Klotho Supplementation Reverses Renal Dysfunction and Interstitial Fibrosis in Remnant Kidney.

INTRODUCTION: While recent investigations show that klotho exerts renoprotective actions, it has not been fully addressed whether klotho protein supplementation reverses renal damage. METHODS: The impacts of subcutaneous klotho supplementation on rats with subtotal nephrectomy were examined. Animals were divided into 3 groups: group 1 (short remnant [SR]): remnant kidney for 4 weeks, group 2 (long remnant [LR]): remnant kidney for 12 weeks, and group 3 (klotho supplementation [KL]): klotho protein (20 µg/kg/day) supplementation on the remnant kidney. Blood pressure, blood and urine compositions with conventional methods such as enzyme-linked immunosorbent assay and radioimmunoassay, kidney histology, and renal expressions of various genes were analyzed. In vitro studies were also performed to support in vivo findings. RESULTS: Klotho protein supplementation decreased albuminuria (-43%), systolic blood pressure (-16%), fibroblast growth factor (FGF) 23 (-51%) and serum phosphate levels (-19%), renal angiotensin II concentration (-43%), fibrosis index (-70%), renal expressions of collagen I (-55%), and transforming growth factor ß (-59%) (p < 0.05 for all). Klotho supplementation enhanced fractional excretion of phosphate (+45%), glomerular filtration rate (+76%), renal expressions of klotho (+148%), superoxide dismutase (+124%), and bone morphogenetic protein (BMP) 7 (+174%) (p < 0.05 for all). CONCLUSION: Our data indicated that klotho protein supplementation inactivated renal renin-angiotensin system, reducing blood pressure and albuminuria in remnant kidney. Furthermore, exogenous klotho protein supplementation elevated endogenous klotho expression to increase phosphate excretion with resultant reductions in FGF23 and serum phosphate. Finally, klotho supplementation reversed renal dysfunction and fibrosis in association with improved BMP7 in remnant kidney.

Klotho supplementation attenuates blood pressure and albuminuria in murine model of IgA nephropathy.

BACKGROUND: Klotho interacts with various membrane proteins, such as transforming growth factor-ß (TGFß) and insulin-like growth factor (IGF) receptors. The renal expression of klotho is diminished in chronic kidney disease. METHOD: In this study, we assessed the effects of klotho supplementation on a murine model of IgA nephropathy. Twenty-four-week-old hyper serum IgA (HIGA) mice were subcutaneously injected daily with recombinant human klotho protein (20 µg/kg per day) or the vehicle. After 2 months, the mice were killed using an anesthesia overdose and their kidneys were harvested for analysis. RESULTS: Supplementation of exogenous klotho protein reduced SBP, albuminuria, 8-epi-prostaglandin F2α excretion, glomerular filtration rate, renal angiotensin II concentration, and angiotensinogen expression in HIGA mice. Additionally, it enhanced renal expression of superoxide dismutase (SOD) and renal klotho itself. The findings using laser-manipulated microdissection demonstrated that klotho supplementation reduced the glomerular expression of TGFß, fibronectin, and IGF, and increased the glomerular expression of connexin (Cx) 40. CONCLUSION: These results indicate that klotho supplementation reduces blood pressure by suppressing the renin--angiotensin system in HIGA mice. Klotho inhibits IGF signaling to preserve glomerular Cx40 levels, ameliorating albuminuria in HIGA mice. Klotho protein supplementation attenuates mesangial expansion by inhibiting TGFß signaling in HIGA mice.

Klotho Ameliorates Medullary Fibrosis and Pressure Natriuresis in Hypertensive Rat Kidneys.

Renal expression of klotho is reduced in hypertension. Experiments were performed to examine whether exogenous klotho protein supplementation ameliorates pressure natriuresis in early phase of hypertension, using stroke-prone spontaneously hypertensive rats (sp-SHR). The interactions between klotho protein and renal renin-Ang (angiotensin) system were examined with immunoprecipitation and cell culture methods. Uninephrectomy was performed in sp-SHRs to induce nephrosclerosis, and they were treated with exogenous klotho protein or vehicle. Exogenous klotho protein supplementation to sp-SHR decreased blood pressure, renal Ang II levels, AGT (angiotensinogen) expression, HIF (hypoxia-inducible factor)-1α abundance, and medullary fibronectin levels, with increased renal klotho expression and serum and urine klotho levels. Klotho supplementation also reduced kidney weight, renal phosphorylated Akt, and mTOR (mammalian target of rapamycin) abundance. Furthermore, klotho supplementation restored renal autoregulation of glomerular filtration rate and enhanced pressure-induced natriuresis in sp-SHR. Klotho protein bound to AT1R (Ang II type-1 receptor) and decreased the presence of AT1R on HK-2 (human proximal tubular) cells, attenuating inositol triphosphate generation. Klotho protein suppressed Ang II-induced increments of AGT expression in HK-2 cells. Collectively, the present data demonstrate that klotho binds with the AT1R to suppress Ang signal transduction, participating in inactivating renal renin-Ang system. Our results also suggest that exogenous klotho supplementation represses Akt-mTOR signaling to reduce renal hypertrophy and restore the autoregulatory ability of glomerular filtration rate in uninephrectomized sp-SHRs. Finally, the present findings implicate that klotho supplementation inhibits HIF-1α pathway and medullary fibrosis, contributing to enhancements of pressure natriuresis and reduction in blood pressure.

