Hisayo Fujita

Extracellular Signal–Regulated Kinase Inhibition Slows Disease Progression in Mice with Polycystic Kidney Disease

The expression of mitogen-activated protein kinases (MAPK) in DBA/2-pcy/pcy (pcy) mice, a murine model of polycystic kidney disease was investigated. Proliferating cell nuclear antigen-positive cells were recognized in cyst epithelium from embryonic day 14.5 to 25 wk of age. Extracellular signal-regulated kinase (ERK) was expressed in the renal tubules of control and pcy mice, but stronger immunostaining was observed in cyst epithelium. Phosphorylated ERK was detected only in pcy mice and was localized predominantly in the cysts. p38 MAPK (p38) was no longer expressed after birth in controls but was detected in the cyst epithelium and in occasional tubular cells of pcy mice at all stages examined. c-Jun N-terminal kinase (JNK) was expressed in all tubular segments of controls after neonatal day 7, whereas in pcy kidneys, tubules became positive for JNK after 8 wk, and the cysts expressed little JNK. Administration of an oral MAP/ERK kinase inhibitor, PD184352, 400 mg/kg per d, to 10-wk-old pcy mice daily for the first week and then every third day for 6 additional weeks significantly decreased BP, kidney weight, serum creatinine level, and water intake and significantly increased urine osmolality. The cystic index and expression of phosphorylated ERK and ERK were significantly lower in PD184352-treated pcy mice. These results demonstrate that the expression of MAPK is dysregulated in cyst epithelium and that inhibition of ERK slowed the progression of renal disease in pcy mice.

The Lack of Cyclin Kinase Inhibitor p27Kip1 Ameliorates Progression of Diabetic Nephropathy

Cyclin kinase inhibitor p27(Kip(1)) (p27) has been shown to be upregulated in glomeruli of diabetic animals and mesangial cells cultured under high glucose. This study was an investigation of the role of p27 in the progression of diabetic nephropathy. Mice deficient in p27 (p27 -/-) and wild-type mice (p27 +/+) were studied 12 wk after diabetes induction by streptozotocin. Blood glucose and BP were comparable between diabetic p27 +/+ and p27 -/- mice. The kidney weight to body weight ratio and glomerular volume increased in diabetic p27 +/+ mice. In contrast, these parameters did not change in diabetic p27 -/- mice. Similarly, albuminuria developed in diabetic p27 +/+ mice but not in diabetic p27 -/- mice. The mesangial expansion was significantly milder in diabetic p27 -/- mice than that in diabetic p27 +/+ mice. These changes were associated with a similar increase in glomerular TGF-beta expression in diabetic p27 +/+ and p27 -/- mice. However, glomerular protein expression of fibronectin, a target of TGF-beta, increased only in diabetic p27 +/+ mice. In mesangial cells cultured from p27 +/+ mice, exposure to high glucose caused significant increases in total protein content and [(3)H]-leucine incorporation. On the other hand, high glucose caused a significant reduction in these parameters in cells from p27 -/- mice. Phosphorylation of 4E-BP1, the translation inhibitor, increased after exposure to high glucose in p27 +/+ cells. In p27 -/- cells, the level of phosphorylated 4E-BP1 was higher than that in control p27 +/+ cells and decreased under high glucose conditions. In conclusion, renal hypertrophy, glomerular hypertrophy, and albuminuria did not develop, and mesangial expansion was milder in diabetic p27 -/- mice despite glomerular TGF-beta upregulation. These results suggest that controlling p27 function may ameliorate diabetic nephropathy.

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-β in mesangial cells

Eicosapentaenoic acid (EPA), an ω‐3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet‐derived growth factor (PDGF)‐stimulated rat mesangial cell mitogenesis. EPA dose‐dependently inhibited PDGF‐stimulated [3H]‐thymidine incorporation. PDGF‐induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 µg/ml EPA. Similarly, PDGF‐stimulated activation of extracellular signal‐regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin‐dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF‐β secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti‐TGF‐β antibody inhibited the EPA‐induced suppression of [3H]‐thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF‐β, did not affect EPAs growth inhibitory effect. These results demonstrate that EPA inhibits PDGF‐stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF‐β. J. Cell. Physiol. 196: 293–300, 2003.

The Herbal Medicine Sairei-to Inhibits Proliferation of Rat Mesangial Cells

Background: The herbal medicine Sairei-to is efficacious in renal diseases where mesangial proliferation is a key event. We examined whether Sairei-to inhibits proliferation of cultured rat mesangial cells and investigated its mechanism of action. Methods: The effect of Sairei-to on [3H]-thymidine incorporation stimulated by serum was assessed. Cell cycle was analyzed by flowcytometry. Extracellular signal-regulated kinase (ERK) activity was determined by immunecomplex kinase assay. Tyrosine phosphorylation of cellular proteins, and phosphorylation of ERK and Raf-1 were analyzed by immunoblot. Cyclic AMP was measured by radioimmunoassay. Results: Incubation of mesangial cells for 18 h with water-soluble but not insoluble fraction of Sairei-to inhibited serum-stimulated [3H]-thymidine incorporation. In subsequent experiments, water-soluble fraction, at a dose required for a half-maximal response (2 mg/ml), was used. Sairei-to inhibited S-phase entry stimulated by serum. Serum-induced tyrosine phosphorylation of p44 and p42 ERK was inhibited by Sairei-to, but that of other cellular proteins was not affected. Suppression of serum-stimulated ERK activation by Sairei-to was confirmed by immunecomplex kinase assay. Activation of Raf-1, an upstream activator of ERK, was also attenuated by Sairei-to. Incubation of cells with Sairei-to significantly increased the generation of cAMP. Conclusions: Sairei-to inhibits serum-induced DNA synthesis of rat mesangial cells by suppressing Raf-1/ERK cascade probably via cAMP.

