The effects of Tripterygium wilfordii Hook F on renal outcomes in type 2 diabetic kidney disease patients with severe proteinuria: a single-center cohort study.

AIM: Tripterygium wilfordii Hook F (TwHF) has been shown to substantially reduce proteinuria in patients with diabetic kidney disease (DKD); however, the effect of TwHF on renal outcomes in DKD remains unknown. Accordingly, we aimed to establish the effects of TwHF on renal outcomes in patients with DKD. METHODS: Overall, 124 patients with DKD, induced by type 2 diabetes mellitus, with 24-h proteinuria > 2 g, and an estimated glomerular filtration rate > 30 mL/min/1.73 m2 were retrospectively investigated. The renal outcomes were defined as doubling serum creatinine levels or end-stage kidney disease. Kaplan-Meier curves and Cox regression analyses were performed to analyze prognostic factors for renal outcomes. RESULTS: By the end of the follow-up, renal outcomes were observed in 23 and 11 patients in the non-TwHF and TwHF groups, respectively (p = 0.006). TwHF significantly reduced the risk of renal outcomes (adjusted hazard ratio [HR] 0.271, 95% confidence interval [CI] 0.111-0.660, p = 0.004) in patients with chronic kidney disease (CKD) G3 (adjusted HR 0.274, 95%CI 0.081-0.932, p = 0.039). Based on the Kaplan-Meier analysis, 1- and 3-year proportions of patients without renal outcomes were significantly lower in the non-TwHF group than those in the TwHF group (92.8% vs. 95.5% and 47.2% vs. 76.8%, respectively; p = 0.0018). CONCLUSION: In DKD patients with severe proteinuria, TwHF could prevent DKD progression, especially in patients with CKD G3. A randomized clinical trial is needed to elucidate the benefits of TwHF on renal outcomes in patients with DKD.

JAK-STAT signaling is activated in the kidney and peripheral blood cells of patients with focal segmental glomerulosclerosis.

Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT 1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS.

A rare case of Alport syndrome, atypical hemolytic uremic syndrome and Pauci-immune crescentic glomerulonephritis.

BACKGROUND: Renal thrombotic microangiopathy (TMA) is occasionally seen in biopsies with pauci-immune necrotizing crescentic glomerulonephritis (PCGN). Recent study indicated that the complement activation is more prominent in the ANCA-negative glomerulonephritis. CASE PRESENTATION: We report a case of concurrent TMA and PCGN without ANCA positivity. Interestingly, our patient also had biopsy features supportive of Alport syndrome (AS). Genetic studies identified variants and polymorphisms in alternative complement pathway genes that confer substantial risk of developing atypical hemolytic uremic syndrome (aHUS). CONCLUSIONS: Abnormal activation in complement pathway may represent a common pathogenic link between these three distinct entities.

A High Fat Diet During Pregnancy and Lactation Induces Cardiac and Renal Abnormalities in GLUT4 +/- Male Mice.

BACKGROUND/AIMS: Altered nutrients during the in utero (IU) and/or lactation (L) period predispose offspring to cardio-renal diseases in adulthood. This study investigates the effect of a high fat diet (HFD) fed to female mice during IU/L on gene expression patterns associated with heart and kidney failure and hypertension in male offspring. METHODS: Female wild type (WT) mice were fed either a HFD or control chow (C) prior to mating with males with a genetic heterozygous deletion of GLUT4 (G4+/-, a model of peripheral insulin resistance and hypertension) and throughout IU/L. After weaning male offspring were placed on a standard rodent chow until 24 weeks of age. RESULTS: All offspring exposed to a maternal HFD showed increased heart and kidney weight and reduced cardiac insulin responsiveness. G4+/- offspring on a HFD displayed early hypertension associated with increased renal gene expression of renin and the AT1- receptors compared to G4+/- on a C diet. This group showed decreased cardiac expression of key genes involved in fatty acid oxidation compared to WT on a C diet. CONCLUSIONS: These results indicate an interaction between a HFD diet and genotype during early life development that can enhance susceptibility to cardio-renal diseases later in life.

Deletion of Lkb1 in Renal Tubular Epithelial Cells Leads to CKD by Altering Metabolism.

