Constitutive transgenic α-Klotho overexpression enhances resilience to and recovery from murine acute lung injury.

Normal lungs do not express α-Klotho (Klotho) protein but derive cytoprotection from circulating soluble Klotho. It is unclear whether chronic supranormal Klotho levels confer additional benefit. To address this, we tested the age-related effects of modest Klotho overexpression on acute lung injury (ALI) and recovery. Transgenic Klotho-overexpressing (Tg-Kl) and wild-type (WT) mice (2 and 6 mo old) were exposed to hyperoxia (95% O2; 72 h; injury; Hx) then returned to normoxia (21% O2; 24 h; recovery; Hx-R). Control mice were kept in normoxia. Renal and serum Klotho, lung histology, and bronchoalveolar lavage fluid oxidative damage markers were assessed. Effects of hyperoxia on Klotho release were tested in human embryonic kidney cells stably expressing Klotho. A549 lung epithelial cells transfected with Klotho cDNA or vector were exposed to cigarette smoke; lactate dehydrogenase and double-strand DNA breaks were measured. Serum Klotho decreased with age. Hyperoxia suppressed renal Klotho at both ages and serum Klotho at 2 mo of age. Tg-Kl mice at both ages and 2-mo-old WT mice survived Hx-R; 6-mo-old Tg-Kl mice showed lower lung damage than age-matched WT mice. Hyperoxia directly inhibited Klotho expression and release in vitro; Klotho transfection attenuated cigarette smoke-induced cytotoxicity and DNA double-strand breaks in lung epithelial cells. Young animals with chronic high baseline Klotho expression were more resistant to ALI. Chronic constitutive Klotho overexpression in older Tg-Kl animals attenuated hyperoxia-induced lung damage and improves survival and short-term recovery despite an acute reduction in serum Klotho during injury. We conclude that chronic enhancement of Klotho expression increases resilience to ALI.

Alpha-Klotho, a critical protein for lung health, is not expressed in normal lung.

Alpha-Klotho (αKlotho), produced by the kidney and selected organs, is essential for tissue maintenance and protection. Homozygous αKlotho-deficiency leads to premature multi-organ degeneration and death; heterozygous insufficiency leads to apoptosis, oxidative stress, and increased injury susceptibility. There is inconsistent data in the literature regarding whether αKlotho is produced locally in the lung or derived from circulation. We probed murine and human lung by immunohistochemistry (IHC) and immunoblot (IB) using two monoclonal (anti-αKlotho Kl1 and Kl2 domains) and three other common commercial antibodies. Monoclonal anti-Kl1 and anti-Kl2 yielded no labeling in lung on IHC or IB; specific labeling was observed in kidney (positive control) and also murine lungs following tracheal delivery of αKlotho cDNA, demonstrating specificity and ability to detect artificial pulmonary expression. Other commercial antibodies labeled numerous lung structures (IHC) and multiple bands (IB) incompatible with known αKlotho mobility; labeling was not abolished by blocking with purified αKlotho or using lungs from hypomorphic αKlotho-deficient mice, indicating nonspecificity. Results highlight the need for rigorous validation of reagents. The lung lacks native αKlotho expression and derives full-length αKlotho from circulation; findings could explain susceptibility to lung injury in extrapulmonary pathology associated with reduced circulating αKlotho levels, for example, renal failure. Conversely, αKlotho may be artificially expressed in the lung, suggesting therapeutic opportunities.

The relationship between the soluble Klotho protein and the residual renal function among peritoneal dialysis patients.

BACKGROUND: Klotho has been investigated as an anti-aging protein that is predominantly expressed in the distal convoluted tubules in the kidneys and in the choroid plexus of the brain. The purpose of the present study was to determine the relationship between the soluble form of Klotho and renal function in chronic peritoneal dialysis (PD) patients, a relationship which remains poorly understood. METHODS: The soluble Klotho levels in the serum, urine, and peritoneal dialysate obtained from thirty-six PD patients were determined by a sandwich enzyme-linked immunosorbent assay system. RESULTS: The amount of urinary excreted soluble Klotho over 24 h ranged from 1.54 to 1774.4 ng/day (median 303.2 ng/day; interquartile range [IR] 84.1-498.5), while the serum soluble Klotho concentration ranged from 194.4 to 990.4 pg/ml (mean 553.7 ± 210.4 pg/ml). The amount of urinary Klotho excretion was significantly correlated with residual renal function. However, there was no apparent correlation between the serum soluble Klotho levels and the residual renal function. Klotho was also detected in the 24-h dialysate collections. There was a significant correlation between the peritoneal Klotho excretion and the amount of albumin contained in the dialysate collections (r = 0.798, p < 0.01). CONCLUSIONS: The total amount of urinary excreted Klotho, but not the serum level of soluble Klotho, may be a potential biomarker for assessing the residual renal function among PD patients. Whether our findings are also valid for chronic kidney disease patients overall should therefore be evaluated in greater detail.

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice.

