Hirotaka Fukasawa

Urinary angiotensinogen as a marker of intrarenal angiotensin II activity associated with deterioration of renal function in patients with chronic kidney disease.

In chronic kidney disease (CKD), enhanced intrarenal angiotensin II (AngII) is involved in deterioration of renal function, but it is difficult to measure it. For assessment of the potential of urinary angiotensinogen as a marker of intrarenal AngII activity, the correlation of plasma and urinary renin-angiotensin system components, including angiotensinogen, with deterioration of renal function was investigated in 80 patients who had CKD and were not treated with AngII blocking agents. Changes that were induced by 14 d of losartan treatment (25 mg/d) were also measured in 28 patients. Angiotensinogen was measured by RIA of AngI after incubation with renin. Urinary angiotensinogen levels were greater in patients with low estimated GFR and elevated urinary protein and type IV collagen and correlated with renal AngII and type I collagen immunostaining intensities. The risk for deterioration of renal function (i.e., estimated GFR decline of >2.5 ml/min per yr) during a mean follow-up period of 23 mo (maximum 43 mo) was associated with urinary angiotensinogen of >3.0 nmol AngI equivalent per 1 g of urinary creatinine (AngI Eq/g Cre) at enrollment (hazard ratio 3.52). The event-free survival for deterioration of renal function was better in patients with urinary angiotensinogen <3.0 nmol AngI Eq/g Cre than those >3.0 nmol AngI Eq/g Cre. Losartan reduced urinary and plasma angiotensinogen, urinary protein and type IV collagen, and systolic BP, despite concomitant increases in plasma renin and AngII. These data suggest that urinary angiotensinogen is a potentially suitable marker of intrarenal AngII activity associated with increased risk for deterioration of renal function in patients with CKD.

Slit Diaphragms Contain Tight Junction Proteins

Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-β

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor‐β (TGF‐β)‐Smad signaling via ubiquitin‐dependent degradation of Smad2 and Smad7. We found that TGF‐β stimulates Smurf2 expression. TGF‐β activated the Smurf2 promoter in a TGF‐β responsive cell lines, whereas IL‐1α, PDGF and epidermal growth factor did not. TGF‐β‐mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor‐like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad‐binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF‐β‐mediated Smurf2 induction. These results suggest that TGF‐β stimulates Smurf2 expression by Smad‐independent pathway such as PI3K/Akt pathway via TGF‐β receptor.

Ubiquitin-Dependent Degradation of Smad2 Is Increased in the Glomeruli of Rats with Anti-Thymocyte Serum Nephritis

The overexpression of transforming growth factor (TGF)-beta and Smad-mediated intracellular TGF-beta signaling in the kidney underlies the development of renal scarring from pathological matrix accumulation. However, changes in the Smad proteins during the progression of kidney disease are unclear. In this study, we investigated the regulation of Smad proteins in the glomeruli of rats with anti-thymocyte serum nephritis. We found that Smad2 protein decreased markedly in nephritic glomeruli, whereas no significant changes were observed in the levels of Smad3 and Smad4 proteins. In contrast, the level of Smad2 mRNA in nephritic glomeruli did not differ significantly from that in control glomeruli. Based on recent reports of the ubiquitin-mediated degradation of Smad2, we investigated the degradation and ubiquitination activity directed against Smad2 in glomerular extracts. Both the degradation and ubiquitination of Smad2 were markedly increased in glomerular extracts from rats with nephritis. We also found that Smurf2, a ubiquitin ligase for Smad2, was increased in the nephritic glomerular extracts. These data suggest that the decrease in Smad2 resulted from enhanced ubiquitin-dependent degradation of Smad2 mediated by Smurf2, and is involved in the regulation of Smad2-mediated TGF-beta signaling in nephritic glomeruli.

