A proteomic atlas of kidney amyloidosis provides insights into disease pathogenesis.

The mechanisms of tissue damage in kidney amyloidosis are not well described. To investigate this further, we used laser microdissection-mass spectrometry to identify proteins deposited in amyloid plaques (expanded proteome) and proteins overexpressed in plaques compared to controls (plaque-specific proteome). This study encompassed 2650 cases of amyloidosis due to light chain (AL), heavy chain (AH), leukocyte chemotactic factor-2-type (ALECT2), secondary (AA), fibrinogen (AFib), apo AIV (AApoAIV), apo CII (AApoCII) and 14 normal/disease controls. We found that AFib, AA, and AApoCII have the most distinct proteomes predominantly driven by increased complement pathway proteins. Clustering of cases based on the expanded proteome identified two ALECT2 and seven AL subtypes. The main differences within the AL and ALECT2 subtypes were driven by complement proteins and, for AL only, 14-3-3 family proteins (a family of structurally similar phospho-binding proteins that regulate major cellular functions) widely implicated in kidney tissue dysfunction. The kidney AL plaque-specific proteome consisted of 24 proteins, including those implicated in kidney damage (α1 antitrypsin and heat shock protein ß1). Hierarchical clustering of AL cases based on their plaque-specific proteome identified four clusters, of which one was associated with improved kidney survival and was characterized by higher overall proteomic content and 14-3-3 proteins but lower levels of light chains and most signature proteins. Thus, our results suggest that there is significant heterogeneity across and within amyloid types, driven predominantly by complement proteins, and that the plaque protein burden does not correlate with amyloid toxicity.

Pathological characteristics of light chain crystalline podocytopathy.

Monoclonal immunoglobulin light chain (LC) crystalline inclusions within podocytes are rare, poorly characterized entities. To provide more insight, we now present the first clinicopathologic series of LC crystalline podocytopathy (LCCP) encompassing 25 patients (68% male, median age 56 years). Most (80%) patients presented with proteinuria and chronic kidney disease, with nephrotic syndrome in 28%. Crystalline keratopathy and Fanconi syndrome were present in 22% and 10%, respectively. The hematologic condition was monoclonal gammopathy of renal significance (MGRS) in 55% and multiple myeloma in 45%. The serum monoclonal immunoglobulin was IgG κappa in 86%. Histologically, 60% exhibited focal segmental glomerulosclerosis (FSGS), often collapsing. Ultrastructurally, podocyte LC crystals were numerous with variable effacement of foot processes. Crystals were also present in proximal tubular cells as light chain proximal tubulopathy (LCPT) in 80% and in interstitial histiocytes in 36%. Significantly, frozen-section immunofluorescence failed to reveal the LC composition of crystals in 88%, requiring paraffin-immunofluorescence or immunohistochemistry, with identification of kappa LC in 87%. The LC variable region gene segment, determined by mass spectrometry of glomeruli or bone marrow plasma cell sequencing, was IGKV1-33 in four and IGKV3-20 in one. Among 21 patients who received anti-plasma cell-directed chemotherapy, 50% achieved a kidney response, which depended on a deep hematologic response. After a median follow-up of 36 months, 26% progressed to kidney failure and 17% died. The mean kidney failure-free survival was 57.6 months and was worse in those with FSGS. In sum, LCCP is rare, mostly associates with IgG κappa MGRS, and frequently has concurrent LCPT, although Fanconi syndrome is uncommon. Paraffin-immunofluorescence and electron microscopy are essential to prevent misdiagnosis as primary FSGS since kidney survival depends on early diagnosis and subsequent clone-directed therapy.

Light Chain-Only Immunotactoid Glomerulopathy: A Case Report.

The monotypic variant of immunotactoid glomerulopathy (ITG), strongly associated with low-grade lymphoproliferative disorders, is characterized histologically by glomerulonephritis and microtubular deposits of monoclonal immunoglobulin G (IgG). We report a patient with high-risk κ light chain multiple myeloma who presented with acute kidney injury, hematuria, proteinuria, and hypocomplementemia. Kidney biopsy revealed immunotactoid glomerulopathy concomitant with κ light chain myeloma cast nephropathy. The glomerular microtubular deposits stained for κ light chain and C3 only. Proteomic analysis of glomeruli and atypical casts detected κ light chain constant domain and a single VL variability subgroup (IGKV3) in both glomeruli and casts (without γ, α, or µ heavy chain or λ light chain). C3, C5, C6, C7, and C9 were detected in glomeruli. No autoantibodies against alternative pathway of complement proteins were detected. Despite clone-directed chemotherapy, the patient remained on dialysis treatment. For this light chain-only variant of immunotactoid glomerulopathy, pathogenesis potentially involves activation of the alternative pathway of complement by a nephrotoxic κ light chain.