Renal tubule epithelial cells are high-energy demanding polarized epithelial cells. Liver kinase B1 (LKB1) is a key regulator of polarity, proliferation, and cell metabolism in epithelial cells, but the function of LKB1 in the kidney is unclear. Our unbiased gene expression studies of human control and CKD kidney samples identified lower expression of LKB1 and regulatory proteins in CKD. Mice with distal tubule epithelial-specific Lkb1 deletion (Ksp-Cre/Lkb1(flox/flox)) exhibited progressive kidney disease characterized by flattened dedifferentiated tubule epithelial cells, interstitial matrix accumulation, and dilated cystic-appearing tubules. Expression of epithelial polarity markers ß-catenin and E-cadherin was not altered even at later stages. However, expression levels of key regulators of metabolism, AMP-activated protein kinase (Ampk), peroxisome proliferative activated receptor gamma coactivator 1-α (Ppargc1a), and Ppara, were significantly lower than those in controls and correlated with fibrosis development. Loss of Lkb1 in cultured epithelial cells resulted in energy depletion, apoptosis, less fatty acid oxidation and glycolysis, and a profibrotic phenotype. Treatment of Lkb1-deficient cells with an AMP-activated protein kinase (AMPK) agonist (A769662) or a peroxisome proliferative activated receptor alpha agonist (fenofibrate) restored the fatty oxidation defect and reduced apoptosis. In conclusion, we show that loss of LKB1 in renal tubular epithelial cells has an important role in kidney disease development by influencing intracellular metabolism.

Endoplasmic Reticulum Stress Predicts Clinical Response to Cyclosporine Treatment in Primary Membranous Nephropathy.

BACKGROUND: Little is known about the endoplasmic reticulum stress (ERS) marker glucose regulated protein 78 (GRP78) and calcineurin in the kidney in primary membranous nephropathy (PMN) and if they could predict post-cyclosporine treatment outcome. METHODS: This is a retrospective study using a dataset of biopsy-confirmed PMN from Peking Union Medical College Hospital from 1996 to 2014. Seventy-six adult patients treated with cyclosporine as primary immunosuppression for at least 6 months were studied. Immunohistochemistry was used to detect GRP78 and calcineurin in the kidney. Serum calcineurin was assayed by ELISA. Patients were grouped into no-remission (NR, n = 17), partial remission (PR, n = 39), or complete remission (CR, n = 20) at the end of 6 months of treatment. RESULTS: There was no difference of initial dose of cyclosporine among NR, PR, and CR groups. Kidney calcineurin expression in PMN was significantly increased compared to that in controls (p < 0.0083). The glomerular GRP78 in NR PMN was higher than that in control, CR and PR patients (p < 0.0083). Kidney calcineurin expression and GRP78 expression was positively correlated. However, there were no differences in either serum calcineurin levels or kidney calcineurin expressions among NR, PR or CR groups. There was a negative correlation between serum calcineurin activity and whole kidney calcineurin expression (p = 0.034) or glomerular calcineurin expression (p = 0.007). Neither kidney calcineurin nor GRP78 expression was correlated with proteinuria. CONCLUSIONS: ERS marker GRP78 in the glomeruli but not serum or kidney calcineurin expression could be a useful marker in PMN to negatively predict the response to cyclosporine treatment at the sixth month.

Differential urinary glycoproteome analysis of type 2 diabetic nephropathy using 2D-LC-MS/MS and iTRAQ quantification.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure and end-stage kidney disease. More accurate and non-invasive test for the diagnosis and monitoring the progression of DN is urgently needed for the better care of such patients. METHODS: In this study we utilized urinary glycoproteome to discover the differential proteins during the course of type 2 DN. The urinary glycoproteins from normal controls, normalbuminuira, microalbuminura, and macroalbuminuria patients were enriched by concanavalin A (ConA) and analyzed by 2DLC/MS/MS and isobaric tags for relative and absolute quantitation quantification. RESULTS: A total of 478 proteins were identified and 408 were annotated as N-linked glycoproteins. A total of 72, 107 and 123 differential proteins were identified in normalbuminuria, microalbuminuria and macroalbuminuria, respectively. By bioinformatics analysis, in normalbuminruia state, cell proliferation and cell movement were activated, which might reflect the compensatory phase during the disease development. In micro- and macro-albuminuria, cell death and apoptosis was activated, which might reflect the de-compensatory phase. Pathway analysis showed acute phase proteins, the member of high density lipoprotein and low density lipoprotein proteins were changed, indicating the role of the inflammatory response and lipid metabolism abnormality in the pathogenesis of DN. Six selected differential proteins were validated by Western Blot. Alpha-1-antitrypsin (SERPINA1) and Ceruloplasmin are the two markers with excellent area under curve values (0.929 and 1.000 respectively) to distinguish the microalbuminuria and normalbuminuria. For the first time, we found pro-epidermal growth factor and prolactin-inducible protein were decreased in macroalbuminuria stage, which might reflect the inhibition of cell viability and the activation of cell death in kidney. CONCLUSIONS: Above data indicated that urinary glycoproteome could be useful to distinguish the differences in protein profiles in different stages in DN, which will help better individualized care of patients in DN.

