Tomoyuki Fujikura

Cell division and phenotypic regression of proximal tubular cells in response to uranyl acetate insult in rats

BACKGROUND We examined whether dedifferentiation is necessary for cell division of proximal tubule (PT) cells after acute PT injury. METHODS Rats were injected with a low (0.2 mg/kg) or high (4 mg/kg) dose of uranyl acetate (UA) to induce acute PT injury. Proliferating PT cells were labelled with bromodeoxyuridine (BrdU) before sacrifice. Renal tissues were examined by double labelling of BrdU and megalin, aquaporin 1 (AQP1), Na(+)-K(+)ATPase or vimentin, and by immunoelectron microscopy for BrdU+ cells. RESULTS Under normal conditions, BrdU+ PT cells were positive for the PT phenotype (megalin-, AQP1- and Na(+)-K(+)ATPase positive and vimentine negative, a mesenchymal marker). Low-dose UA induced focal PT injury, and BrdU+ initially proliferating PT cells were found in the proximal three quarters of the S3 segment of nephron as early as 12 h, which maintained the PT phenotype and were vimentin negative. Proliferating PT cells showed low expression of the PT cell protein phenotype from Day 2 to Day 5 with vimentin expression from Day 2. High-dose UA induced severe PT injury in the proximal three quarters of the S3 segment by Day 5. BrdU+ initially proliferating PT cells, which were found in distal areas of the S3 segment as early as Day 2, showed low expression of the PT protein phenotype but were vimentin positive. Immunoelectron microscopy showed mature PT morphology for BrdU+ PT cells in control rats. BrdU+ initially proliferating PT cells showed a relatively mature phenotype after low-dose UA in- sult but an immature phenotype after high-dose UA insult. CONCLUSIONS PT cells can initiate cell division without de- differentiation after mild PT injury by low-dose UA insult.

Alogliptin improves steroid-induced hyperglycemia in treatment-naïve Japanese patients with chronic kidney disease by decrease of plasma glucagon levels

Background Chronic kidney disease (CKD) is a risk factor for end-stage renal failure and cardiovascular disease, and a strategy to counteract CKD must be established. CKD caused by immunological abnormalities is treated by steroids, frequently resulting in steroid diabetes. Although insulin is the most effective drug against steroid diabetes, administering it to patients can be difficult. Dipeptidyl peptidase-4 (DPP-4) inhibitors were developed for diabetes mellitus with a new mechanism of action. However, their efficacies and mechanisms of action for steroid diabetes are unclear. Material/Methods We studied 11 CKD patients treated with steroids admitted to our hospital (3 men and 8 women; age, 66.0±15.9 years). DPP-4 inhibitor alogliptin was administered for steroid diabetes. Levels of markers related to glucose metabolism were measured before alogliptin treatment and after alogliptin treatment, before the prednisolone dose was reduced. Results Alogliptin treatment significantly increased plasma glucagon-like peptide-1 (GLP-1) levels from 1.16±1.71 pmol/L to 4.48±1.53 pmol/L and significantly reduced levels of plasma glucose recorded 2 h after lunch and hemoglobin A1c (HbA1c). No significant differences were seen in insulin secretory ability of homeostasis model assessment (HOMA) (HOMA-β) and insulin resistance index of HOMA (HOMA-R) before and after alogliptin treatment. In contrast, alogliptin treatment significantly decreased plasma glucagon levels, from 116.1±38.7 pg/mL to 89.6±17.3 pg/mL. Moreover, there were significant correlations among HbA1c, GLP-1, and glucagon levels. Conclusions Alogliptin improves steroid-induced hyperglycemia by decrease of glucagon levels through an increase in plasma GLP-1 levels.