The characteristics of patients with kidney light chain deposition disease concurrent with light chain amyloidosis.

The type of monoclonal light chain nephropathy is thought to be largely a function of the structural and physiochemical properties of light chains; hence most affected patients have only one light chain kidney disease type. Here, we report the first series of kidney light chain deposition disease (LCDD) concomitant with light chain amyloidosis (LCDD+AL), with or without light chain cast nephropathy (LCCN). Our LCDD+AL cohort consisted of 37 patients (54% females, median age 70 years (range 40-86)). All cases showed Congo red-positive amyloid deposits staining for one light chain isotype on immunofluorescence (62% lambda), and LCDD with diffuse linear staining of glomerular and tubular basement membranes for one light chain isotype (97% same isotype as the amyloidogenic light chain) and ultrastructural non-fibrillar punctate deposits. Twelve of 37 cases (about 1/3 of patients) had concomitant LCCN of same light chain isotype. Proteomic analysis of amyloid and/or LCDD deposits in eight revealed a single light chain variable domain mutable subgroup in all cases (including three with separate microdissections of LCDD and amyloid light chain deposits). Clinical data on 21 patients showed proteinuria (100%), hematuria (75%), kidney insufficiency and nephrotic syndrome (55%). Extra-kidney involvement was present in 43% of the patients. Multiple myeloma occurred in 68% (about 2/3) of these patients; none had lymphoma. On follow up (median 16 months), 63% developed kidney failure and 56% died. The median kidney and patient survivals were 12 and 32 months, respectively. LCDD+AL mainly affected patients 60 years of age or older. Thus, LCDD+AL could be caused by two pathological light chains produced by subclones stemming from one immunoglobulin light chain lambda or kappa rearrangement, with a distinct mutated complementary determining region.

Immunoglobulin-Negative DNAJB9-Associated Fibrillary Glomerulonephritis: A Report of 9 Cases.

Fibrillary glomerulonephritis (FGN) was previously defined by glomerular deposition of haphazardly oriented fibrils that stain with antisera to immunoglobulins but do not stain with Congo red. We report what is to our knowledge the first series of immunoglobulin-negative FGN, consisting of 9 adults (7 women and 2 men) with a mean age at diagnosis of 66 years. Patients presented with proteinuria (100%; mean protein excretion, 3g/d), hematuria (100%), and elevated serum creatinine level (100%). Comorbid conditions included carcinoma in 3 and hepatitis C virus infection in 2; no patient had hypocomplementemia or monoclonal gammopathy. Histologically, glomeruli were positive for DNAJB9, showed mostly mild mesangial hypercellularity and/or sclerosis, and were negative for immunoglobulins by immunofluorescence on frozen and paraffin tissue. Ultrastructurally, randomly oriented fibrils measuring 13 to 20nm in diameter were seen intermingling with mesangial matrix in all and infiltrating glomerular basement membranes in 5. On follow-up (mean duration, 21 months), 2 had disease remission, 4 had persistently elevated serum creatinine levels and proteinuria, and 3 required kidney replacement therapy. Thus, rare cases of FGN are not associated with glomerular immunoglobulin deposition, and the diagnosis of FGN in these cases can be confirmed by DNAJB9 immunostaining. Pathogenesis remains to be elucidated.

The novel form of amyloidosis derived from EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) preferentially affects the lower gastrointestinal tract of elderly femalesa.

