non - diabetic kidney disease in patients with diabetes is identified in biopsy - based studies with frequencies ranging 1060% [ 13 ] .
indications for kidney biopsy in patients with diabetes are the presence of features atypical for diabetic nephropathy ( dn ) ; glomerular haematuria , acute kidney deterioration or severe proteinuria [ 2 , 3 ] .
membranous nephropathy ( mn ) and iga nephropathy ( igan ) , superimposed on dn , are most prevalent in communities with a relatively high incident of disease , respectively [ 1 , 2 ] .
mn alongside of dn can present with severe proteinuria and progress quickly to dialysis - dependent end - stage kidney disease ( eskd ) .
concomitant anti - glomerular basement membrane ( gbm ) glomerulonephritis ( gn ) and dn is rare , with poor kidney outcomes reported [ 58 ] .
the simultaneous or sequential occurrence of anti - gbm gn and mn is well recognized .
however , this is the first report of concurrent seronegative anti - gbm gn , mn and dn .
a 22-year - old caucasian male with type 1 diabetes mellitus ( t1 dm ) , presented with acute kidney injury ( aki ) , serum creatinine ( scr ) 387 mol / l and nephrotic syndrome ( timed urinary protein excretion 14 g / day , serum albumin 23
g / l ( 3550 g / l ) , underwent a diagnostic kidney biopsy . clinical assessment
1 year prior indicated minimal proteinuria ( albumin - to - creatinine ratio 1.5 mg / mmol ) and normal kidney function ( egfr   > 90 ml / min/1.73 m ) at that time .
antineutrophil cytoplasmic , extractable nuclear antigen ( ena ) and anti - gbm antibodies were undetected .
urine studies showed protein - to - creatinine ratio 1.133 g / mmol , dysmorphic erythrocytes ( > 1000  10/l ) and lipiduria .
the patient weighed 100 kg and had smoked 10 cigarettes per day for the past 7 years .
there was no history of retinopathy or hypertension , illicit or non - steroidal anti - inflammatory drug use or alcohol dependence .
he had been commenced on frusemide and irbesartan / hydrochlorothiazide 3 weeks earlier to treat oedema .
the kidney biopsy demonstrated a focal segmental necrotizing gn with cellular and fibrocellular crescents involving 29 of 40 glomeruli ; another 6 glomeruli were sclerosed .
there was moderate interstitial fibrosis and tubular atrophy with a patchy interstitial mixed chronic inflammatory cell infiltrate ( see figures 1a and b and 2a and b ) .
weaker anti - albumin staining localized in a linear fashion to the gbm , bowman 's capsule ( bc ) and tubular basement membrane ( tbm ) staining ( figure 3a and b ) . under oil immersion ( 1000 magnification ) , a dual pattern of linear and granular glomerular peripheral capillary wall staining was seen .
electron microscopy showed stage 1 mn with small subepithelial electron - dense  immune - type deposits with early gbm spike formation ( figure 5a and b ) .
the overall diagnosis was that of anti - gbm gn and stage 1 mn superimposed on class iii dn with kimmelstiel  wilson lesions . 

( b ) this glomerulus shows segmental necrosis ( arrow ) and large cellular crescent ( asterisk ) .
( b ) this glomerulus shows a large cellular crescent ( arrows ) . periodic acid silver stain .
( b ) in contrast , albumin shows strong staining of gbm , bc and tbm .
c ) direct immunofluorescence at a higher magnification shows a dual pattern of igg immunofluorescence with linear gbm staining together with granular staining on the outside or urinary space aspect of the gbm ( arrows ) .
images ( b ) and ( c ) were acquired on a nikon a1rsi confocal microscope with a z - slice of 0.6 m . 

5.(a and b ) transmission electron microscopy shows gbm thickening with small subepithelial electron - dense deposits ( arrows ) with early gbm spike formation ( double arrows ) . original magnification 10 000 .
