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Chronic kidney disease (CKD) can progress to kidney failure and require dialysis or transplantation, while early diagnosis can alter the course of disease and lead to better outcomes in both pediatric and adult patients. Significant CKD comorbidities include the manifestation of cardiovascular disease, heart failure, coronary disease, and hypertension. The pathogenesis of chronic kidney diseases can present as subtle and especially difficult to distinguish between different glomerular pathologies. Early detection of adult and pediatric CKD and detailed mechanistic understanding of the kidney damage can be helpful in delaying or curtailing disease progression via precise intervention toward diagnosis and prognosis. Clinically, serum creatinine and albumin levels can be indicative of CKD, but often are a lagging indicator only significantly affected once kidney function has severely diminished. The evolution of proteomics and mass spectrometry technologies has begun to provide a powerful research tool in defining these mechanisms and identifying novel biomarkers of CKD. Many of the same challenges and advances in proteomics apply to adult and pediatric patient populations. Additionally, proteomic analysis of adult CKD patients can be transferred directly toward advancing our knowledge of pediatric CKD as well. In this review, we highlight applications of proteomics that have yielded such biomarkers as PLA2R, SEMA3B, and other markers of membranous nephropathy as well as KIM-1, MCP-1, and NGAL in lupus nephritis among other potential diagnostic and prognostic markers. The potential for improving the clinical toolkit toward better treatment of pediatric kidney diseases is significantly aided by current and future development of proteomic applications.

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase–associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P

Background: Lupus nephritis (LN) is a prevalent and severe complication of systemic lupus erythematosus (SLE). Non-invasive diagnostics are limited, and current therapies have inadequate response rates. Expression of the chemokine Interferon-γ-induced protein 10 (IP-10) is regulated by Interferon-γ signaling and NF-κB, and its molecular activity and enhanced urine concentrations are implicated in LN, but its utility as a diagnostic marker and association with demographic, clinical, or pathologic features is not defined. Methods: 38 LN patients and 11 patients with non-LN glomerular diseases (GD) with active disease were included. Eighteen of the LN patients had achieved remission at one follow-up during the study time. Serum and urine were obtained from these samples, and the IP-10 levels were measured. Results: Serum and urine IP-10 levels are significantly enhanced in LN patients with active disease as compared with normal individuals (serum average 179.7 pg/mL vs. 7.2 pg/mL, p < 0.0001; urine average 28.7 pg/mg vs. 1.6 pg/mg, p = 0.0019) and patients with other forms of glomerular disease (serum average 179.7 pg/mL vs. 84.9 pg/mL, p = 0.0176; urine average 28.7 pg/mg vs. 0.18 pg/mg, p = 0.0011). Urine IP-10 levels are significantly higher in patients with proliferative LN (PLN) than those with membranous LN (MLN) (average 32.8 pg/mg vs. 7.6 pg/mg, p = 0.0155). Urine IP-10 levels are also higher in MLN versus primary membranous nephropathy (MN) (average 7.6 pg/mg vs. 0.2 pg/mg, p = 0.0193). Importantly, serum IP-10 levels remain elevated during active LN and LN remission, but urine IP-10 levels are decreased from active LN to remission in 72% of our patients. Lastly, serum, but not urine IP-10 levels are significantly higher in African American than White American LN patients in active LN (average 227.8 pg/mL vs. 103.4 pg/mL, p = 0.0309) and during LN remission (average 254.6 pg/mL vs. 89.2 pg/mL, p = 0.0399). Conclusions: Our findings suggest that serum and urine IP-10 measurements provide promising tests for monitoring LN activity, differentiation between classifications of LN, and differentiation between LN and other forms of glomerular disease. We also conclude that further assessment of elevated IP-10 levels in the serum and urine of high-risk populations (i.e., African American) could be beneficial in determining why many of these patients have worse outcomes and are non-responsive to standard therapeutics.