A multicolor podocyte reporter highlights heterogeneous podocyte changes in focal segmental glomerulosclerosis.

In contrast to most glomerular diseases, the injury pattern in focal segmental glomerulosclerosis (FSGS) is highly heterogeneous, even though podocytes are genetically identical and exposed to the same environmental factors. To understand changes in individual podocytes, we generated and analyzed a stochastic multicolor Cre-reporter, encoding four fluorescent proteins. In these animals, podocytes were randomly labeled allowing individual cells and their foot processes to be distinguished. In healthy animals, podocyte size and structure showed little cell to cell variability. In the doxorubicin-induced FSGS model, fluorescent-labeled glomerular podocyte numbers decreased and fluorescent cells could be recovered from the urine. The size of the remaining podocytes showed a high degree of heterogeneity, some cells remained small, while others enlarged. Both enlarged and non-enlarged podocytes showed alterations in their foot process morphology. Thus, by the virtue of a multicolor cre-reporter, individual podocytes could be viewed in real time at a cellular resolution indicating a heterogeneous podocyte injury response during the pathogenesis of FSGS.

[Expression of calcineurin in podocytes in response to endoplasmic reticulum stress in diabetic nephropathy].

OBJECTIVE: To investigate the regulation of calcineurin (CaN) by endoplasmic reticulum stress (ERS) in podocytes in vitro and in vivo at the stage microalbuminuria in diabetic nehropathy (DN). METHODS: The urinary albumin excretions of C57BLKS/J (Lepr) db/db and db/m mice at the ages of 6, 9, and 12 weeks were measured. The expressions of CaN and synaptopodin of these mice were observed. In immortalized mouse podocytes, the expression of podocyte CaN incubated with different concentrations of paltimate was quantitatively determined by real-time PCR. The changes of CaN incubated with paltimate with or without ursodeoxy-cholic acid (UDCA) were analyzed by confocal microscopy and Western blotting. RESULTS: As urine protein increased, the expression of CaN was enhanced and the expression of synaptopodin was reduced in early stage DN db/db mice potocytes. In immortalized mouse podocytes, as the concentrations of palmitate increased, CaN mRNA increased. By confocal microscopy, the fluorescence intensity of CaN increased in palmitate treatment group. After co-incubation with palmitate and UDCA, the fluorescence intensity decreased. The similar results were shown by Western blotting. CONCLUSION: At the stage of microalbuminuria in DN, ERS in podocytes up-regulates the expression of CaN.

A Survey for Charting Intake, Output, and Body Weight in the Electronic Medical Record.

BACKGROUND: The accuracy of documentation of body weight and fluid balance in hospitalized patients is frequently questioned. METHODS: We conducted a survey to understand provider perceptions of the accuracy of intake, output, and weight charting in the electronic medical record. We sent a six-item questionnaire to nurses and physicians who provide inpatient service in a community-based teaching hospital of the Northeastern United States. We compared the response difference between nurses and physicians by Fisher exact test. RESULTS: One hundred eight nurses and 39 physicians participated in the survey. Both nurses and physicians responded that the accuracy of documentation is crucial. However, only 25.7% of participating physicians and 38.3% of participating nurses considered that documentation in the electronic medical record is reliable. Both physicians and nurses assumed that the nurses are too busy to collect and document the data, and the variability of non-patient weight and variations in body weight measurement under different conditions account for inaccuracies in the documented body weight. CONCLUSIONS: Assessing the accuracy of documenting intake, output, and body weight in the electronic medical record is warranted. Providers believe that educating patients about fluid balance and volume assessment help to improve the accuracy in charting intake, output, and body weight in the electronic medical record.

Antineutrophil Cytoplasmic Antibody-Associated Vasculitis with Active Kidney Involvement in the United States: 2016-2020.

Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and its subtypes, granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic GPA (EGPA), frequently present with acute kidney injury and can often lead to kidney failure, even with successful induction therapy. Few contemporary, nationally representative studies have described hospital complications of AAV. Methods: Using data from the 2016-2020 National Inpatient Sample, a nationally representative database, we identified hospitalizations from adults with a new diagnosis of AAV (subtype or unspecified) and an inpatient kidney biopsy during the index hospitalization. We described baseline characteristics, associated inpatient procedures and complications, and compared lengths of stay and costs by geographic region, hospital characteristics, and AAV subtype. Results: We identified an average of 1,329 cases of hospitalized AAV with a concurrent kidney biopsy per year over the 5-year period. More than 50% were not designated as having a specific subtype, likely owing to delays in documentation of histopathology. Kidney involvement was severe as the majority of patients developed acute kidney injury, and the proportion of patients who required inpatient dialysis was approximately 24%. Approximately 20% of patients developed hypoxia. Inpatient plasmapheresis was delivered to 20.4% and 20.6% of patients with GPA and MPA, respectively. There were no clinically meaningful or statistically significant differences in adjusted length of stay or inpatient costs among AAV subtypes. Admission in the Midwest region was associated with shorter hospital stays and lower costs than that in the Northeast, South, or West regions of the USA (adjusted p = 0.007 and <0.001, respectively). Conclusion: AAV with acute kidney involvement remains a challenging, high-risk condition. Maintaining a high index of suspicion and a low threshold for kidney biopsy should help ameliorate short- and long-term complications.

Plasmapheresis in ANCA-Associated Vasculitis with Active Kidney Involvement in the United States (2016-2020): A Cross-Sectional Study.

INTRODUCTION: Plasmapheresis is currently recommended when antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) presents with severe kidney and/or lung involvement. This cross-sectional study aimed at describing characteristics of hospitalized patients diagnosed with AAV with severe kidney involvement undergoing plasmapheresis in the US. METHODS: We defined the study population as adults hospitalized for active kidney involvement with a new diagnosis of AAV (by subtype or unspecified). We established the cohort from the 2016-2020 National Inpatient Sample by ICD-10-CM codes. In this cross-sectional study, we described demographic and clinical characteristics, associated inpatient procedures, lengths of stay, hospital costs, and disposition at discharge comparing patients treated and not treated with plasmapheresis. RESULTS: We identified a total of 975 cases of hospitalized AAV with acute kidney involvement in the US treated by plasmapheresis over the 5-year period. Demographic characteristics of patients who received plasmapheresis were similar to those in patients who did not (n=5670). There were no regional differences in the proportion of patients who received plasmapheresis; however, plasmapheresis was deployed more frequently among patients admitted to urban teaching hospitals relative to rural and non-teaching hospitals. Cases treated with plasmapheresis were more likely to have had acute kidney injury (AKI) (96% vs. 90%, p=0.0007), AKI requiring dialysis (52% vs 16%, p<0.001), hypoxia (40% vs. 16%, p<0.0001), and respiratory failure requiring mechanical ventilation (13% vs. 3%, p=0.0003). CONCLUSION: During 2016-2020, plasmapheresis was deployed in approximately 20% of patients being admitted for AAV and acute kidney involvement in the US. As standards of care and practice evolve, the role of plasmapheresis in the management of AAV with acute kidney involvement will require further study.

Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury.

The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cell activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could affect nidogen-1 accessibility. Phosphoproteomics revealed that the 'AKI protector' Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin ß1 to induce Wnt in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.

pSTAT1 Is Activated during the Progression of IgA Nephropathy.

Introduction: IgA nephropathy is the most common primary glomerular disease. Its pathogenesis is still poorly understood. Alterations of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway may play an important role in IgA nephropathy. Methods: We evaluated the clinical features, pathology, and tissue staining for lymphocytes and phosphorylated STAT1 (pSTAT1) in 43 patients with biopsy proven IgA nephropathy. They were followed to determine their disease outcomes. All had biopsy tissue and multiple laboratory measurements to assess their kidney disease progression. Sixteen patients underwent repeat kidney biopsy to further assess their clinical status. Results: The median eGFR at baseline was 61 mL/min/1.73 m2 and the median proteinuria was 2,600 mg/d. The median follow-up was 5 years with an average annual decline in eGFR of 2.25 mL/min/1.73 m2. There was significant inflammation and atrophy seen in the first biopsy, which progressed among those who undertook a 2nd biopsy. Compared to healthy kidney tissue, glomeruli and tubulointerstitium demonstrated increased lymphocyte (CD3+) infiltrates and increased pSTAT1 staining by immunohistochemistry. Increased CD3 (p = 0.001) staining and increased pSTAT1 (p = 0.03) correlated with reduced eGFR levels. In repeat biopsy samples, increasing pSTAT1 staining correlated with loss of eGFR over time (p = 0.02). Conclusion: These findings support the hypothesis that pSTAT1 is activated in IgA nephropathy and may play a role in the progression toward kidney failure.

Urinary complement proteins in IgA nephropathy progression from a relative quantitative proteomic analysis.