Analysis of intra-GBM microstructures in a SLE case with glomerulopathy associated with podocytic infolding

BackgroundSystemically podocytic infolding into the GBM which causes nonargyrophilic holes in the GBM in association with intra-GBM microstructures has been considered as a new pathological entity. However, its pathomechanisms are largely unknown.MethodsWe analyzed intra-GBM microstructures in an SLE patient with glomerulopathy associated with podocytic infolding by immunoelectron microscopy for vimentin (a marker for both podocyte and endothelium) and C5b-9 and by 3D reconstruction of transmission electron microscopy (TEM) images by computer tomography method.ResultsImmunofluorescent study showed immunoglobulin deposition in a diffuse, capillary pattern; however, electron-dense deposits like stage 3 membranous nephropathy could be found only in some capillary loops by TEM in spite of the systemic existence of podocytic infolding and the intra-GBM microstructures. Three-dimensional reconstructed images of the TEM images revealed that some of the intra-GBM microstructures made connections with the podocyte. The clustered microstructures underneath the podocyte and their surroundings looked as a whole like the degraded part of podocyte in 3D reconstructed images. Immunoelectron microscopy showed that vimentin was positive in most intra-GBM microstructures. C5b-9 was positive along the entire epithelial side of the GBM and in some microstructures, suggesting that the podocytes may be attacked by C5b-9 and that the microstructures may contain C5b-9 bound cellular membranes.ConclusionIntra-GBM microstructures may be originated mainly from the podocyte. Podotyte and GBM injuries caused by C5b-9 attack to podocytes might contribute in part to podocytic infolding and intra-GBM microstructures in this case.

Plasma Soluble (Pro)renin Receptor Reflects Renal Damage.

Background (Pro)renin receptor [(P)RR], a specific receptor for renin and prorenin, was identified as a member of the renin-angiotensin system (RAS). (P)RR is cleaved by furin, and soluble (P)RR [s(P)RR] is secreted into the extracellular space. Previous reports have indicated that plasma s(P)RR levels show a significant positive relationship with urinary protein levels, which represent renal damage. However, it is not fully known whether plasma s(P)RR reflects renal damage. Methods We recruited 25 patients who were admitted to our hospital to undergo heminephrectomy. Plasma s(P)RR levels were examined from blood samples drawn before nephrectomy. The extent of renal damage was evaluated by the levels of tubulointerstitial fibrosis. Immunohistochemical analysis of intrarenal (P)RR and cell surface markers (cluster of differentiation [CD]3, CD19, and CD68) was performed on samples taken from the removed kidney. Moreover, double staining of (P)RR and cell surface markers was also performed. Results There were significant positive relationships between plasma s(P)RR and tubulointerstitial fibrosis in all the patients and those not receiving RAS blocker therapy. Significant positive relationships were found between plasma s(P)RR levels and the extent of tubulointerstitial fibrosis after adjustment for age, sex, body weight, blood pressure, and plasma angiotensin II, in all the patients and those not receiving RAS blockers. Moreover, (P)RR expression was elevated in infiltrated mononuclear cells but not connecting tubules or collecting ducts and vessels. Infiltrated cells positive for (P)RR consisted of CD3 and CD68 but not CD19. Conclusions These data suggest that plasma s(P)RR levels may reflect (P)RR expression levels in infiltrated mononuclear cells, which can be a surrogate marker of renal damage.

Acquired resistance to rechallenge injury in rats that recovered from mild renal damage induced by uranyl acetate: accelerated proliferation and hepatocyte growth factor/c-Met axis.

