A Case of Colon Cancer and Pauci-Immune Crescentic Glomerulonephritis.

The association between membranous nephropathy and cancer has been well documented. Crescentic glomerulonephritis (GN) has also been associated with various types of cancers. To our knowledge, there has only been one previously documented case of seronegative pauci-immune crescentic glomerulonephritis associated with colon cancer. We present a case of a 51-year-old male with newly diagnosed high-grade poorly-differentiated colon carcinoma who was found to have seronegative pauci-immune crescentic GN with 70% involvement of glomeruli on renal biopsy. The patient was treated with pulse steroids and rituximab with resolution of acute kidney injury.

IgA-dominant infection-associated glomerulonephritis in the pediatric population.

BACKGROUND: IgA-dominant infection-associated glomerulonephritis is well-documented in adults but has not been studied in depth in children. We assessed the incidence of pediatric IgA-dominant infection-associated glomerulonephritis and clinical and kidney biopsy findings. METHODS: Pediatric native kidney biopsies over a 10-year period with IgA dominance, strong C3, and findings indicative of infection-associated etiology were identified. RESULTS: We identified 9 cases of IgA-dominant infection-associated glomerulonephritis, 0.8% of pediatric native kidney biopsies. Seven patients presented with elevated creatinine. All had hematuria and proteinuria. Eight patients had clinical evidence of infection: one each with central port infection by methicillin-sensitive Staphylococcus aureus, recurrent streptococcal pharyngitis and recent otitis media, streptococcal pharyngitis demonstrated 8 months after biopsy, suspected streptococcal scalded skin syndrome, and viral gastroenteritis, and three with serologic evidence of Streptococcal infection but no identified site of infection. All but one patient experienced short-term normalization of creatinine and resolution of proteinuria, though two eventually progressed to kidney failure: one 3 years later due to progressive disease and one 11 years later due to focal segmental glomerulosclerosis without concurrent immune deposits. CONCLUSIONS: Pediatric IgA-dominant infection-associated glomerulonephritis is rare, and generally has a favorable prognosis, contrasting that seen in adults with severe comorbidities. A higher resolution version of the Graphical abstract is available as Supplementary.

Case Report: Rare Presentation of Mixed Germ Cell Tumor in an Infant.

The estimated incidence of pediatric testis tumor is 0.5-2.0 per 100,000 children, accounting for 1-2% of all pediatric tumors. Mixed germ cell tumors (MGCT) in prepubertal males are exceedingly rare, with only one previous case report found in the literature. We report a case of a MGCT in an infant. For prepubertal males, GCTs typically present with a painless scrotal mass, though trauma, testis torsion and hydrocele are also common presentations. Similar to such tumors in postpubertal males, ultrasonography, computed tomography, and tumor markers are integral to determine the best treatment. The patient described in this report presented with a painless scrotal mass. Following orchiectomy, the patient was found to have MGCT that was limited to the testis. With prudent management, these patients tend to have favorable prognoses.

Na/H Exchange Regulatory Factor 1 Deficient Mice Show Evidence of Oxidative Stress and Altered Cisplatin Pharmacokinetics.

(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.

Patients with Proliferative Lupus Nephritis Have Autoantibodies That React to Moesin and Demonstrate Increased Glomerular Moesin Expression.

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 < 0.01). By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against moesin (p < 0.01). This suggests that moesin is a target glomerular antigen in lupus nephritis.

Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

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.

Loss of the Na+/H+ Exchange Regulatory Factor 1 Increases Susceptibility to Cisplatin-Induced Acute Kidney Injury.

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 < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.

Angiomatoid Fibrous Histiocytoma With Paraneoplastic Platelet Storage Pool Deficiency.