Aim: IgA nephropathy (IgAN) is one of the leading causes of end-stage renal disease (ESRD). Urine testing is a non-invasive way to track the biomarkers used for measuring renal injury. This study aimed to analyse urinary complement proteins during IgAN progression using quantitative proteomics. Methods: In the discovery phase, we analysed 22 IgAN patients who were divided into three groups (IgAN 1-3) according to their estimated glomerular filtration rate (eGFR). Eight patients with primary membranous nephropathy (pMN) were used as controls. Isobaric tags for relative and absolute quantitation (iTRAQ) labelling, coupled with liquid chromatography-tandem mass spectrometry, was used to analyse global urinary protein expression. In the validation phase, western blotting and parallel reaction monitoring (PRM) were used to verify the iTRAQ results in an independent cohort (N = 64). Results: In the discovery phase, 747 proteins were identified in the urine of IgAN and pMN patients. There were different urine protein profiles in IgAN and pMN patients, and the bioinformatics analysis revealed that the complement and coagulation pathways were most activated. We identified a total of 27 urinary complement proteins related to IgAN. The relative abundance of C3, the membrane attack complex (MAC), the complement regulatory proteins of the alternative pathway (AP), and MBL (mannose-binding lectin) and MASP1 (MBL associated serine protease 2) in the lectin pathway (LP) increased during IgAN progression. This was especially true for MAC, which was found to be involved prominently in disease progression. Alpha-N-acetylglucosaminidase (NAGLU) and α-galactosidase A (GLA) were validated by western blot and the results were consistent with the iTRAQ results. Ten proteins were validated in a PRM analysis, and these results were also consistent with the iTRAQ results. Complement factor B (CFB) and complement component C8 alpha chain (C8A) both increased with the progression of IgAN. The combination of CFB and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) also showed potential as a urinary biomarker for monitoring IgAN development. Conclusion: There were abundant complement components in the urine of IgAN patients, indicating that the activation of AP and LP is involved in IgAN progression. Urinary complement proteins may be used as biomarkers for evaluating IgAN progression in the future.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis.

In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy; however, how cellular mechanics and metabolism are connected remains unclear. Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, Calponin 2 (CNN2) is predominantly expressed in fibroblasts and pericytes. CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway results in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. In patients, increased serum CNN2 levels are correlated with lipid content. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2) binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro , ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis.

OBJECTIVE: In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear. METHODS: A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed. RESULT: Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. CONCLUSIONS: Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Correlations of podocyte injury with glucose regulated protein 78 expression and proteinuria in patients with diabetic nephropathy.

OBJECTIVE: To explore the podocyte injury in patients with diabetic nephropathy (DN) and analyze its relationship with glucose regulated protein 78 (GRP78) and proteinuria. METHODS: The clinical data of 48 patients diagnosed as DN by renal biopsy were reviewed. All patients were divided into two groups according to proteinuria (>3.5 g/d, n=31 and 3.5 g/d, n=17). The density of podocytes was illustrated by immunohistochemistry staining of Wilms tumor-1 (WT-1), and the immunofluorescence double-staining results of synaptopodin and GRP78 in podocytes were detected. RESULTS: The podocyte dentistry of urine protein > 3.5 g/d group was significantly lower than that of urine protein>3.5 g/d group urine protein<3.5 g/d group(P=0.003), and it was negatively correlated with proteinuria (P=0.005). The expressions of synaptopodin and GRP78 in podocytes were also negatively correlated with proteinuria (P=0.004 and P=0.001). CONCLUSION: The podocyte injury is aggravated with increased proteinuria in DN patients, along with the decrease of the adaptive ability of endoplasmic reticulum to stress.

Th17/Treg imbalance in adult patients with minimal change nephrotic syndrome.

To determine whether Th17/Treg balance was abnormal in adult patients with minimal change nephrotic syndrome (MCNS), we studied 25 patients with new-onset MCNS and 20 normal persons. The results showed that MCNS patients exhibited a significant increase in Th17 number, Th17-related cytokines (IL-17 and IL-23), and transcription factor (RORγt) levels, as well as an obvious decrease in Treg number, Treg-related cytokines (TGF-ß1 and IL-10), and transcription factor (Foxp3) levels. The Th17/Treg ratios increased along with increased proteinuria and decreased albumin levels in patients with MCNS. IL-17 protein expression was also detected in the renal biopsy tissue of MCNS patients, particularly in patients with acute renal failure. Further, Th17/Treg balance returned to normal after effective corticosteroids therapy in 16 MCNS patients. These results indicated that Th17/Treg imbalance existed in MCNS patients, suggesting a potential role of Th17/Treg imbalance in the pathogenesis of MCNS.

[Effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells].

OBJECTIVE: To investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells. METHODS: The in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot. RESULTS: Oil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group). CONCLUSIONS: By downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.