Impairment of Proteasome Function in Podocytes Leads to CKD.

BACKGROUND: The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system (APLS) are major intracellular degradation procedures. The importance of the APLS in podocytes is established, but the role of the UPS is not well understood. METHODS: To investigate the role of the UPS in podocytes, mice were generated that had deletion of Rpt3 (Rpt3pdKO), which encodes an essential regulatory subunit required for construction of the 26S proteasome and its deubiquitinating function. RESULTS: Rpt3pdKO mice showed albuminuria and glomerulosclerosis, leading to CKD. Impairment of proteasome function caused accumulation of ubiquitinated proteins and of oxidative modified proteins, and it induced podocyte apoptosis. Although impairment of proteasome function normally induces autophagic activity, the number of autophagosomes was lower in podocytes of Rpt3pdKO mice than in control mice, suggesting the autophagic activity was suppressed in podocytes with impairment of proteasome function. In an in vitro study, antioxidant apocynin and autophagy activator rapamycin suppressed podocyte apoptosis induced by proteasome inhibition. Moreover, rapamycin ameliorated the glomerular injury in the Rpt3pdKO mice. The accumulation of ubiquitinated proteins and of oxidative modified proteins, which were detected in the podocytes of Rpt3pdKO mice, is a characteristic feature of aging. An aging marker was increased in the podocytes of Rpt3pdKO mice, suggesting that impairment of proteasome function promoted signs of aging in podocytes. CONCLUSIONS: Impairment of proteasome function in podocytes led to CKD, and antioxidants and autophagy activators can be therapeutic agents for age-dependent CKD.

MAGI-2 orchestrates the localization of backbone proteins in the slit diaphragm of podocytes.

Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained.

Glomerulosclerosis Induced by Deficiency of Membrane-Associated Guanylate Kinase Inverted 2 in Kidney Podocytes.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a component of the slit diaphragm (SD) of glomerular podocytes. Here, we investigated the podocyte-specific function of MAGI-2 using newly generated podocyte-specific MAGI-2-knockout (MAGI-2-KO) mice. Compared with podocytes from wild-type mice, podocytes from MAGI-2-KO mice exhibited SD disruption, morphologic abnormalities of foot processes, and podocyte apoptosis leading to podocyte loss. These pathologic changes manifested as massive albuminuria by 8 weeks of age and glomerulosclerosis and significantly higher plasma creatinine levels at 12 weeks of age; all MAGI-2-KO mice died by 20 weeks of age. Loss of MAGI-2 in podocytes associated with decreased expression and nuclear translocation of dendrin, which is also a component of the SD complex. Dendrin translocates from the SD to the nucleus of injured podocytes, promoting apoptosis. Our coimmunoprecipitation and in vitro reconstitution studies showed that dendrin is phosphorylated by Fyn and dephosphorylated by PTP1B, and that Fyn-induced phosphorylation prevents Nedd4-2-mediated ubiquitination of dendrin. Under physiologic conditions in vivo, phosphorylated dendrin localized at the SDs; in the absence of MAGI-2, dephosphorylated dendrin accumulated in the nucleus. Furthermore, induction of experimental GN in rats led to the downregulation of MAGI-2 expression and the nuclear accumulation of dendrin in podocytes. In summary, MAGI-2 and Fyn protect dendrin from Nedd4-2-mediated ubiquitination and from nuclear translocation, thereby maintaining the physiologic homeostasis of podocytes, and the lack of MAGI-2 in podocytes results in FSGS.

Re-expression of Sall1 in podocytes protects against adriamycin-induced nephrosis.

The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KOp°d°/p°d°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes. Under physiological conditions, Sall1 KOp°d°/p°d° mice exhibited no proteinuria during their lifetime, but foot-process effacement was detected in some of the podocytes. To elucidate the role of Sall1 in injured podocytes, we used an adriamycin (ADR)-induced model of nephrosis and glomerulosclerosis. Surprisingly, the expression of Sall1 was elevated in control mice on day 14 after ADR injection. On day 28 after ADR injection, Sall1 KOp°d°/p°d° mice exhibited significantly higher levels of proteinuria and higher numbers of sclerotic glomeruli. Differentiated Sall1 KD podocytes showed a loss of synaptopodin, suppressed stress fiber formation, and, ultimately, impaired directed cell migration. In addition, the loss of Sall1 increased the number of apoptotic podocytes following ADR treatment. These results indicated that Sall1 has a protective role in podocytes; thus, we investigated the endoplasmic reticulum stress marker GRP78. GRP78 expression was higher in ADR-treated Sall1 KOp°d°/p°d° mice than in control mice. Sall1 appeared to influence the expression of GRP78 in injured podocytes. These results suggest that Sall1 is associated with actin reorganization, endoplasmic reticulum stress, and apoptosis in injured podocytes. These protective aspects of Sall1 re-expression in injured podocytes may have the potential to reduce apoptosis and possibly glomerulosclerosis.