Angiomatoid fibrous histiocytoma is a rare soft tissue tumor usually discovered in young individuals. This tumor is often mistaken for a hematoma and typically misdiagnosed. It is commonly found in the extremities and may be associated with a site of recent or previous trauma. Characteristic histology includes nodules of histiocytoid spindle cells with pseudoangiomatoid spaces, fibrous pseudocapsules, and lymphoid cuffing. We describe the case of an 8-year-old girl who presented after incision and drainage of a superficial thigh lesion and experienced subsequent chronic bleeding of her wound. Her initial presentation was concerning for an underlying bleeding disorder, and laboratory analysis uncovered a paraneoplastic platelet function disorder that resolved with therapy of the primary tumor.

Renal Protection by Genetic Deletion of the Atypical Chemokine Receptor ACKR2 in Diabetic OVE Mice.

In diabetic nephropathy (DN) proinflammatory chemokines and leukocyte infiltration correlate with tubulointerstitial injury and declining renal function. The atypical chemokine receptor ACKR2 is a chemokine scavenger receptor which binds and sequesters many inflammatory CC chemokines but does not transduce typical G-protein mediated signaling events. ACKR2 is known to regulate diverse inflammatory diseases but its role in DN has not been tested. In this study, we utilized ACKR2(-/-) mice to test whether ACKR2 elimination alters progression of diabetic kidney disease. Elimination of ACKR2 greatly reduced DN in OVE26 mice, an established DN model. Albuminuria was significantly lower at 2, 4, and 6 months of age. ACKR2 deletion did not affect diabetic blood glucose levels but significantly decreased parameters of renal inflammation including leukocyte infiltration and fibrosis. Activation of pathways that increase inflammatory gene expression was attenuated. Human biopsies stained with ACKR2 antibody revealed increased staining in diabetic kidney, especially in some tubule and interstitial cells. The results demonstrate a significant interaction between diabetes and ACKR2 protein in the kidney. Unexpectedly, ACKR2 deletion reduced renal inflammation in diabetes and the ultimate response was a high degree of protection from diabetic nephropathy.

Differential expression of endoplasmic reticulum stress-response proteins in different renal tubule subtypes of OVE26 diabetic mice.

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-responseinduced 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 proapoptotic 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.

Significance of C4d Immunostaining in Placental Chronic Intervillositis.

Deposition of the complement split product C4d is a phenomenon studied extensively as a marker for complement activation in antibody-mediated transplant rejection. C4d also is observed in placental disease processes including spontaneous abortion, infarct, and villitis of unknown origins. Massive chronic intervillositis is a rare placental abnormality associated with increased risk of growth restriction, fetal death, and recurrent fetal loss. In this study, we evaluated C4d immunostaining in placentas with accumulation of intervillous monocytes with and without villitis. Archived placentas from Kosair Children's Hospital (Louisville, KY) and Seattle Children's Hospital (Seattle, WA) were selected and divided into 4 groups, 16 cases of intervillositis with complicated pregnancy, 15 cases of uncomplicated intervillositis, 20 cases of complicated villitis, and 13 cases of uncomplicated villitis, all with varying degrees of monocytic cells in the intervillous space. Representative specimen blocks were immunohistochemically stained for C4d. The percentage of positive staining of the microvillous surface of the syncytiotrophoblast was scored by five pathologists, and the following consensus score was determined: 0  =  0% to 5%; 1  =  5% to 25%; 2  =  25% to 75%; and 3 ≥ 75%. C4d immunostain localized to the microvillous border of syncytiotrophoblast in many of the placentas. C4d staining was more strongly associated with intervillositis than with villitis (odds ratio: 6.3; confidence interval: 2.1-18.7; P  =  0.001).

Filifactor alocis infection and inflammatory responses in the mouse subcutaneous chamber model.

Recent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10(9) F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.

Follow-up study of lympho-histiocytic villitis and incidental retroplacental hematoma.