AIMS: To characterise the clinicopathological features of amyloidosis due to EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), a newly described amyloid type. METHODS AND RESULTS: We identified cases by searching the Mayo Clinic amyloid liquid chromatography and tandem mass spectrometry typing database for specimens with the universal amyloid signature proteins, abundant EFEMP1 spectra and absence of other specific amyloid precursor proteins. We also developed an immunohistochemical stain for EFEMP1 applicable to formalin-fixed tissue sections and performed electron microscopy in one case. We identified 33 specimens from 32 patients with EFEMP1 amyloid. Most patients were female (91%) with a mean age of 75 years, and most specimens (94%) were from the bowel. EFEMP1 amyloid was incidentally identified in specimens biopsied/resected for a variety of clinical indications. In bowel specimens, EFEMP1 amyloid involved blood vessels and interstitium of the lamina propria, submucosa and/or muscularis propria. Although the EFEMP1 deposits were weakly to moderately Congo red-positive with absent to weak birefringence, they were strongly positive for EFEMP1 by immunohistochemistry, had the characteristic fibrillar ultrastructure of amyloid and were readily identified by mass spectrometry. CONCLUSIONS: EFEMP1 amyloid is a recently described novel amyloid type that predominantly affects the bowel of elderly females. Because EFEMP1 amyloid is only weakly Congo red-positive, it may be overlooked without a high index of suspicion. However, its characteristic microanatomical distribution is highlighted by immunohistochemistry and its identity is readily confirmed by mass spectrometry. Based on its distinctive features, we propose that EFEMP1 amyloidosis be considered a new amyloid type.

Light chain only variant of proliferative glomerulonephritis with monoclonal immunoglobulin deposits is associated with a high detection rate of the pathogenic plasma cell clone.

IgG (mainly IgG3) is the most commonly involved isotype in proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). Here we describe the first series of PGNMID with deposition of monoclonal immunoglobulin light chain only (PGNMID-light chain). This multicenter cohort of 17 patients presented with nephritic or nephrotic syndrome with underlying hematologic conditions of monoclonal gammopathy of renal significance (71%) or multiple myeloma (29%). Monoclonal immunoglobulin was identified by serum and urine immunofixation in 65% and 73%, respectively, with abnormal serum free light chain in 83%, and a detectable bone marrow plasma cell clone in 88% of patients. Renal biopsy showed a membranoproliferative pattern in most patients. By immunofluorescence, deposits were restricted to glomeruli and composed of restricted light chain (kappa in 71%) and C3, with granular appearance and subendothelial, mesangial and subepithelial distribution by electron microscopy. Proteomic analysis in four cases of kappa PGNMID-light chain revealed spectra for kappa constant and variable domains, without evidence of Ig heavy chains; spectra for proteins of the alternative pathway of complement and terminal complex were detected in three. The classical pathway was not detected in three cases. After median follow up of 70 months, the renal response was dependent on a hematologic response and occurred in six of ten patients treated with plasma cell-directed chemotherapy but none of five patients receiving other therapies. Thus, PGNMID-light chain differs from PGNMID-IgG by higher frequency of a detectable pathogenic plasma cell clone. Hence, proper recognition is crucial as anti-myeloma agents may improve renal prognosis. Activation of an alternative pathway of complement by monoclonal immunoglobulin light chain likely plays a role in its pathogenesis.

Amyloidosis in surgically resected atrial appendages: a study of 345 consecutive cases with clinical implications.

Histomorphologic parameters of atrial appendages removed during the Cox-Maze procedure have been shown to correlate with recurrence of atrial fibrillation. While amyloid deposition has been noted within atrial appendages, the incidence and significance remains incompletely understood. More accurate amyloid typing methodologies and targeted pharmacotherapeutics have recently been developed, prompting pathologists to provide more detailed information about the type of amyloid identified in such samples. This study sought to fully characterize the morphologic characteristics of atrial amyloid as well as its incidence and clinical significance. Tissue archives were queried for atrial appendages removed during the cardiac surgeries (2010-2014). Patient demographics, imaging features, and salient clinical findings were recorded. Pattern and extent of amyloid deposition were recorded. Typing of the amyloid protein, when present, was performed on a subset of cases by laser capture microdissection with mass spectrometry-based proteomic analysis. A total of 383 atrial appendages from 345 consecutive patients were included in the study (mean age, 69 years; range, 26-92 years). Amyloid was present in 46% of patients. A linear relationship was observed between age and presence of atrial amyloidosis. Women were more likely to have atrial amyloidosis. Two distinct morphologies of amyloid were observed: filamentous and nonfilamentous, and correlated perfectly with amyloid type (filamentous = AANF-type amyloid; nonfilamentous = ATTR-type amyloid). Filamentous deposits were observed in 91% of those with amyloid. Amyloid was more likely to be found in the left atrial appendage than the right. Patients with atrial amyloid, irrespective of type, were more likely to have experienced stroke or TIA and more likely to have atrial arrhythmia preoperatively. Postoperatively, those with atrial amyloid are more likely to experience recurrence of arrhythmia than those who did not have atrial amyloid. Understanding the morphologic characteristics of AANF-type amyloid will allow for identification by the light microscopy and obviates the need for expensive ancillary typing techniques. The finding of nonfilamentous amyloid, should still prompt confirmation of amyloid type so that targeted therapy may be employed.