BackgroundRats that recovered from mild proximal tubule (PT) injury without renal dysfunction by subtoxic insult, developed partial resistance to subsequent nephrotoxic insult. This partial resistance was associated with reduced renal dysfunction and accelerated PT cell proliferation compared with vehicle treatment as the first insult. Here we assessed the role and potential mechanisms of accelerated PT proliferation in this acquired resistance model.MethodsRats at 14 days after recovering from prior mild renal damage induced by 0.2 mg/kg uranyl acetate (UA) (subtoxic dose) were rechallenged with 4 mg/kg UA (nephrotoxic dose) to establish the acquired resistance model. Cell cycle was inhibited by colchicine to examine the contribution of accelerated PT cell proliferation evaluated by in vivo bromodeoxyuridine (BrdU) labeling on acquired resistance to subsequent nephrotoxic insult. Hepatocyte growth factor (HGF)/c-Met axis and other related factors of cell cycle were analyzed.ResultsThe acquired resistance to rechallenge injury with nephrotoxic dose of UA in rats recovered from mild renal injury was associated with an earlier increase in BrdU-positive PT cells, accelerated upregulation of HGF mRNA, c-Met mRNA/protein, cyclin D1, phospho-Rb and an earlier phenotypic change of PT cells. Colchicine inhibited PT cell proliferation, reduced the upregulated cyclin D1 and phospho-Rb in the kidney, completely abolishing acquired resistance.ConclusionsCell cycle progression with upregulated renal HGF/c-Met axis may contribute to the accelerated recovery from acute renal failure in rats that recovered from prior mild renal damage, followed by nephrotoxic insult, resulting in partial acquired resistance.

A high ratio of G1 to G0 phase cells and an accumulation of G1 phase cells before S phase progression after injurious stimuli in the proximal tubule.

Proximal tubule (PT) cells can proliferate explosively after injurious stimuli. To investigate this proliferative capacity, we examined cell cycle status and the expression of cyclin‐dependent kinase inhibitor p27, a G1 phase mediator, in PT cells after a proliferative or injurious stimulus. Rats were treated with lead acetate (proliferative stimulus) or uranyl acetate (UA; injurious stimulus). Isolated tubular cells were separated into PT and distal tubule (DT) cells by density‐gradient centrifugation. Cell cycle status was analyzed with flow cytometry by using the Hoechst 33342/pyronin Y method. Most PT and DT cells from control rats were in G0/G1 phase, with a higher percentage of PT cells than DT cells in G1 phase. Lead acetate and UA administration promoted the G0‐G1 transition and the accumulation of G1 phase cells before S phase progression. In PT cells from rats treated with lead acetate or a subnephrotoxic dose of UA, p27 levels increased or did not change, possibly reflecting G1 arrest. In contrast, p27 became undetectable before the appearance of apoptotic cells in rats treated with a nephrotoxic dose of UA. The decrease in p27 might facilitate rapid cell cycling. The decreased number of p27‐positive cells was associated with PT cell proliferation in renal tissues after a proliferative or injurious stimulus. The findings suggest that a high ratio of G1 to G0 phase cells and a rapid accumulation of G1 phase cells before S phase progression in the PT is a biological strategy for safe, timely, and explosive cell proliferation in response to injurious stimuli.

Intrarenal renin–angiotensin system activity is augmented after initiation of dialysis

Circulating renin–angiotensin system (RAS) activation is maintained after renal function has deteriorated. The activation of the intrarenal RAS plays a critical role in the pathophysiology of chronic kidney disease (CKD), independently of the circulating RAS. However, the activation of intrarenal RAS and the chymase-dependent pathway after initiation of dialysis has not been clarified. We recruited 19 CKD patients (10 without dialysis and 9 with dialysis) who underwent a heminephrectomy. Circulating RAS was investigated before nephrectomy. The levels of intrarenal RAS components and chymase-positive cells were investigated using radioimmunoassay or immunoblot analysis on samples collected from the removed kidney. Renal damage was evaluated by the extent of tubulointerstitial fibrosis. No significant differences in circulating RAS between nondialysis and dialysis patients were found. However, intrarenal angiotensin II (AngII) and the extent of tubulointerstitial fibrosis in dialysis patients were significantly increased when compared with nondialysis patients. Prorenin and angiotensin-converting enzyme (ACE) levels were dramatically decreased in accordance with renal dysfunction. On the other hand, chymase-positive cells and AngII type 1 receptor (AT1R) expression was significantly increased in dialysis patients when compared with nondialysis patients. In multiple linear regression analyses, there were significant positive and negative relationships between the extent of interstitial fibrosis and angiotensinogen (β=0.45, P=0.042) and prorenin levels (β=−0.85, P<0.01), respectively. In summary, a decrease in prorenin and ACE expression and an increase in chymase, angiotensinogen and AT1R expression in the kidney may augment the intrarenal RAS activation and be associated with renal damage, even after initiation of dialysis.

