Michihiro Mitobe

Oxidative Stress Decreases Klotho Expression in a Mouse Kidney Cell Line

Background/Aims: Defects in klotho gene expression in the mouse result in a syndrome that resembles human aging. We recently identified expression of klotho in a mouse inner medullary collecting duct (mIMCD3) cell line for the first time, and in the present study we explored the physiological relevance of the regulation of klotho expression in the presence of oxidant stress injury. Methods: Klotho expression was analyzed by real-time PCR, Western blot, and immuocytochemical staining during exposure to hydrogen peroxide (H2O2). Overexpression of the klotho gene was induced by klotho adenoviruses, and the number of apoptotic cells was counted by flowcytometry. Results: Oxidant stress injury by H2O2 dose-dependently reduced klotho expression and diminished klotho staining. There were fewer apoptotic cells among the klotho-transfected cells than among the control cells. Conclusion: Klotho is expressed in cell line mIMCD3, and the klotho gene may be involved in the process of oxidative stress injury and apoptosis in this cell line.

Reduced klotho expression level in kidney aggravates renal interstitial fibrosis

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β(1) (TGF-β(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.

Klotho reduces apoptosis in experimental ischaemic acute kidney injury via HSP-70

BACKGROUND High Klotho expression has been detected in the kidney, and since the results of a recent study suggested that Klotho induction mitigates ischaemic damage in the kidney, in the present study we explored the mechanism by which Klotho expression reduces renal ischaemia-reperfusion injury (IRI). METHODS Male mice were subjected to bilateral renal ischaemia for 30 min and reperfusion for 24 h, or to a sham operation. Both the IRI group and the sham group were intravenously injected with an adenovirus harbouring the mouse Klotho gene (ad-kl) before renal IRI. In addition, mIMCD3 cells induced to overexpress Klotho by transferring the Klotho gene with ad-kl were analysed by DNA microarray and real-time PCR. Renal expression of Klotho and several genes selected by DNA microarray were assessed by real-time PCR or Western blotting, and TUNEL staining was performed to assess apoptosis. RESULTS Prior administration of ad-kl to the mice resulted in robust induction of Klotho mRNA in the kidney and liver. Ad-kl transfer improved the plasma creatinine values and mitigated the histological damage and apoptosis induced by IRI. Expression of several genes was altered in mIMCD3 cells as a result of the change in Klotho expression, and expression of heat shock protein 70 (HSP70), in particular, was up-regulated in ad-kl mouse kidneys and HK2 cells. CONCLUSION The results suggest that Klotho is involved in the pathophysiology of IRI. Klotho mitigates apoptosis in experimental ischaemic acute kidney injury via expression of HSP70.

ATF3 Protects against Renal Ischemia-Reperfusion Injury

Oxidative stress-induced cell death plays a major role in the progression of ischemic acute renal failure. Using microarrays, we sought to identify a stress-induced gene that may be a therapeutic candidate. Human proximal tubule (HK2) cells were treated with hydrogen peroxide (H2O2) and RNA was applied to an Affymetrix gene chip. Five genes were markedly induced in a parallel time-dependent manner by cluster analysis, including activating transcription factor 3 (ATF3), p21(WAF1/CiP1) (p21), CHOP/GADD153, dual-specificity protein phosphatase, and heme oxygenase-1. H2O2 rapidly induced ATF3 approximately 12-fold in HK2 cells and approximately 6.5-fold in a mouse model of renal ischemia-reperfusion injury. Adenovirus-mediated expression of ATF3 protected HK2 cells against H2O2-induced cell death, and this was associated with a decrease of p53 mRNA and an increase of p21 mRNA. Moreover, when ATF3 was overexpressed in mice via adenovirus-mediated gene transfer, ischemia-reperfusion injury was reduced. In conclusion, ATF3 plays a protective role in renal ischemia-reperfusion injury and the mechanism of the protection may involve suppression of p53 and induction of p21.

Clinical outcome and prognosis of anti-neutrophil cytoplasmic antibody-associated vasculitis in Japan.

Background/Aims: We conducted a broad survey of 99 patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and investigated both prognosis and outcomes. Methods: Clinical data evaluated were age, sex, patient survival, renal survival, serum albumin, serum creatinine, urinary protein, hematuria, C-reactive protein (CRP), ANCA titer, IgG and the Birmingham Vasculitis Activity Score (BVAS). Results: The patient survival rate at 6 months after onset was 84.8%, and that at 2 years after onset was 82.0%. Most deaths were within 6 months of onset. Infection accounted for 9 deaths (60.0%). Infection together with pulmonary involvement of active vasculitis accounted for 2 deaths (13.3%). Organ-specific involvement of active vasculitis alone caused 3 deaths (20.0%). Others died of cardiac events. At 1 and 3 months after onset, BVAS (p < 0.0001, p = 0.002), albumin (p = 0.006, p = 0.0004) and CRP (p = 0.04, p = 0.0002) were also associated with patient death. Conclusion: To improve the prognosis of those with ANCA-associated vasculitis, the intensity of initial treatment should be aimed at disease severity. Employing BVAS improved the ability to evaluate therapeutic responses. Finally, prescription with sulfamethoxazole-trimethoprim during the induction therapy with immunosuppressive agents may be advised.