DnaJ Heat Shock Protein Family B Member 9 Is a Novel Biomarker for Fibrillary GN.

Fibrillary GN (FGN) is a rare primary glomerular disease. Histologic and histochemical features of FGN overlap with those of other glomerular diseases, and no unique histologic biomarkers for diagnosing FGN have been identified. We analyzed the proteomic content of glomeruli in patient biopsy specimens and detected DnaJ heat shock protein family (Hsp40) member B9 (DNAJB9) as the fourth most abundant protein in FGN glomeruli. Compared with amyloidosis glomeruli, FGN glomeruli exhibited a >6-fold overexpression of DNAJB9 protein. Sanger sequencing and protein sequence coverage maps showed that the DNAJB9 protein deposited in FGN glomeruli did not have any major sequence or structural alterations. Notably, we detected DNAJB9 in all patients with FGN but not in healthy glomeruli or in 19 types of non-FGN glomerular diseases. We also observed the codeposition of DNAJB9 and Ig-γ Overall, these findings indicate that DNAJB9 is an FGN marker with 100% sensitivity and 100% specificity. The magnitude and specificity of DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN pathogenesis. With this evidence, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsy specimens.

Congophilic Fibrillary Glomerulonephritis: A Case Series.

RATIONALE & OBJECTIVE: Congo Red positivity with birefringence under polarized light has traditionally permitted classification of organized glomerular deposits as from amyloid or nonamyloid diseases. The absence of congophilia has been used to differentiate fibrillary glomerulonephritis (GN) from amyloidosis. We describe a series of fibrillary GN cases in which the deposits are Congo Red-positive (congophilic fibrillary GN) and discuss the role of DNAJB9 in distinguishing congophilic fibrillary GN from amyloidosis. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Analysis of the clinicopathologic characteristics of 18 cases of congophilic fibrillary GN. Mass spectrometry was performed and compared with 24 cases of Congo Red-negative fibrillary GN, 145 cases of amyloidosis, and 12 apparently healthy individuals. DNAJB9 immunohistochemistry was obtained for a subset of cases. RESULTS: The proteomic signature of amyloid was not detected using mass spectrometry among cases of congophilic fibrillary GN. DNAJB9, a recently discovered proteomic marker for fibrillary GN, was detected using mass spectrometry in all cases of fibrillary GN regardless of congophilia and was absent in cases of amyloidosis and in healthy individuals. DNAJB9 immunohistochemistry confirmed the mass spectrometry findings. The congophilic fibrillary GN cases included 11 men and 7 women with a mean age at diagnosis of 65 years. Concomitant monoclonal gammopathy, hepatitis C virus infection, malignancy, or autoimmune disease was present in 35%, 22%, 17%, and 11% of patients, respectively. No patient had evidence of extrarenal amyloidosis. Patients presented with proteinuria (100%), nephrotic syndrome (47%), hematuria (78%), and chronic kidney disease (83%). After a mean follow-up of 23 months, 31% of patients progressed to end-stage kidney disease and the remaining 69% had persistently reduced kidney function. LIMITATIONS: Retrospective nature. Blinded pathology evaluations were not performed. CONCLUSIONS: The congophilic properties of organized fibrillary deposits should not be solely relied on in differentiating fibrillary GN from renal amyloidosis. Mass spectrometry and DNAJB9 immunohistochemistry can be useful in making this distinction.

Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII.

Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited nodular glomerular involvement. Although prior in vitro studies have shown that Apo-CII can form amyloid fibrils and that certain mutations in this protein promote amyloid fibrillogenesis, there are no reports of this type of amyloidosis in humans. We propose that this study reveals a new form of hereditary amyloidosis (AApoCII) that is derived from the Apo-CII protein and appears to manifest in the elderly and preferentially affect the kidneys.

Hereditary Lysozyme Amyloidosis Variant p.Leu102Ser Associates with Unique Phenotype.

