Tomohiro Kikuta

Noninvasive Evaluation of Kidney Hypoxia and Fibrosis Using Magnetic Resonance Imaging

Interstitial fibrosis and hypoxia accelerate the progression of CKD, but clinical tools to quantitate these factors in patients are lacking. Here, we evaluated the use of two magnetic resonance imaging (MRI) techniques, diffusion-weighted (DW)-MRI and blood oxygen level-dependent (BOLD)-MRI, to assess kidney fibrosis and hypoxia of the cortex in 142 patients with either diabetic nephropathy (n = 43), CKD without diabetes (n = 76), or acute kidney injury (AKI) (n = 23). Apparent diffusion coefficient (ADC) values of DW-MRI correlated with estimated glomerular filtration rates (eGFR) in the diabetic nephropathy and CKD groups (r(2) = 0.56 and r(2) = 0.46, respectively). Although the T2* values of BOLD-MRI and eGFR displayed good correlation in the CKD group (r(2) = 0.38), we did not observe a significant correlation between these values in the diabetic nephropathy group, suggesting that factors other than tubulointerstitial alteration determine the degree of hypoxia in the renal cortex. In the AKI group, neither the T2* nor ADC values correlated with eGFR. Renal biopsies from patients with CKD demonstrated that the T2* and ADC MRI values correlated with renal pathology. Taken together, ADC and T2* values appear to serve as accurate indices for evaluating renal tubulointerstitial alterations and parenchymal hypoxia, respectively, in the cortex. Functional MRI can thus contribute to multilateral, noninvasive, in vivo assessment of kidney function.

Connective Tissue Growth Factor Expressed in Tubular Epithelium Plays a Pivotal Role in Renal Fibrogenesis

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-beta. However, its precise role in renal interstitial fibrogenesis has not yet been clarified. It was demonstrated previously that CTGF was expressed in tubular epithelial cells that had been engulfed by interstitial fibrosis in the remnant kidney of the subtotal nephrectomy (SNx) model. In the present study, co-cultures of tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) that mimic the subepithelial mesenchyme in the kidney were used to study the profibrotic effects of TGF-beta1-induced CTGF. In these co-cultures, TGF-beta1 treatment resulted in significantly increased mRNA levels of type I collagen and fibronectin in the TFB. These effects were both direct and indirect, with the latter being mediated by CTGF derived from the co-cultured mProx24. Then TGF-beta1 transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, and their kidneys were analyzed for fibrosis. Intravenous administration of CTGF antisense oligodeoxynucleotide significantly blocked CTGF expression in the proximal tubular epithelial cells in the remnant kidney of these animals despite the sustained level of TGF-beta1 mRNA. This reduction in CTGF mRNA level paralleled a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1, suppressing renal interstitial fibrogenesis. In conclusion, tubular CTGF acts as a downstream mediator of the profibrotic effects of TGF-beta1 in the remnant kidney, which is a promising target for antifibrotic drugs designed to treat TGF-beta1-dependent interstitial fibrosis.

Bradykinin Decreases Plasminogen Activator Inhibitor-1 Expression and Facilitates Matrix Degradation in the Renal Tubulointerstitium under Angiotensin-Converting Enzyme Blockade

A number of experimental and clinical investigations support the notion that angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II type 1 receptor blocker (ARB) compounds attenuate renal fibrosis. Fibrosis can be attenuated by either suppressing matrix formation or facilitating matrix degradation. In this study, drugs of ACEi and ARB classes were tested for their ability to facilitate matrix degradation in the kidney. A murine model system in which cyclosporin A (CsA) treatment for a specified period caused interstitial matrix deposition in the kidney was used. CsA was then discontinued, and experimental procedures were initiated to investigate matrix degradation. Benazepril, an ACEi, facilitated matrix degradation via the bradykinin (BK) B2 receptor on tubular epithelial cells in the kidney, whereas CGP-48933, an ARB, did not. In this murine model of CsA nephropathy under ACE blockade, plasminogen activator inhibitor-1 (PAI-1) expression was decreased in tubular epithelial cells, possibly leading to conversion of plasminogen to plasmin by plasminogen activator and subsequent activation of matrix metalloproteinases. These findings were confirmed in this study by measurements of plasmin activity, collagenolytic activity, and matrix metalloproteinase activities in the kidneys. In tubular epithelial cells stimulated in vitro, BK suppressed PAI-1 gene expression. All of these results suggest that ACEi can decrease PAI-1 expression via BK, thereby facilitating matrix degradation via activation of degradative enzymes to reduce interstitial matrix deposition.

