Calpain 1 inhibitor BDA-410 ameliorates α-klotho-deficiency phenotypes resembling human aging-related syndromes.

Taking good care of elderly is a major challenge of our society, and thus identification of potential drug targets to reduce age-associated disease burden is desirable. α-klotho(-/-) (α-kl) is a short-lived mouse model that displays multiple phenotypes resembling human aging-related syndromes. Such ageing phenotype of α-kl(-/-) mice is associated with activation of a proteolytic enzyme, Calpain-1. We hypothesized that uncontrolled activation of calpain-1 might be causing age-related phenotypes in α-kl-deficient mice. We found that daily administration of BDA-410, a calpain-1 inhibitor, strikingly ameliorated multiple aging-related phenotypes. Treated mice showed recovery of reproductive ability, increased body weight, reduced organ atrophy, and suppression of ectopic calcifications, bone mineral density reduction, pulmonary emphysema and senile atrophy of skin. We also observed ectopic expression of FGF23 in calcified arteries of α-kl(-/-) mice, which might account for the clinically observed association of increased FGF23 level with increased risk of cardiovascular mortality. These findings allow us to propose that modulation of calpain-1 activity is a potential therapeutic option for delaying age-associated organ pathology, particularly caused by the dysregulation of mineral ion homeostasis.

The role of Klotho in energy metabolism.

A disproportionate expansion of white adipose tissue and abnormal recruitment of adipogenic precursor cells can not only lead to obesity but also impair glucose metabolism, which are both common causes of insulin resistance and diabetes mellitus. The development of novel and effective therapeutic strategies to slow the progression of obesity, diabetes mellitus and their associated complications will require improved understanding of adipogenesis and glucose metabolism. Klotho might have a role in adipocyte maturation and systemic glucose metabolism. Klotho increases adipocyte differentiation in vitro, and mice that lack Klotho activity are lean owing to reduced white adipose tissue accumulation; moreover, mice that lack the Kl gene (which encodes Klotho) are resistant to obesity induced by a high-fat diet. Knockout of Kl in leptin-deficient Lep(ob/ob) mice reduces obesity and increases insulin sensitivity, which lowers blood glucose levels. Energy metabolism might also be influenced by Klotho. However, further studies are needed to explore the possibility that Klotho could be a novel therapeutic target to reduce obesity and related complications, and to determine whether and how Klotho might influence the regulation and function of a related protein, ß-Klotho, which is also involved in energy metabolism.

FGF23, klotho and vitamin D interactions: What have we learned from in vivo mouse genetics studies?

The molecular interactions of fibroblast growth factor 23 (FGF23), klotho and vitamin D coordinate to regulate the delicate phosphate levels of the body. Vitamin D can induce both FGF23 and klotho synthesis to influence renal phosphate balance. In the presence of klotho, FGF23 protein gains bioactivity to influence systemic phosphate homeostasis. Experimental studies have convincingly shown that in the absence of klotho, FGF23 is unable to regulate in vivo phosphate homeostasis. Furthermore, genetic inactivation of FGF23, klotho or both of the genes have resulted in markedly increased renal expression of 1-alpha hydroxylase [1α(OH)ase] and concomitant elevated serum levels of 1,25, dihydroxyvitamin D [1,25(OH)(2)D] in the mutant mice. Vitamin D can induce the expression of both FGF23 and klotho while, FGF23 can suppress renal expression of 1α(OH)ase to reduce 1,25(OH)(2)D activity. In this brief chapter, I will summarize the possible in vivo interactions of FGF23, klotho and vitamin D, in the light of recent mouse genetics studies.

Genetic induction of phosphate toxicity significantly reduces the survival of hypercholesterolemic obese mice.