Lysozyme amyloidosis (ALys) is a rare form of hereditary amyloidosis that typically manifests with renal impairment, gastrointestinal (GI) symptoms, and sicca syndrome, whereas cardiac involvement is exceedingly rare and neuropathy has not been reported. Here, we describe a 40-year-old man with renal impairment, cardiac and GI symptoms, and peripheral neuropathy. Renal biopsy specimen analysis revealed amyloidosis with extensive involvement of glomeruli, vessels, and medulla. Amyloid was also detected in the GI tract. Echocardiographic and electrocardiographic findings were consistent with cardiac involvement. Proteomic analysis of Congo red-positive renal and GI amyloid deposits detected abundant lysozyme C protein. DNA sequencing of the lysozyme gene in the patient and his mother detected a heterozygous c.305T>C alteration in exon 3, which causes a leucine to serine substitution at codon 102 (Human Genome Variation Society nomenclature: p.Leu102Ser; legacy designation: L84S). We also detected the mutant peptide in the proband's renal and GI amyloid deposits. PolyPhen analysis predicted that the mutation damages the encoded protein. Molecular dynamics simulations suggested that the pathogenesis of ALys p.Leu102Ser is mediated by shifting the position of the central ß-hairpin coordinated with an antiparallel motion of the C-terminal helix, which may alter the native-state structural ensemble of the molecule, leading to aggregation-prone intermediates.

Clinical, biopsy, and mass spectrometry findings of renal gelsolin amyloidosis.

Gelsolin amyloidosis is a rare type of amyloidosis typically involving the cranial and peripheral nerves, but rarely the kidney. Here we report the clinical, kidney biopsy, and mass spectrometry findings in 12 cases of renal gelsolin amyloidosis. Of the 12 patients, five were men and seven were women with mean age at diagnosis of 63.8 years. Gelsolin amyloidosis was most common in Caucasians (six patients) and Asians (four patients), and included one each African-American and Hispanic patients. Nephrotic syndrome was the most common cause of biopsy, although most patients also had progressive loss of kidney function. Hematological and serological evaluation was negative in 11 patients, while one patient had a monoclonal gammopathy. The renal biopsy showed large amounts of pale eosinophilic Congo red-positive amyloid deposits typically restricted to the glomeruli. Immunofluorescence studies were negative for immunoglobulins in nine cases with three cases of smudgy glomerular staining for IgG. Electron microscopy showed mostly random arrangement of amyloid fibrils with focally parallel bundles/sheets of amyloid fibrils present. Laser microdissection of the amyloid deposits followed by mass spectrometry showed large spectra numbers for gelsolin, serum amyloid P component, and apolipoproteins E and AIV. Furthermore, the p. Asn211Lys gelsolin mutation on mass spectrometry studies was detected in three patients by mass spectrometry, which appears to represent a renal-limited form of gelsolin amyloidosis. Thus, renal gelsolin amyloidosis is seen in older patients, presents with nephrotic syndrome and progressive chronic kidney disease, and histologically exhibits glomerular involvement. The diagnosis can be confirmed by mass spectrometry studies.

Characterization of C3 in C3 glomerulopathy.

Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3ß), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3ß that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3ß. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3ß amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.

Complement activation in pauci-immune necrotizing and crescentic glomerulonephritis: results of a proteomic analysis.

BACKGROUND: Complement activation plays an important role in the pathophysiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), although it remains unclear which pathway is activated. Whether pauci-immune necrotizing crescentic glomerulonephritis (pauci-immune GN) with negative ANCA serology is part of the spectrum of AAV or a different disease entity is essentially unknown. METHODS: We used proteomic analysis to delineate the complement profile in a series of 13 kidney biopsies of patients with pauci-immune GN, with either proteinase 3 (PR3) (five patients) or myeloperoxidase (MPO) antibodies (four patients) or with consistently negative ANCA serology (four patients). Immunofluorescence staining of glomeruli was essentially negative in the PR3-ANCA and MPO-ANCA groups, while a mild staining for C3 was seen in the ANCA-negative cases. No electron-dense deposits were found in the PR3-ANCA and MPO-ANCA groups, but mesangial and few subepithelial deposits were clearly present in the ANCA-negative specimens. RESULTS: Mass spectrometry revealed low spectra numbers for C3 and immunoglobulins in both PR3-positive and MPO-positive patients with minimal or no C4 and C9. In contrast, larger spectra numbers for C3, moderate spectra numbers for C9, complement factor H-related protein-1 and low spectra numbers for C4, C5 and immunoglobulins were found in the ANCA-negative cases. CONCLUSION: While complement activation is noted in AAV, the complement activation appears to be more prominent in the ANCA-negative glomerulonephritis. The larger amount of C3 and moderate amount of C9 in the ANCA-negative glomerulonephritis implies activation of the alternate and terminal pathway of complement, suggesting that this entity may be caused or promoted by a genetic or acquired defect in the alternative pathway.

