9-mediated repression to increase endothelin-1 transcription. Spironolactone ameliorates kidney injury in Streptozotocin-induced diabetic rats, possibly by restoring Dot1a-Af9-mediated repression to reduce endothelin-1 expression. Therefore, Dot1a and Af9 as aldosterone-downregulated targets are negative regulators of endothelin-1 transcription in vitro and in vivo, and may be considered as new potential therapeutic targets of kidney injury in diabetes.
Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys.
RESEARCH DESIGN AND METHODS
Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536.
Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding.
These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domain of RelA.
Aging is characterized by a gradual functional decrease of all systems including the kidneys. Growing evidence links altered lipid protein redox-homeostasis with renal dysfunction. The effect of sexual dimorphism on the lipid protein redox-homeostasis mechanisms in the aging kidney is obscure. In the current study, we aimed to investigate redox homeostasis as it related to sexual dimorphism on protein oxidation and lipid peroxidation parameters, as protein carbonyl (PCO), total thiol (T-SH), advanced oxidation protein products (AOPP), malondialdehyde, glutathione (GSH), and superoxide dismutase (SOD) activity, as potential aging biomarkers, which may contribute to an analysis of the free radical theory of aging.
Materials and methods
The study was carried out with 16 naturally aged rats (24 months old; eight males and eight females) and their corresponding young rat groups as controls (6 months old; eight males and eight females). All of the aforementioned parameters (PCO, T-SH, AOPP, MDA, GSH, SOD) were measured manually instead of automated devices or ELISA kits.
PCO, AOPP, and malondialdehyde levels in aged rats were significantly higher in the older rat group than in the younger rat group, whereas SOD activities were significantly lower in old rats. T-SH levels were not significantly different in male groups; however, T-SH levels were lower in the aged female group than in the young female control group. In addition, GSH levels were significantly different between the aged rat group and the corresponding young control group for both genders.
With respect to PCO and AOPP, impaired redox homeostasis is substantially more prominent in males than females. The decrease of G-SH levels in male groups could be attributed to stabilizing the redox status of protein thiol groups by the depletion of the GSH groups. Considering the results, the renal tissue proteins and lipids in different genders may have different susceptibilities to oxidative damage.
lipid peroxidation; protein oxidation; radicals; renal aging
FGF23 is a bone-derived hormone that regulates systemic phosphate homeostasis, vitamin D metabolism and α-klotho expression through a novel bone-kidney axis. FGF23 inhibits renal tubular reabsorption of phosphate through mechanisms independent of PTH as well as reduces circulating 1, 25(OH)2D through its dual effects to suppress Cyp27b1 production and to stimulate Cyp24 catabolism of 1,25(OH)2D. 1,25(OH)2D and other factors regulating bone remodeling/mineralization are the major physiological regulators of FGF23 expression. FGF23 also suppresses the gene transcription of α-klotho by the kidney, which exists as a membrane and soluble protein. Membrane Klotho acts as a coreceptor for and dictates organ specificity of FGF23, whereas soluble Klotho act as an endocrine factor that regulates activity of cell surface glycoproteins and receptors in multiple tissues. Elevated FGF23 levels are responsible for several hereditary and acquired hypophosphatemic rickets disorders. FGF23 and Klotho deficiency have similar phenotypes characterized by hyperphosphatemia, elevated 1,25(OH)2D and tumoral calcinosis. FGF23 levels progressively increase during chronic kidney disease (CKD). FGF23 has been proposed to be the initial adaptive response leading to reductions in 1,25(OH)2D and secondary hyperparathyroidism (HPT) in CKD. The overall biological effect of this initial step may be to orchestrate a coordinated adaptation to protect the organism from the adverse effects of excess phosphate retention. The second step involves the effects of PTH on bone remodeling that further stimulates FGF23 production through both direct and indirect mechanisms related to alterations in extracellular matrix factors. PTH further amplifies FGF23 expression in later stages of CKD to compensate for the increased phosphate efflux from bone caused by excessive bone turnover. While many aspects of the regulation and functions of FGF23 remain to be established, the idea that FGF23 hormone is the initial adaptive hormonal response in CKD that suppresses 1,25(OH)2D, reduces gastrointestinal calcium and phosphate absorption and leads to a secondary HPT represents a paradigm shift in the conceptualization of the pathogenesis of secondary hyperparathyroidism. In addition, the prevalent thought that CKD is a functional “vitamin D deficient state” requiring therapy with 1,25(OH)2D analogues is challenged by effects of FGF23 to potentially lower both 25(OH)D and 1,25(OH)D by induction of Cyp24-mediated degradation. Finally, increments in FGF23 are associated with increased cardiovascular mortality in CKD. Whether these effects represent direct effects of FGF23 or represent a marker of other abnormalities in CKD remain to be determined.
1,25(OH)2D; bone; kidney; calcium; osteoblasts; osteocytes; cyp27b1; cyp24; klotho
Declining cognitive performance is associated with increasing age, even in the absence of overt pathological processes. We and others have reported that declining cognitive performance is associated with age-related changes in brain glucose utilization, long-term potentiation and paired-pulse facilitation, protein expression, neurotransmitter levels, and trophic factors. However, it is unclear whether these changes are causes or symptoms of the underlying alterations in dendritic and synaptic morphology that occur with age. In this study, we examined the hippocampal proteome for age- and cognition-associated changes in behaviorally stratified young and old rats, using 2-DIGE and MS/MS-MS. Comparison of old cognitively intact with old cognitively impaired animals revealed additional changes that would not have been detected otherwise. Interestingly, not all age-related changes in protein expression were associated with cognitive decline, and distinct differences in protein expression were found when comparing old cognitively intact with old cognitively impaired rats. A large number of protein changes with age were related to the glycolysis/gluconeogenesis pathway. In total, the proteomic changes suggest that age-related alterations act synergistically with other perturbations to result in cognitive decline. This study also demonstrates the importance of examining behaviorally-defined animals in proteomic studies, as comparison of young to old animals regardless of behavioral performance would have failed to detect many cognitive impairment-specific protein expression changes evident when behavioral stratification data was used.
protein; hippocampus; behavior; aging; cognitive decline
Klotho is a putative aging suppressor gene encoding a single-pass transmembrane co-receptor that makes the fibroblast growth factor (FGF) receptor specific for FGF-23. In addition to multiple endocrine organs, Klotho is expressed in kidney distal convoluted tubules and parathyroid cells, mediating the role of FGF-23 in bone–kidney–parathyroid control of phosphate and calcium. Klotho–/– mice display premature aging and chronic kidney disease-associated mineral and bone disorder (CKD-MBD)-like phenotypes mediated by hyperphosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter). CKD can be seen as a state of hyperphosphatemia-induced accelerated aging associated with Klotho deficiency. Humans with CKD experience decreased Klotho expression as early as stage 1 CKD; Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases, and hypovitaminosis D. Secreted Klotho protein, formed by extracellular clipping, exerts FGF-23-independent phosphaturic and calcium-conserving effects through its paracrine action on the proximal and distal tubules, respectively. We contend that decreased Klotho expression is the earliest biomarker of CKD and the initiator of CKD-MBD pathophysiology. Maintaining normal phosphate levels with phosphate binders in patients with CKD with declining Klotho expression is expected to reduce mineral and vascular derangements.
biomarkers of CKD; FGF-23; hyperphosphatemia; Klotho; secreted Klotho
Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D3 ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (−31 kb to −46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D3 are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.