OBJECTIVE: The adverse effects of metabolic disorders in obesity have been extensively studied; however, the pathologic effects of hyperphosphatemia or phosphate toxicity in obesity have not been studied in similar depth and detail, chiefly because such an association is thought to be uncommon. Studies have established that the incidence of obesity-associated nephropathy is increasing. Because hyperphosphatemia is a major consequence of renal impairment, this study determines the in vivo effects of hyperphosphatemia in obesity. METHODS AND RESULTS: We genetically induced hyperphosphatemia in leptin-deficient obese (ob/ob) mice by generating ob/ob and klotho double knockout [ob/ob-klotho(-/-)] mice. As a control, we made ob/ob mice with hypophosphatemia by generating ob/ob and 1-alpha hydroxylase double knockout [ob/ob-1α(OH)ase(-/-)] mice. Compared to the wild-type mice, all three obese background mice, namely ob/ob, ob/ob-klotho(-/-), and ob/ob-1α(OH)ase(-/-) mice developed hypercholesterolemia. In addition, the hyperphosphatemic, ob/ob-klotho(-/-) genetic background induced generalized tissue atrophy and widespread soft-tissue and vascular calcifications, which led to a shorter lifespan; no such changes were observed in the hypophosphatemic, ob/ob-1α(OH)ase(-/-) mice. Significantly, in contrast to the reduced survival of the ob/ob-klotho(-/-) mice, lowering serum phosphate levels in ob/ob-1α(OH)ase(-/-) mice showed no such compromised survival, despite both mice being hypercholesterolemic. CONCLUSION: These genetic manipulation studies suggest phosphate toxicity is an important risk factor in obesity that can adversely affect survival.

Dietary and genetic evidence for enhancing glucose metabolism and reducing obesity by inhibiting klotho functions.

Klotho is a multifunctional protein involved in numerous biological functions, ranging from mineral ion metabolism to signaling activities. Recent studies have identified klotho as a target gene for peroxisome proliferator-activated receptor-γ (PPAR-γ), a master regulator of adipocyte differentiation, and an adipogenesis-promoting factor. In a similar line of observation, eliminating klotho function from mice resulted in the generation of lean mice with almost no detectable fat tissue. In contrast to the klotho-knockout mice (11.7±0.3 g at 9 wk), leptin-deficient (ob/ob) mice are severely obese (49.3±0.6 g at 9 wk), due to excessive fat accumulation. To study the in vivo role of klotho in obesity, we have generated and characterized ob/ob mice lacking klotho activity [ob/ob-klotho double-knockout (DKO) mice]. The ob/ob mice started to get bigger from 3 wk onward and gained almost 2 times more weight than their wild-type (WT) counterparts (WT vs. ob/ob: 34.8±1.3 vs. 65.5±1.2 g at 21 wk). The generated ob/ob-klotho DKO mice were not only viable throughout their adulthood but also showed markedly reduced fat tissue accumulation compared to their ob/ob littermates. The ob/ob-klotho DKO mice had significantly (P<0.01) less retroperitoneal, mesenteric, and epididymal fat accumulation, compared to their ob/ob counterparts. Similarly, the fatty liver that was consistently observed in the ob/ob mice was eliminated in the ob/ob-klotho DKO mice. Such structural improvement in the liver was also evident from markedly reduced fasting blood glucose levels in ob/ob-klotho DKO mice, compared to their ob/ob counterparts (ob/ob vs. ob/ob-klotho DKO: 266 ± 36 vs. 65±2 mg/dl). Finally, to study whether the absence of klotho can induce resistance to high-fat-diet-induced obesity, we provided a high-fat (60%) diet to klotho-knockout mice and compared them with normal-fat (20%) diet-fed klotho-knockout mice. No significant difference in body weight was detected in klotho-knockout mice fed either the normal-fat diet or high-fat diet, while WT mice fed the high-fat diet gradually gained body weight, compared to the normal-fat-diet-fed counterparts. The results of our dietary and genetic manipulation studies provide in vivo evidence for a role of klotho in obesity and offer a novel target to manipulate obesity and associated complications.

The dualistic role of vitamin D in vascular calcifications.