Relationship between urinary fractional excretion of sodium and life prognosis in liver cirrhosis patients

Renal vasoconstriction in generalized vasodilatation with blood pooling and the consequent reduction in effective arterial volume is the pathophysiological basis of liver cirrhosis (LC). Low levels of fractional excretion of sodium (FENa) are an effective marker of hypoperfusion of the renal artery. However, the relationship between levels of FENa, LC severity and life prognosis has not yet been elucidated.

Cytoresistance after acute kidney injury is limited to the recovery period of proximal tubule integrity and possibly involves Hippo‐YAP signaling

Rat proximal tubule (PT) cells that have recovered from severe acute kidney injury induced by uranyl acetate (UA) develop cytoresistance to subsequent UA treatments. We reported that enhanced G1 arrest might contribute to cytoresistance. Herein, we examined these mechanisms by investigating Yes‐associated protein (YAP), a regulator of cell number, and survivin, a downstream mediator of YAP that inhibits apoptosis. Rats pretreated with saline (vehicle group) or UA (AKI group) were injected with UA 2 weeks, 2 months, or 6 months after treatment. Cytoresistance, evaluated by serum creatinine, was observed at 2 weeks, was attenuated at 2 months, and was lost at 6 months in the AKI group. Based on immunohistochemistry, overexpressed YAP/survivin in PT cells and an increased number of PT cells was found before the second insult at 2 weeks, regressed gradually, and returned to a normal value by 6 months in the AKI group. Cell cycle status, assessed by flow cytometry, was equivalent in all groups before the second insult. However, early G1 phase (cyclin D1−) and p27+ PT cells increased in the AKI group compared to those in the vehicle group until 2 months, but were comparable to those in the vehicle group at 6 months. p21+ PT cells increased at 2 weeks, but normalized by 2 months. Thus, PT cells that have recovered from AKI transiently overexpress YAP/survivin, probably inhibiting apoptosis and resulting in acquired cytoresistance. This effect occurs until PT remodeling is complete, subceullular PT integrity is restored, and cell numbers are normalized.

Acquired resistance to rechallenge injury after acute kidney injury in rats is associated with cell cycle arrest in proximal tubule cells

Rats that have recovered from severe proximal tubule (PT) injury induced by uranyl acetate (UA), a toxic stimulus, developed resistance to subsequent UA treatment. We investigated cell cycle status and progression in PT cells in relation to this acquired resistance. Fourteen days after pretreatment with saline (vehicle group) or UA [acute kidney injury (AKI) group], rats were injected with UA or lead acetate (a proliferative stimulus). Cell cycle status (G0/G1/S/G2/M) was analyzed by flow cytometry. The expression of cell cycle markers, cyclin-dependent kinase inhibitors, and phenotypic markers were examined by immunohistochemistry. Cell cycle status in PT cells in the AKI group was comparable to those of the vehicle group. However, more early G1-phase cells (cyclin D1- or Ki67-) and p21+ or p27+ cells were found in the PT of the AKI group than in that of the vehicle group. UA induced G1 arrest and inhibited S phase progression with earlier dedifferentiation and less apoptosis in PT cells of the AKI group. Lead acetate induced proliferation without dedifferentiation but with delayed G0-G1 transition and inhibited S phase progression in PT cells in the AKI group. Sustained p21 and increased p27 expression in PT cells were found in the AKI group in response to UA and lead acetate. PT cells in the AKI group inhibited cell cycle progression by enhanced G1 arrest, probably via p21/p27 modulation as an injury or proliferation response, resulting in cytoresistance to rechallenge injury.