Cathepsin D in Podocytes Is Important in the Pathogenesis of Proteinuria and CKD.

Studies have revealed many analogies between podocytes and neurons, and these analogies may be key to elucidating the pathogenesis of podocyte injury. Cathepsin D (CD) is a representative aspartic proteinase in lysosomes. Central nervous system neurons in CD-deficient mice exhibit a form of lysosomal storage disease with a phenotype resembling neuronal ceroid lipofuscinoses. In the kidney, the role of CD in podocytes has not been fully explored. Herein, we generated podocyte-specific CD-knockout mice that developed proteinuria at 5 months of age and ESRD by 20-22 months of age. Immunohistochemical analysis of these mice showed apoptotic podocyte death followed by proteinuria and glomerulosclerosis with aging. Using electron microscopy, we identified, in podocytes, granular osmiophilic deposits (GRODs), autophagosome/autolysosome-like bodies, and fingerprint profiles, typical hallmarks of CD-deficient neurons. CD deficiency in podocytes also led to the cessation of autolysosomal degradation and accumulation of proteins indicative of autophagy impairment and the mitochondrial ATP synthase subunit c accumulation in the GRODs, again similar to changes reported in CD-deficient neurons. Furthermore, both podocin and nephrin, two essential components of the slit diaphragm, translocated to Rab7- and lysosome-associated membrane glycoprotein 1-positive amphisomes/autolysosomes that accumulated in podocyte cell bodies in podocyte-specific CD-knockout mice. We hypothesize that defective lysosomal activity resulting in foot process effacement caused this accumulation of podocin and nephrin. Overall, our results suggest that loss of CD in podocytes causes autophagy impairment, triggering the accumulation of toxic subunit c-positive lipofuscins as well as slit diaphragm proteins followed by apoptotic cell death.

Transient increase in proteinuria, poly-ubiquitylated proteins and ER stress markers in podocyte-specific autophagy-deficient mice following unilateral nephrectomy.

Previous studies have revealed that podocytes normally can be associated with a very high degree of autophagic activity, and that a lack of autophagic activity in podocytes is associated with susceptibility to disease and to late-onset glomerulosclerosis. In the present study, we conducted unilateral nephrectomy as a surgical model for acute nephron reduction. First, using GFP-LC3 transgenic mice to monitor autophagy, we found that glomerular autophagy could be transiently suppressed by surgery, but that it was restored quickly. To further explore the significance of podocyte autophagy after unilateral nephrectomy, we investigated podocyte-specific Atg7-deficient mice. The knockout mice exhibited no pathological phenotype compared with wild-type mice before nephrectomy. However, 1 day after nephrectomy, significantly higher levels of proteinuria and ultrastructural changes that included foot process effacement and a significant reduction in podocyte number were detected in mice harboring Atg7-deficient podocytes. Moreover, biochemical and immunohistochemical analyses showed a robust increase in polyubiquitin levels and ER stress markers in the glomeruli of the mice with autophagy-deficient podocytes. These results show the importance of the autophagic process in podocytes for maintaining a normal degree of filtration function during the adaptation to compensatory kidney hypertrophy following unilateral nephrectomy.

A Rare Case of Drug Reaction with Eosinophilia and Systemic Symptoms Syndrome Leading to Hemodialysis and Multiple Infections: A Case Report and Literature Review.

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a severe drug eruption that causes multiple organ damage. The renal impairment in these patients usually improves with immunosuppressants, but subsequent infections often develop. We herein report a rare case of DRESS syndrome leading to hemodialysis and multiple infections with Pneumocystis pneumonia, cytomegalovirus and Aspergillus despite the administration of low-dose prednisolone. We also present a literature review of cases requiring dialysis after DRESS syndrome. In patients with chronic kidney disease, it is important to be alert for not only the development of DRESS syndrome but also subsequent infections.

Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-µm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Intractable membranous lupus nephritis showing selective improvement of subepithelial deposits with tacrolimus therapy: a case report.

A 37-year-old female patient was admitted for evaluation of nephrotic proteinuria refractory to prednisolone and other immunosuppressants in 2004. On admission, urinary protein loss was 16 g/d. Anti-ds DNA antibody was positive and hypocomplementemia was detected. Renal biopsy revealed membranous lupus nephritis. Because 5 cyclophosphamide pulse therapies did not have an effect, tacrolimus was started at 3 mg daily. Proteinuria decreased to 4.8 g/d after 5 months and was < 0.1 g/d in 2009, but antids DNA antibody remained positive and hypocomplementemia persisted. Repeat renal biopsy revealed thinning of the glomerular capillary walls and disappearance of subepithelial electron-dense deposits. However, the subendothelial and mesangial deposits were unchanged. In this patient, proteinuria refractory to various immunosuppressants including cyclosporine A improved after administration of tacrolimus, and selective disappearance of subepithelial deposits was seen histologically. This is the first histological evidence that tacrolimus therapy may cause removal of subepithelial deposits, which are separated from the circulation by the glomerular basement membrane. This finding is supported by experimental data that tacrolimus selectively block the binding of FK-binding protein 12 to transient receptor potential-cation channel 6, resulting in normalization of affected podocytes.

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice.

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165-1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.

Effects of 22-oxa-calcitriol on podocyte injury in adriamycin-induced nephrosis.

BACKGROUND: In various animal studies, vitamin D has been shown to have a significant effect on reduction of proteinuria and the progression of kidney disease. However, little is known on its renoprotective effect in adriamycin (ADR)-induced nephrosis mice. The present study was intended to determine the therapeutic benefit of 22-oxa-calcitriol (OCT), a vitamin D analog, in reducing proteinuria and its renoprotective effect, i.e. preventing podocyte injury on ADR-induced nephrosis mice. METHODS: Three experimental groups were used as follows: (1) nephrosis mice, established by a single intravenous injection of ADR; (2) ADR+OCT mice, nephrosis mice treated with OCT, and (3) mice treated only with OCT as the control group. Podocyte injury was assessed by podocyte apoptosis using the TUNEL assay, podocyte counting, podocyte-specific expressed protein by immunofluorescence and Western blot analysis, and foot process effacement using electron microscopy. RESULTS: Lower proteinuria was observed in ADR+OCT mice. Improvement in glomerulosclerosis and interstitial fibrosis, and prevention of glomerular hyperfiltration were observed in ADR+OCT mice. Immunofluorescence and Western blot analyses showed restoration of downregulated expression of nephrin, CD2AP and podocin. Nevertheless, dendrin expression was not restored. An insignificant reduction in podocyte numbers was found in ADR+OCT mice. Complete foot process effacement was partially prevented in ADR+OCT mice. CONCLUSIONS: The results indicate that OCT reduces podocyte injury and has renoprotective effects in ADR nephrosis mice.

Risk of overestimation of kidney function using GFR-estimating equations in patients with low inulin clearance.

BACKGROUND: Accurate estimation of the glomerular filtration rate (GFR) is very important in clinical practice. Although renal inulin clearance (Cin) is the gold standard for measuring GFR, the procedure for Cin measurement is complicated. Use of GFR-estimating equations has been increasing recently due to their simplicity. The objectives of the present study are to analyze the correlation between Cin and other GFR-estimating parameters and to investigate their clinical usefulness and limitation. METHODS: Seventy-two Japanese patients were enrolled in this study. Cin was measured by the continuous infusion method. Serum creatinine (s-Cr), cystatin C, uric acid (UA), and hemoglobin (Hb) were measured. The Japanese formula of estimated GFR (eGFR) was as follows: eGFR (ml/min/1.73m(2) ) = 194 × s-Cr(-1.094) × Age(-0.287) × 0.739 (if female). The endogenous creatinine clearance test was also performed. RESULTS: Levels of Cin were highly correlated with those of endogenous creatinine clearance (Ccr) (R(2) = 0.7585) and eGFR (R(2) = 0.5659). However, patients with lower Cin showed unexpectedly elevated levels of endogenous Ccr and eGFR. Moreover, the levels of eGFR tended to be unexpectedly increased in patients with low body surface area. CONCLUSION: Although GFR-estimating equations are useful for estimating GFR accurately, they pose a risk of overestimation of kidney function in patients with decreased GFRor a poor physique.