Vitamin D is a multifunctional hormone that can affect many essential biological functions, ranging from the immune regulation to mineral ion metabolism. A close association between altered activity of vitamin D and vascular calcification has been reported in various human diseases, including in patients with atherosclerosis, osteoporosis, and chronic kidney disease (CKD). Vascular calcification is a progressive disorder and is a major determinant of morbidity and mortality of the affected patients. Experimental studies have shown that excessive vitamin D activities can induce vascular calcification, and such vascular pathology can be reversed by reducing vitamin D activities. The human relevance of these experimental studies is not clear, as vitamin D toxicity is relatively rare in the general population. Contrary to the relationship between vitamin D and vascular calcification, in experimental uremic models, low levels of vitamin D were shown to be associated with extensive vascular calcification, a phenomenon that is very similar to the vascular pathology seen in patients with CKD. The current treatment approach of providing vitamin D analogs to patients with CKD often poses a dilemma, as studies linked vitamin D treatment to subsequent vascular calcification. Recent genetic studies, however, have shown that vascular calcification can be prevented by reducing serum phosphate levels, even in the presence of extremely high serum 1,25-dihydroxyvitamin D and calcium levels. This article will briefly summarize the dual effects of vitamin D in vascular calcification and will provide evidence of vitamin D-dependent and -independent vascular calcification.

Phosphate toxicity: new insights into an old problem.

Phosphorus is an essential nutrient required for critical biological reactions that maintain the normal homoeostatic control of the cell. This element is an important component of different cellular structures, including nucleic acids and cell membranes. Adequate phosphorus balance is vital for maintaining basic cellular functions, ranging from energy metabolism to cell signalling. In addition, many intracellular pathways utilize phosphate ions for important cellular reactions; therefore, homoeostatic control of phosphate is one of the most delicate biological regulations. Impaired phosphorus balance can affect the functionality of almost every human system, including musculoskeletal and cardiovascular systems, ultimately leading to an increase in morbidity and mortality of the affected patients. Human and experimental studies have found that delicate balance among circulating factors, like vitamin D, PTH (parathyroid hormone) and FGF23 (fibroblast growth factor 23), are essential for regulation of physiological phosphate balance. Dysregulation of these factors, either alone or in combination, can induce phosphorus imbalance. Recent studies have shown that suppression of the FGF23-klotho system can lead to hyperphosphataemia with extensive tissue damage caused by phosphate toxicity. The cause and consequences of phosphate toxicity will be briefly summarized in the present review.

Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.

Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging.

Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid-base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klotho-knockout mice (klotho(-/-)) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium-phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho(-/-) mice. Genetically reducing serum phosphate levels in klotho(-/-) mice by generating a NaPi2a and klotho double-knockout (NaPi2a(-/-)/klotho(-/-)) strain resulted in amelioration of premature aging-like features. The NaPi2a(-/-)/klotho(-/-) double-knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a(-/-)/klotho(-/-) mice by feeding with a high-phosphate diet, premature aging-like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho(-/-) mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.

In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels.

BACKGROUND: Klotho-knockout mice (klotho(-/-)) have increased renal expression of sodium/phosphate cotransporters (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. CONCLUSIONS: Our in vivo genetic manipulation studies have provided compelling evidence for a pathological 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 patients with chronic kidney disease with cardiovascular calcification.

The FGF23-Klotho axis: endocrine regulation of phosphate homeostasis.

Appropriate levels of phosphate in the body are maintained by the coordinated regulation of the bone-derived growth factor FGF23 and the membrane-bound protein Klotho. The endocrine actions of FGF23, in association with parathyroid hormone and vitamin D, mobilize sodium-phosphate cotransporters that control renal phosphate transport in proximal tubular epithelial cells. The availability of an adequate amount of Klotho is essential for FGF23 to exert its phosphaturic effects in the kidney. In the presence of Klotho, FGF23 activates downstream signaling components that influence the homeostasis of phosphate, whereas in the absence of this membrane protein, it is unable to exert such regulatory effects, as demonstrated convincingly in animal models. Several factors, including phosphate and vitamin D, can regulate the production of both FGF23 and Klotho and influence their functions. In various acquired and genetic human diseases, dysregulation of FGF23 and Klotho is associated with vascular and skeletal anomalies owing to altered phosphate turnover. In this Review, I summarize how the endocrine effects of bone-derived FGF23, in coordination with Klotho, can regulate systemic phosphate homeostasis, and how an inadequate balance of these molecules can lead to complications that are caused by abnormal mineral ion metabolism.

Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model.

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of fibroblast growth factor (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a cofactor of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15-20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase.

Changes in the expression of klotho, a beta-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 1alpha-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 1alpha-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.

In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis.

A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis.

FGF23-mediated regulation of systemic phosphate homeostasis: is Klotho an essential player?

Understanding the physiological regulation of mineral ion metabolism is essential for determining the pathomechanisms of skeletal, vascular, and renal diseases associated with an abnormal regulation of calcium and phosphate homeostasis. Normal calcium and phosphate balance is delicately maintained by endocrine factors that coordinate to influence the functions of the intestine, bone, parathyroid gland, and kidney. Under physiological conditions, the kidneys play an important role in maintaining normal mineral ion balance by fine-tuning the amount of urinary excretion of calcium and phosphate according to the body's needs. Fibroblast growth factor (FGF)23 regulates urinary phosphate excretion to maintain systemic phosphate homeostasis. The exact mode of action of the phosphaturic effects of FGF23 is not fully understood and is an intense area of research. Studies suggest, however, that FGF23, by interacting with FGF receptors, can initiate downstream signaling events and that Klotho, a transmembrane protein, facilitates the interaction of FGF23 with its receptor. FGF23 can inhibit the activities of 1-alpha-hydroxylase and sodium-phosphate cotransporter in the kidney to influence the overall systemic phosphate balance. This article briefly summarizes how FGF23 might coordinately regulate systemic phosphate homeostasis and how Klotho is involved in such regulation.

Does Fgf23-klotho activity influence vascular and soft tissue calcification through regulating mineral ion metabolism?

Recent studies describe a novel role of fibroblast growth factor-23 (Fgf23)-klotho activity in the systemic regulation of calcium and phosphate homeostasis. Both Fgf23 and klotho ablated mice develop extensive vascular and soft tissue calcification. Inability to clear the required amount of phosphate by the kidney, due to the absence of Fgf23-klotho activity, leads to increased accumulation of serum phosphate in these genetically modified mice, causing extensive calcification. Serum calcium and 1,25 hydroxyvitamin D levels are also elevated in both Fgf23 and klotho ablated mice. Moreover, increased sodium phosphate co-transporter activity in both Fgf23 and klotho ablated mice increases renal phosphate reabsorption which in turn can facilitate calcification. Collectively, these observations bring new insights into our understanding of the roles of the Fgf23-klotho axis in the development of vascular and soft tissue calcification.

Genetic suppression of GH-IGF-1 activity, combined with lifelong caloric restriction, prevents age-related renal damage and prolongs the life span in rats.