Kidney involvement in systemic lupus erythematosus (SLE)—termed lupus nephritis (LN)—is a severe manifestation of SLE that can lead to end-stage kidney disease (ESKD). LN is characterized by immune complex deposition and inflammation in the glomerulus. We tested the hypothesis that autoantibodies targeting podocyte and glomerular cell proteins contribute to the development of immune complex formation in LN. We used Western blotting with SLE sera from patients with and without LN to identify target antigens in human glomerular and cultured human-derived podocyte membrane proteins. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified the proteins in the gel regions corresponding to reactive bands observed with sera from LN patients. We identified 102 proteins that were present in both the podocyte and glomerular samples. We identified 10 high-probability candidates, including moesin, using bioinformatic analysis. Confirmation of moesin as a target antigen was conducted using immunohistochemical analysis (IHC) of kidney biopsy tissue and enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies. By IHC, biopsies from patients with proliferative lupus nephritis (PLN, class III/IV) demonstrated significantly increased glomerular expression of moesin (p &lt, 0.01). By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against moesin (p &lt, 0.01). This suggests that moesin is a target glomerular antigen in lupus nephritis.

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

Numerous studies show a direct relation between circulating autoantibodies, characteristic of systemic autoimmune disorders, and primary hypertension in humans. Whether these autoantibodies mechanistically contribute to the development of hypertension remains unclear. Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by aberrant immunoglobulin production, notably pathogenic autoantibodies, and is associated with prevalent hypertension, renal injury, and cardiovascular disease. Because plasma cells produce the majority of serum immunoglobulins and are the primary source of autoantibodies in SLE, we hypothesized that plasma cell depletion using the proteasome inhibitor bortezomib would lower autoantibody production and attenuate hypertension. Thirty-week-old female SLE (NZBWF1) and control (NZW [New Zealand White]) mice were injected IV with vehicle (0.9% saline) or bortezomib (0.75 mg/kg) twice weekly for 4 weeks. Bortezomib treatment significantly lowered the percentage of bone marrow plasma cells in SLE mice. Total plasma IgG and anti-dsDNA IgG levels were higher in SLE mice compared with control mice but were lowered by bortezomib treatment. Mean arterial pressure (mm Hg) measured in conscious mice by carotid artery catheter was higher in SLE mice than in control mice, but mean arterial pressure was significantly lower in bortezomib-treated SLE mice. Bortezomib also attenuated renal injury, as assessed by albuminuria and glomerulosclerosis, and reduced glomerular immunoglobulin deposition and B and T lymphocytes infiltration into the kidneys. Taken together, these data show that the production of autoantibodies by plasma cells mechanistically contributes to autoimmune-associated hypertension and suggests a potential role for patients with primary hypertension who have increased circulating immunoglobulins.

Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor–stimulated enhanced expression and secretion of NF-κB–targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.

Aims Transforming growth factor-β (TGF-β) mediates fibrotic manifestations of diabetic nephropathy. We demonstrated proteasomal degradation of anti-fibrotic protein, nuclear factor-erythroid derived 2 (NF-E2), in TGF-β treated human renal proximal tubule (HK-11) cells and in diabetic mouse kidneys. The current study examined the role of mitogen-activated protein kinase (MAPK) pathways in mediating NF-E2 proteasomal degradation and stimulating profibrotic signaling in HK-11 cells. Main methods HK-11 cells were pretreated with vehicle or appropriate proteasome and MAPK inhibitors, MG132 (0.5 μM), SB203580 (1 μM), PD98059 (25 μM) and SP600125 (10 μM), respectively, followed by treatment with/without TGF-β (10 ng/ml, 24 h). Cell lysates and kidney homogenates from FVB and OVE26 mice treated with/without MG132 were immunoblotted with appropriate antibodies. pUse vector and pUse-NF-E2 cDNA were transfected in HK-11 cells and effects of TGF-β on JNK MAPK phosphorylation (pJNK) was examined. Key findings We demonstrated activation of p38, ERK, and JNK MAPK pathways in TGF-β treated HK-11 cells. Dual p38 and ERK MAPK blockade prevented TGF-β-induced pSer82Hsp27, fibronectin and connective tissue growth factor (CTGF) expression while preserving NF-E2 expression. Blockade of JNK MAPK inhibited TGF-β-induced CTGF expression without preserving NF-E2 expression. MG132 treatment prevented TGF-β-induced pJNK in HK-11 cells and in type 1 diabetic OVE26 mouse kidneys, demonstrating that TGF-β- and diabetes-induced pJNK occurs downstream of proteasome activation. A direct role for NF-E2 in modulating pJNK activation was demonstrated by NF-E2 over-expression. Significance ERK and p38 MAPK promotes NF-E2 proteasomal degradation while proteasome activation promotes pJNK and profibrotic signaling in renal proximal tubule cells.

Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy (DCM), but the mechanism remains largely unclear. We found that diabetic hearts exhibited decreased phosphorylation of total Akt and isoform Akt2 but not Akt1 in wild-type (WT) male FVB mice, which was accompanied by attenuation of Akt downstream glucose metabolic signal. All of these signal changes were not observed in metallothionein cardiac-specific transgenic (MT-TG) hearts. Furthermore, insulin-induced glucose metabolic signals were attenuated only in WT diabetic hearts. In addition, diabetic hearts exhibited increased Akt-negative regulator tribbles pseudokinase 3 (TRB3) expression only in WT mice, suggesting that MT may preserve Akt2 function via inhibiting TRB3. Moreover, MT prevented tert-butyl hydroperoxide (tBHP)–reduced insulin-stimulated Akt2 phosphorylation in MT-TG cardiomyocytes, which was abolished by specific silencing of Akt2. Specific silencing of TRB3 blocked tBHP inhibition of insulin-stimulated Akt2 phosphorylation in WT cardiomyocytes, whereas overexpression of TRB3 in MT-TG cardiomyocytes and hearts abolished MT preservation of insulin-stimulated Akt2 signals and MT prevention of DCM. Most importantly, supplementation of Zn to induce MT preserved cardiac Akt2 signals and prevented DCM. These results suggest that diabetes-inhibited cardiac Akt2 function via TRB3 upregulation leads to aberrant cardiac glucose metabolism. MT preservation of cardiac Akt2 function by inhibition of TRB3 prevents DCM.

Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.

Background A targeted analysis of the 50 kDa C-terminal fragment of insulin-response element binding protein-1 (IRE-BP1) activation of target genes through the insulin receptor substrate receptor/PI-3 kinase/Akt pathway has been demonstrated for the insulin growth factor-1 receptor. The broader effects of 50 kDa C-terminal IRE-BP1 fragment over-expression on protein abundance in pancreatic islet beta cells have not been determined. Results Liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses of replicate lysates of pancreatic islets isolated from background strain animals and transgenic animals, overexpressing IRE-BP1 in pancreatic islet beta cells, demonstrated statistically significant increases in the expression of proteins involved in protein synthesis, endoplasmic reticulum (ER) stress and scaffolding proteins important for protein kinase C signaling; some of which were confirmed by immunoblot analyses. Bioinformatic analysis of protein expression network patterns suggested IRE-BP1 over-expression leads to protein expression patterns indicative of activation of functional protein networks utilized for protein post-translational modification, protein folding, and protein synthesis. Co-immunoprecipitation experiments demonstrate a novel interaction between two differentially regulated proteins receptor for activated protein kinase C (RACK1) and translationally controlled tumor protein (TCTP). Conclusions Proteomic analysis of IRE-BP1 over-expression in pancreatic islet beta cells suggest IRE-BP1 (a) directly or indirectly through establishing hyperglycemia results in increased expression of ribosomal proteins and markers of ER stress and (b) leads to the enhanced and previously un-described interaction of RACK1 and TCTP. Significance This study identified C-terminal 50 kDa domain of IRE-BP1 over-expression results in increased markers of ER-stress and a novel interaction between the scaffolding proteins RACK1 and TCTP.

Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which—Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI—were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.

Background The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. Methods ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. Results Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. Conclusions ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

Aims This study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. Main methods Anti-fibrotic effects of NF-E2 were examined in transforming growth factor-β (TGF-β) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. Key findings NF-E2 expression was significantly decreased in TGF-β treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-β treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-β- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-β treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-β-treated HK-11 cells, respectively. Significance NF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Kidney inflammation or lupus nephritis (LN) is the most common and severe complication of SLE and mechanisms of pathogenesis are not clearly defined and current therapies are toxic and ineffective. We previously reported TNIP1 gene variants as risks for LN. TNIP1 encodes the protein ABIN1, which is a polyubiquitin binding protein that negatively regulates the prominent immune regulatory transcription factor NF-κB. We reported that transgenic mice with impaired ABIN1 ubiquitin binding function (ABIN1[D485N]) spontaneously develop SLE-like autoimmunity and LN and that ABIN1 determines the severity of LN via activation of kidney and immune cell inflammation. Interferon gamma-inducible protein -10 (IP-10) is a pro-inflammatory chemokine and NF-κB target that has been implicated in the pathogenesis and as a diagnostic marker of LN. The current project tested a hypothesis that LN development is mediated by induction of P-10 expression due to loss of cellular ABIN1 ubiquitin binding activity. We found that serum, urine, and kidney cell IP-10 expression is enhanced in ABIN1[D485N] mice. We also found that urinary IP-10 levels are higher in LN patients with TINIP1/ABIN1 variant rs4958881 when compare to LN patients without the TNIP1 variant and healthy controls. Our findings indicate that TNIIP/ABIN1 mutation contributes to the pathogenesis of LN via kidney and immune cell induction of IP-10 secretion and that serum and urinary IP-10 are promising diagnostic markers for LN especially in patients with TNIP1 variants. Further, successful Phase 2 clinical trials with IP-10 mAb for ulcerative colitis indicate its potential for effective LN treatment.

In the article cited above, affiliation 1 was mistakenly included for authors Yi Tan and Lu Cai. The author …

Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-response-induced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced pro-apoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

Rationale: Endothelial progenitor cells (EPCs) respond to stromal cell–derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus–induced EPCs dysfunction remains unknown. Objective: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. Methods and Results: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3β phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. Conclusions: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus–induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3β/Fyn pathway via increased activity of Nrf2.

Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those of Saccharomyces cerevisiae and Ustilago maydis ) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen U. maydis , we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactions in vivo . This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interaction in vivo of an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis in U. maydis .

The mechanisms by which aldosterone increases Na(+), K(+) ATPase and sodium channel activity in cortical collecting duct and distal nephron have been extensively studied. Recent investigations demonstrate that aldosterone increases Na-H exchanger-3 (NHE-3) activity, bicarbonate transport, and H(+) ATPase in proximal tubules. However, the role of aldosterone in regulation of Na(+), K(+) ATPase in proximal tubules is unknown. We hypothesize that aldosterone increases Na(+), K(+) ATPase activity in proximal tubules through activation of the mineralocorticoid receptor (MR). Immunohistochemistry of kidney sections from human, rat, and mouse kidneys revealed that the MR is expressed in the cytosol of tubules staining positively for Lotus tetragonolobus agglutinin and type IIa sodium-phosphate cotransporter (NpT2a), confirming proximal tubule localization. Adrenalectomy in Sprague-Dawley rats decreased expression of MR, ENaC α, Na(+), K(+) ATPase α1, and NHE-1 in all tubules, while supplementation with aldosterone restored expression of above proteins. In human kidney proximal tubule (HKC11) cells, treatment with aldosterone resulted in translocation of MR to the nucleus and phosphorylation of SGK-1. Treatment with aldosterone also increased Na(+), K(+) ATPase-mediated (86)Rb uptake and expression of Na(+), K(+) ATPase α1 subunits in HKC11 cells. The effects of aldosterone on Na(+), K(+) ATPase-mediated (86)Rb uptake were prevented by spironolactone, a competitive inhibitor of aldosterone for the MR, and partially by Mifepristone, a glucocorticoid receptor (GR) inhibitor. These results suggest that aldosterone regulates Na(+), K(+) ATPase in renal proximal tubule cells through an MR-dependent mechanism.