Clostridium difficile infection induced by pregabalin-associated agranulocytosis.

A 33-year-old man who had recently undergone surgery for cervical spondylotic myelopathy was prescribed pregabalin for neuralgia, and the dose was increased to 600 mg/day during hospitalization. However, the patient was diagnosed with a Clostridium difficile infection on day 34 after admission. A complete blood count showed agranulocytosis (neutrophil count: 105/µL). We did not observe any changes in vital signs, a relative increase in band cells, or intestinal edema. The patient's agranulocytosis resolved after withdrawing pregabalin. This is the first reported case of agranulocytosis associated with pregabalin. Periodic monitoring of the white blood cell count is therefore considered to be useful in patients receiving high-dose pregabalin therapy.

Calcitriol supplementation improves endothelium-dependent vasodilation in rat hypertensive renal injury.

BACKGROUND/AIMS: Vitamin D increases renal expression of klotho in normotensive rats. Klotho reduces oxidative stress. METHODS: In this study, we aimed to determine if vitamin D would suppress oxidative stress using 4 groups of hypertensive rats: uninephrectomized, stroke-prone, spontaneously hypertensive rats fed a high-salt (6%) diet (controls; C); those treated with irbesartan (I); those treated with calcitriol (V); and those treated with both irbesartan and calcitriol (I+V). RESULTS: Systolic blood pressure was higher in the C group than in the I and I+V groups. Albuminuria was attenuated in groups I, V, and I+V. Renal angiotensin II (AngII) concentration was lower in groups I and I+V than in group C, and plasma AngII levels of groups I and V were higher and lower than those in group C, respectively. Compared with group C, renal klotho expression, 8-epi-prostaglandin F2α excretion, and acetylcholine-induced decrease in blood pressure improved in the V and I+V groups. CONCLUSIONS: The data indicate that irbesartan effectively decreases blood pressure and renal AngII levels, and improves albuminuria. Our findings indicate that vitamin D enhances klotho expression, suppressing oxidative stress and albuminuria without substantial changes in renal AngII levels. These results suggest that the amelioration of endothelium function by vitamin D involves free klotho.

Close association of vascular and valvular calcification and prognosis of patients on continuous ambulatory peritoneal dialysis.

In the present study, we examined the association between vascular and valvular calcification and the prognosis of patients on continuous ambulatory peritoneal dialysis (CAPD). Data were collected from the records of patients introduced onto CAPD therapy during 1999 - 2006 at the Department of Nephrology, Saitama Medical University. At the start of CAPD, cardiac and vascular echography were used to examine 162 patients (average age: 56 +/- 5 years; 58 men, 104 women; 43 with and 119 without diabetes) for evaluation of vascular and valvular calcification. Both vascular and valvular calcification were found in 32 patients. Vascular calcification was found in 16, and valvular calcification in 11. Over 5 years, 11 patients suffered from cardiovascular disease (7 with stroke, 4 with myocardial infarction). All of these patients had vascular or valvular calcification at the start of CAPD therapy. We also used Cox hazard analysis to examine values for Ca, P, Ca x P, intact parathyroid hormone (iPTH), and lipids. None of these values were independent contributory factors for incidence of cardiovascular disease in patients on CAPD. These data suggest the importance of vascular and valvular echography to evaluate patients on CAPD, especially at the start of CAPD therapy. Vascular and valvular calcification are important factors for determining the prognosis of patients on CAPD.

Differences in corrective mode for divalent ions and parathyroid hormone between standard- and low-calcium dialysate in patients on continuous ambulatory peritoneal dialysis--result of a nationwide survey in Japan.