( b ) this glomerulus shows segmental necrosis ( arrow ) and large cellular crescent ( asterisk ) .
( b ) this glomerulus shows a large cellular crescent ( arrows ) . periodic acid silver stain .
( b ) in contrast , albumin shows strong staining of gbm , bc and tbm .
( a  c ) direct immunofluorescence at a higher magnification shows a dual pattern of igg immunofluorescence with linear gbm staining together with granular staining on the outside or urinary space aspect of the gbm ( arrows ) .
images ( b ) and ( c ) were acquired on a nikon a1rsi confocal microscope with a z - slice of 0.6 m .
( a and b ) transmission electron microscopy shows gbm thickening with small subepithelial electron - dense deposits ( arrows ) with early gbm spike formation ( double arrows ) .
the simultaneous diagnosis of seronegative anti - gbm gn , mn and dn was consistent with clinical presentation of proteinuria , haematuria and aki , although unexpected , as anti - gbm antibody serology was negative ( orgentec , mainz , germany ) .
the patient 's human leucocyte antigens ( hla ) were : a1 , 24 ; b8 , 39 ; dr4 , 16 . screening for serum anti - secretory phospholipase a2 receptor ( pla2r ) antibodies was not detected using an indirect immunofluorescence test ( euroimmun ag , lubeck , germany ) .
in addition , pla2r was not detected by immunofluorescence in the paraffin - embedded kidney biopsy using anti - rabbit pla2r antibody ( atlas antibodies ) .
treatment included corticosteroids ( 3 intravenous methylprednisolone 1 g / day then 75 mg / day oral prednisolone ) , oral cyclophosphamide ( 2 mg / kg / day ) after fertility protection measures and 10 alternate - daily plasmapheresis treatments ( 3 l volume exchanges , replacement albumin 4% ) . he was discharged with mildly improved stable kidney function , with scr 253
one week later , he developed small - volume haemoptysis that settled with oral antibiotics , cessation of smoking and cyclophosphamide reduction ( 1.5 mg / kg / day ) .
prednisolone 60 mg / day was continued in light of further deterioration in kidney function , with scr 383 mol / l and worsening proteinuria at 19 g / day .
another nine alternate - daily plasmapheresis treatments were initiated once neutropaenia resolved , attempting to salvage kidney function .
his clinical course was complicated by h. zoster reactivation ( t7 dermatome ) that resolved with valaciclovir treatment .
progressive renal disease and inability to monitor disease activity serologically led to a repeat kidney biopsy , which showed segmental sclerotic lesions and fibrous crescents in 13 of 22 glomeruli and another 3 glomeruli sclerosed .
immunofluorescence showed a strong dual pattern of staining for igg as described in the first biopsy ( not illustrated ) .
the overall diagnosis of the subsequent biopsy was inactive anti - gbm gn and mn superimposed on dn .
persistent strong linear gbm staining for igg despite lack of active lesions in this biopsy .
the patient commenced maintenance haemodialysis 14 weeks after initial presentation when refractory fluid overload ensued , coupled with no renal recovery .
currently , 5 months after presentation , he is training for home haemodialysis and suitability for combined kidney 
this is an unusual report of the simultaneous diagnosis in a kidney biopsy of seronegative anti - gbm gn , mn and dn .
kidney biopsy was undertaken in this patient with longstanding diabetes and clinical renal parameters not typical of dn .
pulmonary involvement from anti - gbm disease was uncertain , as haemoptysis was minimal , settled with antibiotics and occurred in the context of smoking and recent immunosuppression .