The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na+-K+-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-Picotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.

This study was to investigate whether sulforaphane (SFN) can prevent diabetic cardiomyopathy. Type 1 diabetes was induced in FVB mice by multiple intraperitoneal injections with low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with or without SFN at 0.5mg/kg daily in five days of each week for 3 months and then kept until 6 months. At 3 and 6 months of diabetes, blood pressure and cardiac function were assessed. Cardiac fibrosis, inflammation, and oxidative damage were assessed by Western blot, real-time qPCR, and histopathological examination. SFN significantly prevented diabetes-induced high blood pressure and cardiac dysfunction at both 3 and 6 months, and also prevented diabetes-induced cardiac hypertrophy (increased the ratio of heart weight to tibia length and the expression of atrial natriuretic peptide mRNA and protein) and fibrosis (increased the accumulation of collagen and expression of connective tissue growth factor and tissue growth factor-β). SFN also almost completely prevented diabetes-induced cardiac oxidative damage (increased accumulation of 3-nitrotyrosine and 4-hydroxynonenal) and inflammation (increased tumor necrotic factor-α and plasminogen activator inhibitor 1 expression). SFN up-regulated NFE2-related factor 2 (Nrf2) expression and transcription activity that was reflected by increased Nrf2 nuclear accumulation and phosphorylation as well as the mRNA and protein expression of Nrf2 downstream antioxidants. Furthermore, in cultured H9c2 cardiac cells silencing Nrf2 gene with its siRNA abolished the SFN's prevention of high glucose-induced fibrotic response. These results suggest that diabetes-induced cardiomyopathy can be prevented by SFN, which was associated with the up-regulated Nrf2 expression and transcription function.

Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.

Notch proteins (Notch 1-4) are a family of trans-membrane cell surface receptors that are converted into transcriptional regulators when activated by interactions with cell surface ligands on adjacent cells. Ligand-binding stimulates proteolytic cleavage of the trans-membrane domain, releasing an active intracellular domain (ICD) that translocates to the nucleus and impacts transcription. In transit, the ICD may interact with regulatory proteins that modulate the expression and transcriptional activity. We have found that Notch4(ICD) expression is enhanced in the tubule cells of fibrotic kidneys from diabetic mice and humans and identified Notch4(ICD) interacting proteins that could be pertinent to normal and pathological functions. Using proteomic techniques, several components of the Elongin C complex were identified as candidate Notch4(ICD) interactors. Elongin C complexes can function as ubiquitin ligases capable of regulating proteasomal degradation of specific protein substrates. Our studies indicate that ectopic Elongin C expression stimulates Notch4(ICD) degradation and inhibits its transcriptional activity in human kidney tubule HK11 cells. Blocking Elongin C mediated degradation by MG132 indicates the potential for ubiquitin-mediated Elongin C regulation of Notch4(ICD). Functional interaction of Notch4(ICD) and Elongin C provides novel insight into regulation of Notch signaling in epithelial cell biology and disease.

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT–SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT–SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT–SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT–SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT–SNAP-23 inhibited the increase in plasma membrane expression of gp91phox in TNF-α–primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-beta signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-beta signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-beta-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.

