Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 μm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of artery and capillary-sized channels-drives LECT2 fibrillogenesis. To mimic blood flow through the human kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 μm in width. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering the mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Aggregation of the circulating protein leukocyte-cell-derived chemotaxin 2 (LECT2) causes amyloidosis of LECT2 (ALECT2), one of the most prevalent forms of systemic amyloidosis affecting the kidney and liver. The I40V mutation is thought to be necessary but not sufficient for ALECT2, with a second, as-yet undetermined condition being required for the disease. EM, X-ray diffraction, NMR, and fluorescence experiments demonstrate that LECT2 forms amyloid fibrils in vitro in the absence of other proteins. Removal of LECT2\'s single bound Zn2+ appears to be obligatory for fibril formation. Zinc-binding affinity is strongly dependent on pH: 9-13 % of LECT2 is calculated to exist in the zinc-free state over the normal pH range of blood, with this fraction rising to 80 % at pH 6.5. The I40V mutation does not alter zinc-binding affinity or kinetics but destabilizes the zinc-free conformation. These results suggest a mechanism in which loss of zinc together with the I40V mutation leads to ALECT2.

Karyomegalic interstitial nephritis (KIN) is a rare hereditary cause of chronic kidney disease. It typically causes progressive renal impairment with haemoproteinuria requiring renal replacement therapy before 50 years of age. It has been associated with mutations in the Fanconi anaemia-associated nuclease 1 (FAN1) gene and has an autosomal recessive pattern of inheritance. Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is the third most common cause of amyloid nephropathy presenting with chronic kidney disease and variable proteinuria. We report a novel mutation in the FAN1 gene causing KIN and to our knowledge, the first case of concurrent KIN and ALECT.
We describe the case of 44 year old Pakistani woman, presenting with stage four non-proteinuric chronic kidney disease, and a brother on dialysis. Renal biopsy demonstrated KIN and concurrent ALECT2. Genetic sequencing identified a novel FAN1 mutation as the cause of her KIN and she is being managed conservatively for chronic kidney disease. Her brother also had KIN with no evidence of amyloidosis and is being worked up for kidney transplantation.
This case highlights two rare causes of chronic kidney disease considered underdiagnosed in the wider population due to their lack of proteinuria, and may contribute to the cohort of patients reaching end stage renal disease without a renal biopsy. We report a novel mutation of the FAN1 gene causing KIN, and report the first case of concurrent KIN and ALECT2. This case highlights the importance of renal biopsy in chronic kidney disease of unclear aetiology which has resulted in a diagnosis with implications for kidney transplantation and family planning.

Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a recently described form of systemic amyloidosis, which most commonly affects the kidney and liver. The LECT2 protein is produced during inflammatory processes, but its precise function in renal diseases in unclear. ALECT2, however, is known to be a relatively common form of renal amyloidosis, after amyloid light chain and serum amyloid A types and is most often seen in patients of Hispanic ethnicity. ALECT2 can occur de novo or as recurrent disease in kidney transplants. We present the first case, to our knowledge, of de novo ALECT2 in a pediatric kidney transplant patient, 15 years post-transplant.

Amyloidosis is a disorder characterized by the deposition of abnormal protein fibrils in tissues. Leukocyte cell-derived chemotaxin 2-associated amyloidosis is a recently recognized entity and is characterized by a distinctive clinicopathologic type of amyloid deposition manifested in adults by varying degrees of impaired kidney function and proteinuria. There are only a limited number of cases reported in the literature. We present a 64-year-old Hispanic female with a history of hypertension who was referred for chronic kidney disease management. The review of her laboratory tests revealed a serum creatinine of 1.5-1.8 mg/dL and microalbuminuria (in the presence of a bland urine sediment) in the past year. She denied any history of diabetes, rheumatologic disorders or exposure to intravenous contrast, nonsteroidal anti-inflammatory drugs, herbals, and heavy metals. Serological workup was negative. A renal biopsy showed diffuse infiltration of glomerulus with pale eosinophilic material strongly positive for Congo red stain and a similar eosinophilic material in the interstitium, muscular arteries, and arterioles. Electron microscopy showed marked infiltration of the mesangium, capillary loops, and interstitium with haphazardly arranged fibrillary deposits (9.8 nm thick). Liquid chromatography tandem mass spectrometry confirmed leukocyte cell-derived chemotaxin 2 (LECT2) amyloid deposition. LECT2 amyloidosis (ALECT2) should be suspected in renal biopsy specimens exhibiting extensive and strong mesangial as well as interstitial congophilia. Individuals with LECT2 renal amyloidosis have a varying prognosis. Therapeutic options include supportive measures and consideration of a kidney transplant for those with end-stage renal disease.

Renal amyloidosis is characterized by acellular Congo red positive deposits in the glomeruli, interstitium and/or arteries. Light chain restriction on immunofluorescence studies is present in AL-amyloidosis, the most common type of amyloidosis involving the kidney. The detection of Congo red positive deposits coupled with negative immunofluorescence studies is highly suggestive of non-AL amyloidosis. Some of the non-AL amyloidosis are common while others are relatively rare. The clinical features, laboratory and renal pathology findings are helpful in the diagnosis and typing of non-AL amyloidosis. Thus, ALECT2 amyloidosis is characterized by diffuse cortical interstitial amyloid deposits, AA amyloidosis shows vascular deposits in addition to the glomerular deposits, AFib amyloidosis is characterized by massive amyloid accumulation limited to the glomeruli resulting in the obliteration of glomerular architecture, AApoA1 and AApoAIV are characterized by large amyloid deposits restricted to the medulla, and AGel shows swirling patterns of amyloid fibrils on electron microscopy. While light microscopy is very helpful, accurate typing of non-AL amyloidosis then requires immunohistochemical or laser microdissection/mass spectrometry studies of the Congo red positive deposits. Immunohistochemical studies are available for some of the non-AL amyloidosis. On the other hand, mass spectrometry analysis is a one stop methodology for confirmation and typing of amyloidosis. The diagnosis and typing of amyloidosis by mass spectrometry is based on finding the signature amyloid peptides, apolipoprotein E and serum amyloid-P component, followed by detection of precursor amyloidogenic protein such as LECT2, fibrinogen-α, gelsolin, etc. To, summarize, non-AL amyloidosis is a group of amyloidosis with distinctive clinical, laboratory and renal pathology findings. Typing of the amyloidosis is best performed using mass spectrometry methodology. Accurate typing of non-AL amyloidosis is imperative for correct management, prognosis, and genetic counseling.

Amyloidoses are a spectrum of disorders caused by abnormal folding and extracellular deposition of proteins. The deposits lead to tissue damage and organ dysfunction, particularly in the heart, kidneys, and nerves. There are at least 30 different proteins that can cause amyloidosis. The clinical management depends entirely on the type of protein deposited, and thus on the underlying pathogenesis, and often requires high-risk therapeutic intervention. Application of mass spectrometry-based proteomic technologies for analysis of amyloid plaques has transformed the way amyloidosis is diagnosed and classified. Proteomic assays have been extensively used for clinical management of patients with amyloidosis, providing unprecedented diagnostic and biological information. They have shed light on the pathogenesis of different amyloid types and have led to identification of numerous new amyloid types, including ALECT2 amyloidosis, which is now recognized as one of the most common causes of systemic amyloidosis in North America.