Reduction of transforming growth factor-β type II receptor is caused by the enhanced ubiquitin-dependent degradation in human renal cell carcinoma

Although dysregulation of transforming growth factor‐β (TGF‐β) signaling is implicated in renal carcinogenesis, its precise mechanism is unknown in renal cell carcinoma (RCC). In our study, we investigated Smad‐mediated TGF‐β signaling pathway and its regulatory mechanisms in surgical samples from patients with RCC. We found that immunoreactivity for nuclear phosphorylated Smad2 was significantly decreased in RCC compared to normal renal tissues, thereby TGF‐β signaling was suggested to be attenuated in RCC tissues. In accordance with the result, transcriptional downregulation of Smad4 and post‐transcriptional downregulation of TGF‐β type II receptor (TβR‐II) were frequently found in RCC tissues compared to normal renal tissues. Next, to clarify the reason why the protein level of TβR‐II was decreased in RCC, we investigated the activities of degradation and ubiquitination of TβR‐II. We found that both proteasome‐mediated degradation and ubiquitination of TβR‐II were markedly enhanced in RCC tissues. Moreover, we found that the level of Smad‐ubiquitination regulatory factor 2 (Smurf2), the E3 ligase for TβR‐II, was increased in RCC tissues of the patients with higher clinical stages compared to the normal tissues and was inversely correlated with the level of TβR‐II. Our results suggest that the low TβR‐II protein level is due to augmented ubiquitin‐dependent degradation via Smurf2 and might be involved in the attenuation of TGF‐β signaling pathway in RCC.

Intrarenal RAS activity and urinary angiotensinogen excretion in anti-thymocyte serum nephritis rats

The differential roles of circulating and intrarenal renin-angiotensin system (RAS) in glomerulonephritis have not been elucidated. In this study, we investigated the levels of circulating and intrarenal RAS activity and urinary angiotensinogen (AGT) excretion in anti-thymocyte serum (ATS) nephritis induced by an ATS injection (ATS group). The effect of olmesartan, an angiotensin II (ANG II) type 1 receptor blocker (ARB), on the development of nephritis was also examined (ATS+ARB group). In addition, the rats received a saline injection instead of ATS (control group). Mesangial proliferation with transient proteinuria, which peaked at day 7, was significantly increased in the ATS group compared with the control group. The levels of glomerular AGT mRNA, intrarenal ANG II, and urinary AGT excretion in the ATS group were increased significantly at day 7 compared with the control group. Administration of olmesartan (ATS+ARB group) significantly decreased the levels of renal lesions, proteinuria, and intrarenal RAS activity compared with the ATS group. In addition, the levels of urinary AGT excretion correlated with the levels of glomerular damage, urinary protein excretion, and immunoreactivity for AGT and ANG II in kidney. On the other hand, plasma renin activity was significantly lower in the ATS group compared with the control group and significantly higher in the ATS+ARB group than in the ATS group. These data suggest that an increase in kidney-specific RAS activity, which parallels urinary AGT excretion, plays an important role in the development of ATS nephritis.

Decrease in Tumor Necrosis Factor-α Receptor-Associated Death Domain Results from Ubiquitin-Dependent Degradation in Obstructive Renal Injury in Rats

Increased expression levels of tumor necrosis factor-alpha (TNFalpha) is involved in tubulointerstitial cell proliferation and apoptosis in obstructive renal injury. Two TNFalpha receptors (TNFRs), TNFR1 and TNFR2, are known to exist. On TNFalpha binding, TNFR1 recruits TNFR-associated death domain (TRADD), an assembly platform to mediate TNFR1 signaling. We investigated postreceptor TRADD regulation in rat kidneys with unilateral ureteral obstruction (UUO). Whereas UUO was associated with increased expression levels of TNFalpha, TNFR1, TNFR2, and TRADD mRNAs, it resulted in the marked decrease of TRADD protein levels (which appeared at day 1 and persisted thereafter) and a slight decrease in TNFR1 protein levels at days 7 and 14. Both ubiquitination and degradation of TRADD were increased in UUO kidneys, degradation of TRADD was stimulated by TNFalpha in HK-2 cells, and TRADD degradation was suppressed by proteasome inhibitor. Inhibition of TNFalpha by soluble TNFR2, etanercept, reduced significantly, although transiently, tubular and interstitial cell proliferation, fibronectin expression, and apoptosis in UUO kidneys, and also suppressed TRADD degradation. These data suggest that the decrease in TRADD resulting from enhanced ubiquitin-dependent degradation is involved in obstructive renal injury. Since TRADD is not incorporated into TNFR2-mediated TNFalpha signaling, the persistent decrease in TRADD, associated with a mild decrease in TNFR1 levels, may function, at least in part, to divert TNFalpha signals toward a TNFR2-mediated pathway in UUO kidneys.