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-ß receptor and inhibits TGF-ß1 binding to cell surface receptors, thereby inhibiting TGF-ß1 signaling. Klotho suppresses TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-ß1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.

CLN3P, the Batten's disease protein, is a novel palmitoyl-protein Delta-9 desaturase.

OBJECTIVE: Batten's disease, one of the most common recessively inherited, untreatable, neurodegenerative diseases of humans, is characterized by progressive neuronal loss and intraneuronal proteolipid storage. Although the gene for the disorder was cloned more than a decade ago, the function of the encoded protein, CLN3P, has not been defined thus far. METHODS: Sequence analysis using the Pfam server identified a low stringency match to a fatty acid desaturase domain in the N-terminal sequence of CLN3P. We developed a fatty acid desaturase assay based on measurement of desaturase products by gas chromatography/mass spectrometry. RESULTS: We show that CLN3P is a novel palmitoyl-protein Delta-9 desaturase, which converts membrane-associated palmitoylated proteins to their respective palmitoleated derivatives. We have further demonstrated that this palmitoyl-protein Delta-9 desaturase activity is deficient in cln3(-/-) mouse pancreas and is completely ablated in neuroblastoma cells by RNA inhibition. INTERPRETATION: We propose that palmitoyl-protein desaturation defines a new mechanism of proteolipid modification, and that deficiency of this process leads to the signs and symptoms of Batten's disease.

Over-expression of CLN3P, the Batten disease protein, inhibits PANDER-induced apoptosis in neuroblastoma cells: further evidence that CLN3P has anti-apoptotic properties.

Juvenile neuronal ceroid-lipofuscinosis (JNCL) or Batten/Spielmeyer-Vogt-Sjogren disease (OMIM #204200) is one of a group of nine clinically related inherited neurodegenerative disorders (CLN1-9). JNCL results from mutations in CLN3 on chromosome 16p12.1. The neuronal loss in Batten disease has been shown to be due to a combination of apoptosis and autophagy suggesting that CLN3P, the defective protein, may have an anti-neuronal death function. PANDER (PANcreatic-DERived factor) is a novel cytokine that was recently cloned from pancreatic islet cells. PANDER is specifically expressed in the pancreatic islets, small intestine, testis, prostate, and neurons of the central nervous system, and has been demonstrated to induce apoptosis. In this study, we over-expressed CLN3P in SH-SY5Y neuroblastoma cells and monitored the effects on PANDER-induced apoptosis. CLN3P significantly increased the survival rate of the SH-SY5Y cells in this system. This study provides additional evidence that the function of CLN3P is related to preventing neuronal apoptosis.

Regulation of oxidative stress by the anti-aging hormone klotho.

klotho is an aging suppressor gene and extends life span when overexpressed in mice. Klotho protein was recently demonstrated to function as a hormone that inhibits insulin/insulin-like growth factor-1 (IGF-1) signaling. Here we show that Klotho protein increases resistance to oxidative stress at the cellular and organismal level in mammals. Klotho protein activates the FoxO forkhead transcription factors that are negatively regulated by insulin/IGF-1 signaling, thereby inducing expression of manganese superoxide dismutase. This in turn facilitates removal of reactive oxygen species and confers oxidative stress resistance. Thus, Klotho-induced inhibition of insulin/IGF-1 signaling is associated with increased resistance to oxidative stress, which potentially contributes to the anti-aging properties of klotho.

Suppression of aging in mice by the hormone Klotho.

A defect in Klotho gene expression in mice accelerates the degeneration of multiple age-sensitive traits. Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.

CLN3L, a novel protein related to the Batten disease protein, is overexpressed in Cln3-/- mice and in Batten disease.

Batten disease is a severe autosomal recessive neurodegenerative disease which results from mutations in CLN3. Although the gene was cloned in 1995, the tissue distribution and subcellular localization of the CLN3 protein (CLN3P) remains inconclusive. We have demonstrated the presence of a novel 33 kDa protein in both normal human and wild-type mouse brain. This 33 kDa protein, which is overexpressed in brains of patients with Batten disease and in Cln3-/- mouse brain, binds to the antibody raised against the peptide sequence of CLN3P and results in aberrant CLN3P localization studies. We expressed a novel 33 kDa protein that is highly similar to CLN3P. We showed that the 33 kDa protein is identical to that recognized in Batten disease and Cln3-/- brain. These studies strongly suggest the presence of an alternative CLN3-like (CLN3L) product in Batten disease. Previous studies of CLN3P tissue distribution and intracellular localization will require extensive reanalysis in order to determine the true expression of CLN3P.

CLN3P, the Batten disease protein, localizes to membrane lipid rafts (detergent-resistant membranes).

Juvenile neuronal ceroid lipofuscinosis is an inherited pediatric neurodegenerative disorder, which occurs as a result of mutations in the CLN3 gene that is located on chromosome 16p12.1. The encoded protein, CLN3P, is a putative transmembrane protein with no known function. In this study, we demonstrate that CLN3P resides on membrane lipid raft domains (detergent-resistant membranes) and provide important new data towards possible functions of the protein.