Placentas are usually submitted for pathologic examination based on obstetrical indications. We hypothesized that the placenta may have diagnostic value to the infant independent of obstetrical events. We specifically tested whether lymphohistiocytic villitis (noninfectious) would predict autoimmune or alloimmune disease based on transfer of activated maternal T-cells to the fetus and whether clinically silent placental separations (retroplacental hematomas, RPH) would predict neurologic injury in the infant. All placentas from consecutive deliveries had a routine pathologic examination of the placenta. The infants with placentas demonstrating inflammation of >1% of villi or RPH >2 cm and matched controls had their hospital charts reviewed and parental interviews by telephone at 5 to 7 years of age. The children of consented patients were also searched for in the office visits of the University of Louisville Pediatric Neurology and Rheumatology divisions. One thousand six hundred eighty-four patients consented to the follow-up study. We found no cases of autoimmune disease among 17 children with villitis >1%. Of 16 infants with RPH, 1 had cerebral palsy but with other placental findings, 1 had lethal hydranenecephaly, and the remainder had no adverse outcome. Of 15 children seen by a pediatric neurologist, none had the same placental lesion. The specific lesions of lymphohistiocytic villitis or asymptomatic RPH do not predict significant pediatric disease by 7 years of age. At least for these 2 lesions, the placenta does not have diagnostic value to the infant.

ABIN1 dysfunction as a genetic basis for lupus nephritis.

The genetic factors underlying the pathogenesis of lupus nephritis associated with systemic lupus erythematosus are largely unknown, although animal studies indicate that nuclear factor (NF)-κB is involved. We reported previously that a knockin mouse expressing an inactive form of ABIN1 (ABIN1[D485N]) develops lupus-like autoimmune disease and demonstrates enhanced activation of NF-κB and mitogen-activated protein kinases in immune cells after toll-like receptor stimulation. In the current study, we show that ABIN1[D485N] mice develop progressive GN similar to class III and IV lupus nephritis in humans. To investigate the clinical relevance of ABIN1 dysfunction, we genotyped five single-nucleotide polymorphisms in the gene encoding ABIN1, TNIP1, in samples from European-American, African American, Asian, Gullah, and Hispanic participants in the Large Lupus Association Study 2. Comparing cases of systemic lupus erythematosus with nephritis and cases of systemic lupus erythematosus without nephritis revealed strong associations with lupus nephritis at rs7708392 in European Americans and rs4958881 in African Americans. Comparing cases of systemic lupus erythematosus with nephritis and healthy controls revealed a stronger association at rs7708392 in European Americans but not at rs4958881 in African Americans. Our data suggest that variants in the TNIP1 gene are associated with the risk for lupus nephritis and could be mechanistically involved in disease development via aberrant regulation of NF-κB and mitogen-activated protein kinase activity.

Associations between structural and functional changes to the kidney in diabetic humans and mice.

Type 1 and Type 2 diabetic patients are at high risk of developing diabetic nephropathy (DN). Renal functional decline is gradual and there is high variability between patients, though the reason for the variability is unknown. Enough diabetic patients progress to end stage renal disease to make diabetes the leading cause of renal failure. The first symptoms of DN do not appear for years or decades after the onset of diabetes. During and after the asymptomatic period structural changes develop in the diabetic kidney. Typically, but not always, the first symptom of DN is albuminuria. Loss of renal filtration rate develops later. This review examines the structural abnormalities of diabetic kidneys that are associated with and possibly the basis for advancing albuminuria and declining GFR. Mouse models of diabetes and genetic manipulations of these models have become central to research into mechanisms underlying DN. This article also looks at the value of these mouse models to understanding human DN as well as potential pitfalls in translating the mouse results to humans.

Isocitrate dehydrogenase 1 R132H mutation is not detected in angiocentric glioma.