Enhanced intrarenal receptor-mediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake

Despite suppression of the circulating renin-angiotensin system (RAS), high salt intake (HSI) aggravates kidney injury in chronic kidney disease. To elucidate the effect of HSI on intrarenal RAS, we investigated the levels of intrarenal prorenin, renin, (pro)renin receptor (PRR), receptor-mediated prorenin activation, and ANG II in chronic anti-thymocyte serum (ATS) nephritic rats on HSI. Kidney fibrosis grew more severe in the nephritic rats on HSI than normal salt intake. Despite suppression of plasma renin and ANG II, marked increases in tubular prorenin and renin proteins without concomitant rises in renin mRNA, non-proteolytically activated prorenin, and ANG II were noted in the nephritic rats on HSI. Redistribution of PRR from the cytoplasm to the apical membrane, along with elevated non-proteolytically activated prorenin and ANG II, was observed in the collecting ducts and connecting tubules in the nephritic rats on HSI. Olmesartan decreased cortical prorenin, non-proteolytically activated prorenin and ANG II, and apical membranous PRR in the collecting ducts and connecting tubules, and attenuated the renal lesions. Cell surface trafficking of PRR was enhanced by ANG II and was suppressed by olmesartan in Madin-Darby canine kidney cells. These data suggest the involvement of the ANG II-dependent increase in apical membrane PRR in the augmentation of intrarenal binding of prorenin and renin, followed by nonproteolytic activation of prorenin, enhancement of renin catalytic activity, ANG II generation, and progression of kidney fibrosis in the nephritic rat kidneys on HSI. The origin of the increased tubular prorenin and renin remains to be clarified. Further studies measuring the urinary prorenin and renin are needed.

Tuberculous Arthritis Mimicking Neoplasm in a Hemodialysis Patient

Hemodialysis patients are known to develop the complication of extrapulmonary tuberculosis more frequently than the general population. Tuberculous arthritis is a rare form of extrapulmonary tuberculosis and is reported to occur in approximately 1% of cases in nonuremic patients. Only 3 cases in dialysis patients, who were not proven by a bacterial culture or had died before treatment, have been reported. We report herein a culture-proven case of tuberculous arthritis developing at the sternoclavicular joint, which initially mimicked an apparent neoplasm in a hemodialysis patient. A favorable outcome was obtained after antituberculous therapy. Tuberculosis must be considered one of the most significant diagnoses in hemodialysis patients who present with a tumor-like lesion.

The role of the ubiquitin-proteasome system in kidney diseases.

Proteins in mammalian cells are continually being degraded and synthesized. Protein degradation via the ubiquitin–proteasome system (UPS) is the major pathway for non-lysosomal proteolysis of intracellular proteins and plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, differentiation, apoptosis, sodium channel function, and modulation of inflammatory responses. The central element of this system is the covalent linkage of ubiquitins to targeted proteins, which are then recognized by the 26S proteasome composed of adenosine triphosphate-dependent, multi-catalytic proteases. Damaged or misfolded proteins, as well as regulatory proteins that control many critical cellular functions, are among the targets of this degradation process. Consequently, aberration of the system leads to dysregulation of cellular homeostasis and development of many diseases. Based on the findings, it is not surprising that abnormalities of the system are also associated with the pathogenesis of kidney diseases. In this review, I discuss (1) the basic mechanism of the UPS, and (2) the association between the pathogenesis of kidney diseases and the UPS. Diverse roles of the UPS are implicated in the development of kidney diseases, and further studies on this system may reveal new strategies for overcoming kidney diseases.