Recombinant Human Erythropoietin Mitigates Reductions in Renal Klotho Expression

Background: Erythropoietin (EPO) and Klotho expression have both been detected in the kidney. Since a recent study suggested that both EPO and Klotho mitigate kidney damage, we explored the relation between EPO and Klotho in a doxorubicin hydrochloride (DXR)-induced rat nephropathy model treated with recombinant human erythropoietin (rhEPO). Methods: Male Sprague-Dawley rats were subjected to DXR-induced nephropathy. The rhEPO group was intracutaneously injected with rhEPO twice weekly at 4–16 weeks after the DXR injection. The rats were sacrificed at 16 weeks after the DXR administration. Expression of renal Klotho, HSP70, α-smooth-muscle actin and E-cadherin were assessed using real-time PCR or western blotting. The hematocrit, plasma creatinine and phosphate levels were also determined. Immunohistochemical studies and Masson-trichrome staining were performed. Results: The renal Klotho mRNA and Klotho protein expressions were significantly reduced in the DXR nephropathy group. Treatment with rhEPO improved the serum creatinine, phosphate level and histological changes observed in the DXR nephropathy group. The reduction in Klotho expression induced by DXR nephropathy was mitigated by rhEPO administration. Conclusion: rhEPO is involved in the pathophysiology of DXR nephropathy. rhEPO mitigated elevated plasma phosphate concentrations in an experimental model of chronic kidney disease via the expression of Klotho.

Glomerulopathy with Homozygous Apolipoprotein E2: A Report of Three Cases and Review of the Literature

Most cases of type III hyperlipoproteinemia are accounted for by apolipoprotein E2 (apoE2) homozygotes, a genetic mutation of apoE (Arg158Cys). Glomerulopathy with homozygous apoE2 is rare and characterized by marked foam cell infiltration in the glomerular capillaries and mesangium. Here, we report 3 cases of apoE2 homozygote glomerulopathy diagnosed by renal biopsy and DNA analysis. All 3 cases were middle-aged or elderly males complicated with diabetes for at least a decade. The kidney biopsies showed massive foam cell infiltration in the glomerular capillaries and expanded mesangium accompanied by histological findings of diabetic glomerulosclerosis. The lipid profiles showed type III hyperlipoproteinemia and phenotypic/genetic analyses revealed homozygosity of apoE2. Two of the cases showed nephrotic proteinuria and progressed to renal failure in 3 and 8 years after the diagnosis of kidney disease.

Elevation of the serum liver enzyme levels during tolvaptan treatment in patients with autosomal dominant polycystic kidney disease (ADPKD)

BackgroundIn 2014, tolvaptan, a vasopressin receptor antagonist, was approved for the treatment of autosomal dominant polycystic kidney disease (ADPKD) in Japan. Clinical trials of tolvaptan revealed frequent occurrence of the liver function abnormality. According to the package insert in Japan, liver function tests should be performed once a month in patients receiving tolvaptan. Furthermore, immediate discontinuation of tolvaptan is recommended in the appearance of liver function abnormalities.MethodsSeven patients of ADPKD who was discontinued tolvaptan because of elevation of the serum liver enzyme levels were described in detail and analyzed.ResultsNone of them fulfilled the criteria for applicability of Hy’s law, which predicts a high risk of severe, potentially fatal, drug-induced liver injury (DILI). In our patients, the rate of increase of total kidney volume (TKV) significantly decreased during tolvaptan administration, but increased after discontinuation; in Cases 1–5, mean annual growth rate of TKV during administration was − 10.15%/year, and during discontinuation was + 23.72%/year. After the serum liver enzyme levels returned to normal range, tolvaptan was resumed in six patients with informed consent. Except one patient, tolvaptan has been continued without increase of the serum liver enzyme levels.ConclusionIn patients with mild elevation of the serum liver enzyme, as is less than three times the upper limit of normal (ULN), resumption of tolvaptan may be considered after the serum liver enzyme levels return to normal range.

Successful steroid treatment of a patient with autosomal dominant polycystic kidney disease complicated by sarcoidosis

The patient was a 54-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) who developed complicating systemic sarcoidosis. Hypercalcemia and an abrupt increase in serum creatinine levels were observed during the clinical course. Steroid therapy was initiated and produced a distinct improvement in renal function. A kidney biopsy was not feasible because ADPKD is a contraindication for renal needle biopsy. The clinical findings strongly suggested renal disorder secondary to tubulointerstitial nephritis caused by renal sarcoidosis with complicating hypercalcemia. In addition to controlling hypertension and improving the hypercalcemia and dehydration, steroid therapy also improved the renal function in this patient.

Acute renal failure in a patient with hemophagocytic syndrome

Abstract Acute renal failure (ARF) occurred in a 47-year-old man with hemophagocytic syndrome. Histological findings of the kidney revealed diffuse infiltration of interstitium by phagocytosing cells mixed with atypical lymphoid cells of varying size. The cytological features of the lymphoid population in liver and spleen were consistent with a diagnosis of peripheral T-cell lymphoma. We believe that this ARF could have been exacerbated by the interstitial infiltration of phagocytosing cells, reactive lymphoid cells, and T-cell lymphoma cells.