AIM: The aim of this study was to determine the effects of kidney pathology on overall survival and longevity and the combined effects of chronic suppression of growth hormone (GH)/insulin-like growth factor-1 (IGF-1) activity and lifelong caloric restriction on age-associated nephropathy. METHODS: We analyzed the kidneys of rats with suppressed GH activity through genetic manipulation with an antisense GH transgene. Rats were fed normally or with a 30% calorie-restricted diet for 24-26 months. The kidneys of male wild-type young (6 months) and old (24-26 months) rats were compared with male hemizygote transgenic young (6 months) and old (24-26 months) rats fed with either regular diet or 30% calorie-restricted diet for their entire life span. RESULTS: The transgenic rats had relatively less pituitary GH-secreting cells, and the plasma levels of IGF-1 were decreased by 53% in homozygote rats (tg/tg) and by 28% in hemizygote rats (tg/wt) compared to wild-type rats (wt/wt) of the same age (6 months). Wild-type rats fed the regular diet developed age-associated nephropathy as they aged, showing severe inflammatory cell infiltration, glomerulosclerosis, and tubulointerstitial fibrosis. In addition, about 83% of the wild-type rats allowed to survive naturally showed signs of nephropathy. In contrast, only 26% of the naturally surviving hemizygote rats showed features of nephropathy, despite the fact that these rats lived 8% longer (maximum survival 171 weeks) than the wild-type rats (maximum survival 158 weeks). When chronic suppression of GH/IGF-1 activity was combined with lifelong caloric restriction, however, age- associated nephropathy was nonexistent in hemizygote transgenic rats, and they showed about 30% increase in survival (maximum survival 204 weeks). There was no significant difference in the rate of neoplastic or nonneoplastic lesions (other than in the kidney) in the regularly fed wild-type rats or in the calorie-restricted hemizygote transgenic rats that survived longer. CONCLUSION: We concluded that kidney pathology is an important determinant of overall survival, and that prevention of kidney pathology by dietary restriction, combined with chronic suppression of GH/IGF-1 activity, significantly extends overall survival and longevity.

Low-dose local kidney irradiation inhibits progression of experimental crescentic nephritis by promoting apoptosis.

BACKGROUND: Crescentic glomerulonephritis is a rapidly progressive form of nephritis and is usually resistant to therapeutic intervention. Apoptosis plays a role in the resolution of glomerulonephritis. We investigated the effects of local kidney irradiation on the progression of experimental crescentic glomerulonephritis. METHODS: The following three experimental rat groups were generated: (1) Group I, sham-operated control (n = 12); (2) Group II, rats injected intravenously with rabbit anti-rat GBM antibody (nephrotoxic serum, NTS) (n = 23), and (3) Group III, a single low-dose irradiation of 0.5 Gy X-ray to both kidneys at days 6, 13, 20, and 27 after NTS injection (n = 55). Renal function and blood leukocyte count were examined in different groups of rats at various time points. Kidneys obtained at various time points were analyzed to determine the effects of radiation in experimental nephritis. RESULTS: Radiation of the kidneys reduced the levels of blood urea nitrogen and serum creatinine compared with Group II nephritic rats of similar age (p < 0.05 or 0.001). No apparent changes in blood leukocyte counts were noted in various experimental groups. Glomerular hypercellularity, crescents, global sclerosis and tubulointerstitial damage developed gradually in Group II rats, but were decreased (p < 0.05 or 0.001) after radiation treatment. The extent of tubulointerstitial damage was also reduced, and radiation-associated histological improvements were accompanied by reduced infiltration of macrophages in the glomeruli and interstitium. The numbers of PCNA- and ED1-positive cells were reduced in the kidneys at 1 day post-irradiation, of rats irradiated at 6 and 13 days after NTS injection, compared with Group II at similar time intervals (p < 0.05). A larger numbers of TUNEL-positive cells were noted at 1 day post-irradiation in rats irradiated at 6 and 13 days after NTS injection, compared with Group II at similar time intervals (p < 0.05). Immunostaining for macrophages ED1 and TUNEL staining of serial sections of irradiated nephritic kidneys showed few ED1-positive macrophages stained for TUNEL. Overexpression of active caspases 3 and 7 was noted in irradiated kidneys, compared with the corresponding Group II rats at similar time intervals. Western blot analysis showed marked increase in active caspase 3 and active caspase 7 expression in irradiated kidneys compared with NTS injection only. A marked increase in the expression of p53 protein, which is closely related to radiation-induced apoptosis, was also observed in irradiated kidneys compared with NTS injection only. CONCLUSION: Our study showed that renal radiation can alter acute glomerular inflammation by inducing apoptosis of intrinsic and infiltrating cells in the kidney in a rat model of crescentic glomerulonephritis. Low-dose kidney irradiation can inhibit the progression of experimental nephritis through inducing apoptosis.