BACKGROUND: In patients on continuous ambulatory peritoneal dialysis (CAPD), dialysate calcium concentration has a strong influence on correction of serum calcium, phosphorus, and parathyroid hormone (PTH); however, the optimal concentration of Ca in PD solution is still uncertain. The aim of the survey reported here was to evaluate the prevalence of patients treated with standard- [SCD (approximately 3.25 - 4.0 mEq/L)] or low-calcium [LCD (approximately 1.8 - 2.5 mEq/L)] dialysate and differences in the clinical effects for correction of abnormalities in divalent ions and PTH. MATERIALS AND METHODS: We used a questionnaire to survey 333 peritoneal dialysis facilities nationwide in Japan. Then, we analyzed serum Ca, P, and PTH levels and the prescription rates for CaCO(3) as a P binder and for vitamin D (VitD) analogs. RESULTS: The 2384 CAPD patients enrolled in this analysis had a mean age of 60.5 +/- 14.2 years and a mean duration of CAPD of 44.1 +/- 39.2 months. The prevalences of SCD, LCD, and combination of SCD and LCD were, respectively, 49%, 50%, and 1% at initiation, and 40%, 38%, and 22% at the time of the survey. In 735 and 876 patients respectively, LCD and SCD had been prescribed from initiation to the time of the survey. In these two groups, we observed no difference in initiation and current serum levels of Ca and P. But prescription rates for CaCO(3) and VitD analogs were higher in the LCD group than in the SCD group, and PTH levels were higher in the LCD group than in the SCD group. CONCLUSIONS: A beneficial effect of LCD was revealed in the increased doses of CaCO(3) and VitD analogs seen in that group without the occurrence of hypercalcemia; however, PTH levels in that group were not maintained within an acceptable range. The survey suggests that more serious attention should be paid to the Ca concentration in peritoneal dialysate so as to lessen mineral and PTH disorders in CAPD.

Young hemodialysis patients are exposed to hyperhomocysteinemia.

OBJECTIVE: Homocysteine is one of the cardiovascular risk factors in hemodialysis (HD) patients. Studies were performed to assess the effects of folic acid on pulse wave velocity (PWV) in HD patients. METHODS: In a cross-sectional study, plasma total homocysteine (tHcy) was measured in 49 patients on maintenance HD. Ten HD patients younger than 45 years old entered the prospective study. Monthly changes in PWV were compared before and during folic acid treatment. RESULTS: Younger HD patients had higher tHcy (r = -0.53, n = 49, P < .001). Patients who manifested myocardial ischemia (37 +/- 3 nmol/mL) possessed higher tHcy than those who did not (30 +/- 3 nmol/mL, P < .05). In prospective study, folic acid treatment (10 to 20 mg/d) failed to alter blood pressure and biochemical parameters, including lipids, calcium, phosphate, and parathormone. However, in association with a decrease in tHcy (46 +/- 5 to 27 +/- 3 nmol/mL, n = 10, P < .005), progressive increases in PWV (33 +/- 8 to 3 +/- 6 cm/sec/month, P < .01) were stopped. CONCLUSIONS: The present findings indicate that young HD patients are exposed to severe hyperhomocysteinemia, and suggest that relatively large doses of folic acid attenuate progressive increases in PWV of young or middle-age HD patients.

Peritoneal dialysis versus hemodialysis: a five-year comparison of survival and effects on the cardiovascular system, erythropoiesis, and calcium metabolism.

The influence that the mode of dialysis has on the prognosis of patients with renal disease is controversial. The controversy arises at least in part because of the heterogeneity of patient populations, who may be receiving either continuous ambulatory peritoneal dialysis (CAPD) or hemodialysis (HD). In the absence of randomized trials, epidemiologic investigations present the best method for studying the problem. To determine the influence of the mode of dialysis on prognosis and on the cardiovascular system, erythropoiesis, and calcium metabolism, we selected 36 patients undergoing CAPD and 36 patients undergoing HD for a 3-year follow-up study. Patients were matched for age, sex, and cause of renal disease. Among the HD patients, 8 deaths occurred from congestive heart failure, 1 death from cerebrovascular accident, and 2 deaths from severe infection. In the HD group, the average age was 63 +/- 3 years. Among the CAPD patients, 6 were transferred to HD because of recurrent peritonitis or elevation of serum creatinine. Patients on CAPD had lower blood pressures, and patients on HD had lower total cholesterol levels. Other parameters were not significantly different between the two groups, including dose of erythropoietin and calcium supplements administered. Our study provides evidence that clinical outcome in renal failure may depend to some extent on the mode of dialysis. Our results suggest that blood pressure level and serum cholesterol should be taken into account for patients treated with either CAPD or HD. Blood pressure and cholesterol level are both likely to be important contributors to mortality and morbidity in renal patients.