Connective tissue growth factor mediates the profibrotic effects of transforming growth factor-β produced by tubular epithelial cells in response to high glucose

BackgroundIt was reported that connective tissue growth factor (CTGF) was expressed in the tubular epithelial cells of the diabetic kidney. CTGF has, among other factors, been implicated in mediating the downstream, profibrotic effects of transforming growth factor-β (TGF-β), though is precise role in interstitial fibrogenesis in the diabetic kidney has not yet been clarified.MethodsWe employed a coculture system involving cultured murine proximal tubular epithelial cells (mProx24) and renal fibroblasts (TFB), as a model of the subepithelial mesenchyme in the kidney in order to examine the profibrotic effects of CTGF derived from mProx24 cells in response to high glucose (30 mM).ResultsWe showed that glucose stimulated CTGF expression in cultured mProx24 in both a dose- and a time-dependent manner, and that this effect was mediated by increased levels of TGF-β. We also found that high glucose significantly stimulated TFB cells to produce profibrotic molecules, such as type I collagen, the EIIIA isoform of fibronectin, and plasminogen activator inhibitor-1. The induction of these molecules was both direct and indirect, the latter induction being mediated by mProx24 cell-derived CTGF, which, in turn, was induced by TGF-β that was produced by the mProx24 cells.ConclusionsCTGF plays an important role in mediating renal interstitial fibrogenesis in response to high glucose and, as such, is a reasonable target for anti-fibrotic therapy.

Poly(ADP-Ribose) Polymerase-1 Enhances Transcription of the Profibrotic CCN2 Gene

In the fibrotic kidney, tubular epithelial cells express CCN2, formerly known as connective tissue growth factor. Because little is known about the transcriptional regulation of this profibrotic protein, this study investigated the mechanism underlying epithelial cell-selective upregulation of CCN2 in fibrosis. It was found that a previously unidentified cis-regulatory element located in the promoter of the murine CCN2 gene plays an essential role in basal and TGF-beta1-induced gene transcription in tubular epithelial cells; this element acts in conjunction with the Smad-binding element and the basal control element-1. By protein mass fingerprint analysis and de novo sequencing, poly(ADP-ribose) polymerase-1 (PARP-1) was identified as a trans-acting protein factor that binds to this promoter region, which we termed the PARP-1-binding element. In vivo, knockdown of PARP-1 in proximal tubular epithelial cells significantly reduced CCN2 mRNA levels and attenuated interstitial fibrosis in the obstructed kidney. Thus, the PARP-1/PARP-1 binding element complex functions as a nonspecific, fundamental enhancer of both basal and induced CCN2 gene transcription in tubular epithelial cells. This regulatory complex may be a promising target for antifibrotic therapy.

TGF-β1 and HGF coordinately facilitate collagen turnover in subepithelial mesenchyme

We have employed co-culture of proximal tubular epithelial cells (PTEC) and renal tubulo-interstitial fibroblasts (TFB) to study the role of transforming growth factor-beta1 (TGF-beta1) and hepatocyte growth factor (HGF) in epithelial-mesenchymal interactions. In co-culture, TGF-beta1 stimulated TFB to produce type I collagen (COLI). This effect was both direct and indirect, via connective tissue growth factor (CTGF) produced by the co-cultured PTEC. Co-administration of TGF-beta1 and HGF significantly increased overall COLI production by TFB by 24h. However, in detail, this co-administration enhanced CTGF induction in PTEC during the first 8h, and then decreased its expression, resulting in a rapid decrease in expression of the alpha1(I) procollagen gene in TFB by 24h. Additionally, tissue inhibitor of metalloproteinase-1 was induced in PTEC by TGF-beta1 with or without co-administration of HGF, which contributed to the COLI accumulation. In contrast, HGF alone or co-administered with TGF-beta1 significantly increased collagenolytic activity derived from PTEC. Therefore, TGF-beta1 and HGF seem to coordinately modulate epithelial-mesenchymal interactions to facilitate COLI turnover in subepithelial mesenchyme.