Mutations of isocitrate dehydrogenase-1 gene (IDH1), most commonly resulting in replacement of arginine at position 132 by histidine (R132H), have been described in World Health Organization grade II and III diffuse gliomas and secondary glioblastoma. Immunohistochemistry using a mouse monoclonal antibody has a high specificity and sensitivity for detecting IDH1 R132H mutant protein in sections from formalin-fixed, paraffin-embedded tissue. Angiocentric glioma (AG), a unique neoplasm with mixed phenotypic features of diffuse glioma and ependymoma, has recently been codified as a grade I neoplasm in the 2007 World Health Organization classification of central nervous system tumors. The present study was designed to evaluate IDH1 R132H protein in AG. Three cases of AG were collected, and the diagnoses were confirmed. Expression of mutant IDH1 R132H protein was determined by immunohistochemistry on representative formalin-fixed, paraffin-embedded sections using the antihuman mouse monoclonal antibody IDH1 R132H (Dianova, Hamburg, Germany). Known IDH1 mutation-positive and IDH1 wild-type cases of grade II to IV glioma served as positive and negative controls. All 3 patients were male, aged 3, 5, and 15 years, with intra-axial tumors in the right posterior parietal-occipital lobe, right frontal lobe, and left frontal lobe, respectively. All 3 cases showed characteristic morphologic features of AG, including a monomorphous population of slender bipolar cells that diffusely infiltrated cortical parenchyma and ensheathed cortical blood vessels radially and longitudinally. All 3 cases were negative for the presence of IDH1 R132H mutant protein (0/3). All control cases showed appropriate reactivity. IDH1 R132H mutation has been described as a common molecular signature of grade II and III diffuse gliomas and secondary glioblastoma; however, AG, which exhibits some features of diffuse glioma, has not been evaluated. The absence of mutant IDH1 R132H protein expression in AG may help further distinguish this unique neoplasm from diffuse glioma.

Reassessing the clinical significance of chorionic membrane microcysts and linear necrosis.

Recent studies have suggested that 2 lesions of the fetal membranes, linear necrosis at the choriodecidual junction and chorionic membrane microcysts, are markers of uteroplacental ischemia. To evaluate this hypothesis, we examined 807 placentas from unselected, consecutive deliveries at a single hospital over approximately 6 months with specific recording of the presence of chorionic microcysts or linear membrane necrosis. Clinical factors that might indicate uteroplacental ischemia were abstracted from the pathology report, including small for gestational age, pregnancy-induced hypertension, meconium macrophages in the membranes, infarctions, and small placenta. We found that both chorionic microcysts and linear membrane necrosis are very common lesions in unselected placentas, involving 28% and 18% of all placentas, respectively. There was no correlation between the presence of chorionic membrane microcysts and any marker of uteroplacental ischemia. Linear necrosis correlated only with the presence of meconium macrophages. We conclude that these membrane changes are not a useful marker of ischemia in an unselected population of placentas. We suggest caution in the interpretation of these findings, to avoid overdiagnosing ischemia or other pathologic processes.

Angiocentric glioma with high proliferative index: case report and review of the literature.

With less than 50 cases previously reported in literature, angiocentric glioma (AG) is an uncommon, slow-growing tumor of the CNS that typically exhibits a low proliferative rate and is generally surgically curable. We report the case of a 3-year-old child who presented with seizures and was found to have a non-enhancing parietal lobe lesion with overlying calvarial remodeling. Following complete resection of the tumor, histopathological examination revealed bipolar spindle cells centered on cortical blood vessels, forming pseudorosettes with an ependymomatous appearance. Tumor cells were glial fibrillary acidic protein (GFAP) positive, yet failed to label with synaptophysin. Scattered tumor cells had a "dot-like" cytoplasmic staining with the antibody against epithelial membrane antigen (EMA). Collectively, these features favored a diagnosis of AG. Interestingly, the monoclonal antibody against Ki-67 (MIB-1) labeling rate averaged approximately 10.0%. The child continues to be tumor- and seizure-free ten months postoperatively. Long-term follow up is required to determine if the high proliferative rate observed in this AG translates into altered clinical behavior and/or a worse prognosis.

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel regulator of TGF-beta signaling.

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.