Cyclic stretch induces proliferation and TGF-β1-mediated apoptosis via p38 and ERK in ureteric bud cells

We previously reported that p38 mitogen-activated protein kinase (p38) and phosphorylated ERK are upregulated in cyst epithelium of human renal dysplasia and obstructive uropathy in fetal lambs (Omori S, Fukuzawa R, Hida M, Awazu M. Kidney Int 61: 899-906, 2002; Omori S, Kitagawa H, Koike J, Fujita H, Hida M, Pringle KC, Awazu M. Kidney Int 73: 1031-1037, 2008). Dysplastic epithelium is characterized by proliferation, apoptosis, and upregulation of Pax2 and transforming growth factor (TGF)-beta1. In the present study, we investigated whether cyclic mechanical stretching of ureteric bud cells, a mimic of the hydrodynamic derangement after fetal urinary tract obstruction, reproduces events seen in vivo. Cyclic stretch activated p38 and ERK and upregulated Pax2 expression in a time-dependent manner in ureteric bud cells. Stretch-stimulated Pax2 expression was suppressed by a p38 inhibitor, SB203580, or a MEK inhibitor, PD98059. 5-Deoxyuridine incorporation was increased by stretch at 24 h, which was also abolished by SB203580 or PD98059. On the other hand, apoptosis was not induced at 24 h by stretch but was significantly increased at 48 h. TGF-beta1 secretion was increased by stretch at 24 h, which was inhibited by SB203580 or PD98059. Inhibition of p38 or ERK as well as anti-TGF-beta antibody abolished the stretch-induced apoptosis. Finally, exogenous TGF-beta1 induced apoptosis of ureteric bud cells, which was inhibited by SB203580 and PD98059. In conclusion, cyclic stretch induces Pax2 upregulation, proliferation, and TGF-beta1-mediated apoptosis, features characteristic of dysplastic epithelium, via p38 and ERK in ureteric bud cells.

Activated extracellular signal-regulated kinase correlates with cyst formation and transforming growth factor-β expression in fetal obstructive uropathy

Human renal dysplasia is frequently associated with urinary tract obstruction and the abnormal expression of mitogen-activated protein kinase (MAPK). Here, we determined the renal responses and MAPK expression in developing kidneys that were obstructed in fetal lambs. Kidneys were harvested at various times after obstruction (gestation day 60) through normal term (day 145). Dilation of Bowmans capsule and proximal tubules was seen 2 days after obstruction and involved the whole cortex 18 days later, with numerous cysts present throughout the kidney at term. The proliferation marker Ki-67 and transforming growth factor-beta (TGF-beta) were detected 2 days after obstruction and progressively increased in tubules, cysts, and the interstitium. In control kidneys, p38 was expressed in tubules only during the fetal stage, whereas phosphorylated extracellular signal-regulated kinase (P-ERK) was limited to ureteric buds and collecting ducts at all stages examined. However, Jun-N-terminal kinase (JNK) was absent in the fetal kidney but present in tubules at term. In obstructed kidneys, cyst epithelia were positive for p38 and P-ERK but negative for JNK throughout all stages. These studies show that P-ERK correlated spatially and temporally with Ki-67 and TGF-beta expression, which suggests that ERK may contribute to cyst formation and fibrosis in the obstructed fetal kidney.

Height-specific body mass index reference curves for Japanese children and adolescents 5−17 years of age

Abstract Background : Body mass index (BMI) reference values in consideration of height variation have not previously been reported. This study established height‐specific BMI reference curves for Japanese children and adolescents aged from 5 to 17 years.

Masked Isolated Nocturnal Hypertension in Children and Young Adults

Isolated nocturnal hypertension (INH) is characterized by normal daytime blood pressure (BP) and elevated nighttime BP diagnosed by ambulatory BP monitoring. Masked isolated nocturnal hypertension (MINH) is a subtype of INH in which office BP is normal. We studied the frequency and characteristics of INH and MINH in children and young adults. One hundred and ninety-eight subjects seen by the pediatric nephrology service were studied retrospectively. Isolated nocturnal hypertension (INH) and MINH were diagnosed according to daytime and nighttime ABP and office BP in the case of the latter. One hundred and eighteen subjects (60%) had normotension, 6 (3%) had isolated daytime hypertension, 32 (16%) had INH, and 42 (21%) had day–night hypertension. Sixteen subjects had MINH (8.1%). The underlying diseases of MINH were as follows: no underlying disease 9 (56%), renal disease 6 (38%), and endocrine disease 1 (6%). There was no significant difference in the underlying disease, gender, age, and BMI between MINH and INH with elevated office BP. In conclusion, MINH is present in children and young adults. Since there were no specific features for MINH, screening with ambulatory or home BP monitoring during sleep may be appropriate.