Recent evidence supports the idea that insulin signaling through the insulin receptor substrate/phosphatidyl-inositol 3-kinase/Akt pathway is involved in the maintenance of beta-cell mass and function. We previously identified the insulin-response element binding protein-1 (IRE-BP1) as an effector of insulin-induced Akt signaling in the liver, and showed that the 50-kDa carboxyl fragment confers the transcriptional activity of this factor. In this investigation we found that IRE-BP1 is expressed in the alpha, beta, and delta-cells of the islets of Langerhans, and is localized to the cytoplasm in beta-cells in normal rats, but is reduced and redistributed to the islet cell nuclei in obese Zucker rats. To test whether IRE-BP1 modulates beta-cell function and insulin secretion, we used the rat insulin II promoter to drive expression of the carboxyl fragment in beta-cells. Transgenic expression of IRE-BP1 in FVB mice increases nuclear IRE-BP1 expression, and produces a phenotype similar to that of type 2 diabetes, with hyperinsulinemia, hyperglycemia, and increased body weight. IRE-BP1 increased islet type I IGF receptor expression, potentially contributing to the development of islet hypertrophy. Our findings suggest that increased gene transcription mediated through IRE-BP1 may contribute to beta-cell dysfunction in insulin resistance, and allow for the hypothesis that IRE-BP1 plays a role in the pathophysiology of type 2 diabetes.

OBJECTIVE Despite extensive evidence for genetic susceptibility to diabetic nephropathy, the identification of susceptibility genes and their variants has had limited success. To search for genes that contribute to diabetic nephropathy, a genome-wide association scan was implemented on the Genetics of Kidneys in Diabetes collection. RESEARCH DESIGN AND METHODS We genotyped ∼360,000 single nucleotide polymorphisms (SNPs) in 820 case subjects (284 with proteinuria and 536 with end-stage renal disease) and 885 control subjects with type 1 diabetes. Confirmation of implicated SNPs was sought in 1,304 participants of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, a long-term, prospective investigation of the development of diabetes-associated complications. RESULTS A total of 13 SNPs located in four genomic loci were associated with diabetic nephropathy with P < 1 × 10−5. The strongest association was at the FRMD3 (4.1 protein ezrin, radixin, moesin [FERM] domain containing 3) locus (odds ratio [OR] = 1.45, P = 5.0 × 10−7). A strong association was also identified at the CARS (cysteinyl-tRNA synthetase) locus (OR = 1.36, P = 3.1 × 10−6). Associations between both loci and time to onset of diabetic nephropathy were supported in the DCCT/EDIC study (hazard ratio [HR] = 1.33, P = 0.02, and HR = 1.32, P = 0.01, respectively). We demonstratedexpression of both FRMD3 and CARS in human kidney. CONCLUSIONS We identified genetic associations for susceptibility to diabetic nephropathy at two novel candidate loci near the FRMD3 and CARS genes. Their identification implicates previously unsuspected pathways in the pathogenesis of this important late complication of type 1 diabetes.

Background/Aims: Tubulointerstitial fibrosis (TIF) is a prominent feature of progressive diabetic nephropathy. The goal of this study was to determine if hallmarks of TIF occur in the transgenic OVE26 type 1 diabetic mouse and define signaling events associated with TIF. Methods: The expression patterns of several phenotypic markers of TIF were determined in kidneys of OVE26 diabetic and control mice by immunohistochemistry and immunoblot analysis. Results: Pathological signatures of TIF are an accumulation of myofibroblasts and excessive deposition of extracellular matrix in the tubulointerstitium. Kidneys from OVE26 diabetic animals exhibited an increase in tubulointerstitial myofibroblast marker (α-smooth muscle actin), fibronectin and collagen I staining. Abundance of the pro-fibrotic cytokine TGF-β was also enhanced in diabetic tubules. As injury involving loss of epithelial cell-cell contact promotes tissue fibrosis, we examined expression of the adhesion protein, E-cadherin. The percent of E-cadherin-stained tubules was decreased in diabetic kidneys. Prominent regulators of TGF-β signaling, glycogen synthase kinase-3 (GSK-3) α and β, were also differentially expressed. Conclusions: These results indicate that TGF-β-induced TIF occurs in OVE26 diabetic mice, providing a practical in vivo model for defining novel regulatory events and treatment strategies for diabetes-induced TIF.