Rac1 in podocytes promotes glomerular repair and limits the formation of sclerosis.

Rac1, a Rho family member, is ubiquitously expressed and participates in various biological processes. Rac1 expression is induced early in podocyte injury, but its role in repair is unclear. To investigate the role of Rac1 expression in podocytes under pathological conditions, we used podocyte-specific Rac1 conditional knock-out (cKO) mice administered adriamycin (ADR), which causes nephrosis and glomerulosclerosis. Larger areas of detached podocytes, more adhesion of the GBM to Bowman's capsule, and a higher ratio of sclerotic glomeruli were observed in Rac1 cKO mice than in control mice, whereas no differences were observed in glomerular podocyte numbers in both groups after ADR treatment. The mammalian target of rapamycin (mTOR) pathway, which regulates the cell size, was more strongly suppressed in the podocytes of Rac1 cKO mice than in those of control mice under pathological conditions. In accordance with this result, the volumes of podocytes in Rac1 cKO mice were significantly reduced compared with those of control mice. Experiments using in vitro ADR-administered Rac1 knockdown podocytes also supported that a reduction in Rac1 suppressed mTOR activity in injured podocytes. Taken together, these data indicate that Rac1-associated mTOR activation in podocytes plays an important role in preventing the kidneys from developing glomerulosclerosis.

Significance of urinary full-length megalin in patients with IgA nephropathy.

BACKGROUND AND OBJECTIVES: Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin) assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years) and 5 patients with membranous nephropathy (MN). Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA. RESULTS: Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR = 1.76, 95% CI: 1.04-3.27, P<0.05). There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (ß = 0.33, P = 0.008). The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05). The levels of urinary C-megalin were significantly higher in MN patients compared to the control group. CONCLUSIONS: The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is an independent predictor of disease progression of IgAN. In addition, our results suggest that urinary C-megalin is a marker of glomerular abnormalities in various glomerular diseases as well as IgAN.

Notch2 activation ameliorates nephrosis.

Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases. Here we show that Notch2 prevents podocyte loss and nephrosis. Administration of a Notch2 agonistic monoclonal antibody ameliorates proteinuria and glomerulosclerosis in a mouse model of nephrosis and focal segmental glomerulosclerosis. In vitro, the specific knockdown of Notch2 increases apoptosis in damaged podocytes, while Notch2 agonistic antibodies enhance activation of Akt and protect damaged podocytes from apoptosis. Treatment with triciribine, an inhibitor of Akt pathway, abolishes the protective effect of the Notch2 agonistic antibody. We find a positive linear correlation between the number of podocytes expressing activated Notch2 and the number of residual podocytes in human nephrotic specimens. Hence, specific activation of Notch2 rescues damaged podocytes and activating Notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis.

Clinical and pathological evaluation of hypertensive emergency-related nephropathy.

OBJECTIVE: The prognosis of patients with hypertensive emergencies has recently improved dramatically owing to the development of effective antihypertensive therapy. We examined the histological and clinical features of patients with hypertensive emergency-related nephropathy. METHODS: Twelve patients (11 men and one woman) were diagnosed as having hypertensive emergencies with acute renal failure according to the Joint National Committee-7 classification of blood pressure for adults and underwent renal biopsies at our hospital between 1995 and 2008. These patients were enrolled in this retrospective study. RESULTS: The age of the subjects was 40.1±9.8 years. At presentation, the mean systolic/diastolic blood pressure was 232±32/146±12 mmHg and none of the patients were being treated with antihypertensive drugs, although 10 patients had histories of hypertension. The mean serum creatinine level was 6.1±4.7 mg/dL. All 12 patients showed left ventricular hypertrophy on echocardiography. On light microscopy of the renal biopsy specimens, all 12 patients showed onion skin patterns of the arterioles; however, no fibrinoid necrosis of the small arteries was found. Electron microscopy revealed electron-lucent widening of the subendothelial zone of the glomerular capillary walls in seven patients. One of the 12 patients did not respond to medical therapy and required regular dialysis. The other 11 patients responded to treatment. CONCLUSION: An onion skin pattern of the arterioles is the most frequent histological finding in patients with hypertensive emergency-related nephropathy. Long-standing hypertension might contribute to this arteriolar change, since left ventricular hypertrophy was also seen in these patients. With strict control of hypertension using antihypertensive medications, the prognosis of patients with hypertensive emergency-related nephropathy can be improved.