Long-Term Effects of Calcium Antagonists on Augmentation Index in Hypertensive Patients with Chronic Kidney Disease: A Randomized Controlled Study

Background: Our previous retrospective study showed that benidipine was superior to amlodipine (AM) for reducing proteinuria and preserving the augmentation index (AI) in patients with chronic kidney disease (CKD). Methods: The present study enrolled CKD patients whose blood pressure was not well controlled by an angiotensin receptor blocker (ARB) and a calcium channel blocker other than AM or azelnidipine (AZ). Either AM (5 mg) or AZ (16 mg) was prescribed randomly. Clinical parameters, including proteinuria, serum creatinine, and AI, were measured before initiation of AM or AZ and 1 year later to assess the long-term effect on renal function and central blood pressure. Results: Brachial and central blood pressures were similarly reduced in both groups. However, pulse rate increased in the AM group, but decreased in the AZ group (+3 ± 1 vs. –2 ± 1 bpm, p < 0.0001). The reduction of proteinuria was greater in the AZ group (–29 ± 2 vs. –38 ± 3%, p < 0.01). Improvement of AI adjusted for a pulse rate of 75 bpm was larger in the AZ group than in the AM group (–4 ± 1 vs. –9 ± 1%, p < 0.05). In both groups, estimated GFR remained unchanged throughout the observation period. Conclusion: In hypertensive patients with CKD, combined treatment with AZ and an ARB decreases proteinuria and preferentially improves arterial reflection.

Granulomatous interstitial nephritis in chronic lymphocytic leukaemia

We present a case of granulomatous interstitial nephritis (GIN) associated with chronic lymphocytic leukaemia (CLL). GIN is a rare pathological finding noted in renal biopsy specimens. Furthermore, CLL does not usually cause GIN. In this case, acute renal injury probably resulted from GIN, and urgent dialysis was required, despite sufficient chemotherapy. Immunohistochemical analyses of a biopsy specimen revealed invasion of CD20( +) CLL cells, surrounded by reactive T cells, and granuloma formation. Thus, CLL may induce secondary interstitial nephritis as a granulomatous reaction.

Time for Reflection Predicts the Progression of Renal Dysfunction in Patients with Nondiabetic Chronic Kidney Disease

Our previous data indicated that both home blood pressure and arterial stiffness predicted the progression of renal dysfunction in the patients with chronic kidney diseases. In the present study, we examined both home blood pressure and the parameters of arterial stiffness as the indicator to the progression of chronic kidney diseases. Forty-two nondiabetic chronic kidney disease patients were enrolled and followed for 1 year. Anti-hypertensive therapy was adjusted to achieve office blood pressure below 130/80 mmHg. Home blood pressure was examined twice a day in the morning and evening. Pulse wave velocity (PWV) and augmentation index (AI) were measured as the index of arterial stiffness. The time for reflection (TR) was also determined. The relationship of annual changes in serum creatinine (Scr) with the above parameters was assessed. Multivariate regression analysis revealed that TR inversely correlated to annual increase in Scr (β = −0.03, p < 0.05). Home blood pressure did not correlate to annual changes in Scr in the present study. The present data indicated that arterial stiffness is elevated despite good blood pressure control in chronic kidney disease, especially among the dippings. In addition, our data suggest that PWV and AI correlated to each other, while they were influenced differently by hemodynamic factors. Finally, the present findings provide the evidence that the arterial stiffness parameter is more sensitive than home blood pressure as an indicator to the progression of chronic kidney disease.

Angiotensin II Type 1 and Type 2 Receptors Reciprocally Modulate Pro-inflammatory/ Pro-Fibrotic Reactions in Activated Splenic Lymphocytes

Angiotensin II (Ang II) type 1 receptor (AT₁R) has been confirmed to confer renoprotection in the progressive, immune-mediated nephritis in animal models as well as in humans. However, the relative contributions of direct AT₁R blockade, indirect counteractivation of Ang II type 2 receptor (AT₂R), or both, to renoprotection through AT₁R antagonism remains to be clarified. Immunohistochemical studies in the nephritic kidney revealed that tubular epithelial cells and infiltrating immune cells were positive for AT₁R and AT₂R. In the present study, we investigated the action of Ang II on both receptors on immune cells. A subpopulation of lipopolysaccharide-activated splenic lymphocytes (mixed lymphocyte populations) was positive for AT₁R and AT₂R. Ang II alone could not induce gene expression of a pro-inflammatory chemokine JE or a pro-fibrotic cytokine transforming growth factor-β1 in those cells. However, Ang II could significantly suppress the expression of both genes in those cells under AT₁R blockade, and this action was mediated through AT₂R. Conversely, the pro-inflammatory/pro-fibrotic gene expression could be enhanced by AT₂R blockade, and this was mediated through AT₁R. AT₁R and AT₂R expressed in activated immune cells can modulate pro-inflammatory and pro-fibrotic reactions reciprocally. In advanced immune-mediated nephritic kidneys, AT₁R antagonism likely confers renoprotection via activation of AT₂R.