Clinical, biopsy, and mass spectrometry characteristics of renal apolipoprotein A-IV amyloidosis.

Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis with only a single reported case. Here we describe the clinical, biopsy, and mass spectrometry characteristics of 11 cases of renal AApoAIV amyloidosis encompassing 9 men and 2 women with a mean age at diagnosis of 63.5 years. Progressive chronic kidney disease (mean serum creatinine 2.9 mg/dl) was the most common cause for biopsy with proteinuria absent or minimal in all except one. Hematological and serological evaluation was negative in 9 patients, while 2 had a monoclonal gammopathy. The renal biopsy findings were striking and showed large amounts of eosinophilic Congo-red positive amyloid deposits restricted to the renal medulla with sparing of the renal cortex. In 6 cases, peritubular amyloid was noted in addition to the interstitial involvement. Immunofluorescence studies were negative for immunoglobulins. Electron microscopy showed nonbranching fibrils measuring 7 to 10 nm in diameter. Laser microdissection of the amyloid deposits followed by mass spectrometry showed large spectra number (a semiquantitative measure of abundance) for AApoAIV protein ranging from 49 to 169 (average 85), serum amyloid protein (average 19), and apolipoprotein E (average 48). Importantly, no peptides were detected for any other forms of known amyloidogenic precursor proteins. Thus, renal AApoAIV amyloidosis typically presents with progressive chronic kidney disease and histologically exhibits extensive medullary involvement with sparing of the cortex. The diagnosis is best established by mass spectrometry. Hence, a high degree of suspicion and examination of the renal medulla is required to make the diagnosis.

Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a common form of renal amyloidosis among Egyptians.

Large case series of renal amyloidosis subtypes have recently been published in the United States and Europe showing AL amyloidosis to be the predominant subtype in this part of the world. However, the most common subtypes of renal amyloidosis throughout the rest of the world are unknown. We present here the first large case series detailing the subtypes of renal amyloidosis among Egyptians. In this population, AA amyloidosis was the most common type of amyloidosis on renal biopsy at 48%. The newly described leukocyte chemotactic factor 2 amyloidosis (ALECT2) was the second most common type and represented nearly one-third of renal amyloid cases at 31%. AL accounted for only 20% of cases. The pathologic findings in ALECT2 cases were similar to those previously described in other case series. Thus ALECT2, which was initially thought to affect mainly Hispanics in the United States, appears to represent an important and likely underrecognized etiology of chronic kidney disease among Egyptians and probably in other ethnic groups around the world.

Leukocyte cell-derived chemotaxin 2 (LECT2)-associated amyloidosis is a frequent cause of hepatic amyloidosis in the United States.

Using laser microdissection and mass spectrometry (MS)-based proteomics, we subtyped amyloid deposits from 130 cases of hepatic amyloidosis. Although we confirmed that immunoglobulin light chain amyloidosis was the most frequent cause of hepatic amyloidosis, leukocyte cell-derived chemotaxin 2 (LECT2) amyloidosis (ALect2) accounted for 25% of cases. This novel finding was associated with Hispanic ancestry, incidental discovery of amyloid in liver specimens sampled for other unrelated conditions, and a characteristic pattern of hepatic amyloid deposition. Although ALect2 patients had a common LECT2 polymorphism, pathogenic mutations were not discovered, suggesting that constitutive or compensatory LECT2 overexpression led to ALect2 deposition. These findings indicate that ALect2 is a common cause of hepatic amyloidosis in the population of the United States, and subtyping hepatic amyloid deposits by an accurate analytic method such as MS is required for optimal clinical management of hepatic amyloidosis patients and to avoid incorrect and unnecessarily toxic therapies.

Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury.

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/ß, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.