To attain a profile of protein expression during diabetes, we applied proteomic analysis to glomeruli of 160-day-old db/db diabetic and db/m nondiabetic mice. Glomerular proteins were extracted and separated by two-dimensional gel electrophoresis to construct a proteome map. Matrix-assisted laser desorption and ionization-time of flight mass spectrometry and peptide mass fingerprinting were used to identify 190 proteins. Of 105 analyzed spots, expression of 40 proteins, including the antioxidative enzymes peroxiredoxin 1 and 3, glutathione peroxidase 1, and SOD-1, was increased with diabetes, suggesting an adaptive response to oxidative stress associated with this diabetic model. However, activity of glutathione peroxidase and SOD was unaltered in glomeruli of diabetic mice. Expression of glyoxalase I was increased in glomeruli of diabetic mice. Because the cofactor for glyoxalase I, glutathione, is decreased in renal cortex of db/db mice, renal cortical glyoxalase I activity was measured in vitro with fixed amounts of exogenous glutathione. Glyoxalase I activity was decreased in renal cortex of db/db mice. These data indicate that diabetes-induced decreases in glyoxalase I activity are likely to be due to glutathione-dependent and -independent mechanisms and that increased expression of glyoxalase I may represent an insufficient adaptive response to increased methylglyoxal formation.

Aging animals develop hypertension when challenged with high salt diet due, in part, to desensitization of dopamine receptors (DR) in renal proximal tubules (RPT). We have demonstrated that NHERF1 associates with DR1 and Na-K ATPase (NKA) and is important for regulation of NKA in RPT. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 results in increased blood pressure (BP) and lack of natriuretic response to dopamine (DA) in aging animals. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed diet containing 1% or 8% NaCl for one week and, BP was measured in anesthetized animals using an indwelling left femoral artery catheter. 8% NaCl did not increase BP in 1m or 4 month old rats. By contrast, 8% NaCl diet increased BP in 12m (84.3±3.5 vs 90.8±2.36) and 24m (73.5±7.58 vs 104±1.6) old animals. To determine if lack of NHERF1 is responsible for the increase in BP, we measured BP in 12 m old WT and NHERF1 KO mice. By contrast to WT mice, 8% NaCl diet did not increase BP in NHERF1 KO mice (84±4.9 vs 96.5±3.56 (WT) and 78.2±3.89 vs 81.8±9.2 (NHERF1 KO mice)). To confirm that NHERF1 is required for DA-mediated inhibition of NKA, NKA activity in primary proximal tubule cells (PTC) from young and old mice in culture was measured in the presence or absence of DA. DA decreased NKA activity in PTC from young animals (67.2±3.8 vs 32.7±5.3) but not in PTC from old animals. Transfection of NHERF1 restored NKA regulation by DA in PTC from old rats (58.4±4.2 vs 64.4±4.3 (in untransfected cells) 54.2±3.8 vs 31.1±3.4 (in NHERF1 transfected cells)). We conclude that NHERF1 regulates DA-mediated proximal tubule sodium handling; however, other factors modulate BP response to dietary sodium intake in young and old animals. The contribution of NHERF1 and dopamine signaling to sodium homeostasis requires further study.

The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG⁎) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na + ,K + -ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na + ,K + -ATPase was tested by comparison of PTH regulation of Na + ,K + -ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na + ,K + -ATPase α 1 subunit when compared with OK-WT cells. PTH decreased Na + ,K + -ATPase activity and stimulated phosphorylation of the Na + ,K + -ATPase α 1 in OK-WT cells but not in NHERF-deficient cells. Rubidium ( 86 Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86 Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86 Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86 Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Cα activity and α 1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Cα activity and α 1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na + ,K + -ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.