. anti - gbm kidney disease , characteristically a rapidly progressive crescentic gn associated with pulmonary haemorrhage and circulating anti - gbm antibodies directed against the nc1 domain of type iv collagen 3 chain ( 3(iv)nc1 ) , is commonly diagnosed through a combination of clinical , histological and serological findings .
seronegative anti - gbm disease occurs in 23% of cases , where antibodies not detected by elisa methods are found with other biosensor techniques .
jia et al .   suggest that circulating anti - gbm antibodies undetectable by elisa may recognize cryptic and highly conformation - dependent epitopes restricted on 3(iv)nc1 .
however , the gold standard for anti - gbm gn diagnosis is strong linear gbm histological staining for igg .
occurrence of crescentic gn with dn is uncommon , with five previous reports of anti - gbm gn superimposed on dn [ 58 ] , and only one subject recovered kidney function .
poor prognostic factors for kidney recovery described in anti - gbm gn include oligoanuria , scr >
600 mol / l , high serum anti - gbm antibodies ( elisa ) levels and a high percentage of crescents on biopsy . in biopsy - based studies , mn is a relatively common non - diabetic kidney pathology in diabetic populations [ 1 , 2 ] .
though , severe proteinuria as biopsy indication suggests selection bias , as an autopsy study investigating kidney pathology in diabetes demonstrated no mn cases and only one igan in 210 subjects .
outcomes in those with combined mn and dn are poor , both contributing to worsening proteinuria and acceleration of kidney injury .
conversely , associations between mn and anti - gbm gn are well recognized , and were first described in 1974 .
two recent reviews identified 30 cases , sequential occurrence in one - third ( either anti - gbm gn following mn or vice versa ) , and simultaneous diagnosis in the remainder [ 9 , 20 ] .
reported outcomes varied with kidney recovery in one - third , progression to eskd in the majority and death in four patients .
some have speculated that initial alteration in basement membrane permeability allows further disruption leading to heightened immune system exposure and development of additional disease [ 9 , 21 ] .
seronegative anti - gbm gn and mn have not been reported together , although seronegativity itself is rare .
anti - gbm disease is one of the few human autoimmune diseases where the autoantigen is known   with multiple hla - positive associations , namely hla - dr15 and hla - dr4 [ 23 , 24 ] .
hla typing in our patient was positive for hla - dr4 and -dr16 but negative for -dr15 .
more recently , primary mn has been linked with the hla - dqa1 allele on chromosome 6p21 , and pla2r identified as a major autoantigen .
circulating autoantibodies against m - type pla2r in the podocyte are present in 70% of idiopathic mn patients .
serum pla2r antibody was negative in this patient and pla2r was not detected in the glomeruli on biopsy .
rather secondary mn subsequent to gbm / podocyte injury caused by anti - gbm gn and/or dn is postulated .
t1 dm , long considered a chronic autoimmune disease , results from anti - islet autoimmunity developing in genetically susceptible individuals , where major susceptibility loci map to hla - drb1 and hla - dqb1 , also located on chromosome 6p21 .
immunological tolerance mechanisms prevent these diseases in healthy individuals and occurrence of all three in our patient suggests such tolerance may have broken down .
the biopsy shows weak to moderate linear staining of gbm , bc and tbm for igg and albumin .
however , in this case , there was very strong linear gbm staining for igg with negligible staining in bc and tbm , a pattern diagnostic of anti - gbm gn .
in contrast , albumin showed diffuse moderate staining of gbm , bc and tbm ( illustrated in figure 3a ) . with triple pathology ,
kidney recovery seemed unlikely despite reasonable prognostic markers at presentation , scr < 600 mol / l and urine output > 2 l / day .
subsequent complications , neutropaenia and opportunistic infection , in the setting of worsening histology with less overall activity , resulted in immunosuppression cessation and dialysis commencement , with a view to future combined kidney  pancreas transplantation .
the kidney biopsy in glomerular disease is invaluable , providing diagnosis and important disease information whilst aiding management decisions . despite advances with serological testing
, this case continues to illustrate the importance of the biopsy . such as in this instance
, kidney biopsies of patients with diabetes , particularly those with clinical features not typical of dn , may reveal other potentially treatable kidney pathology .
 in diabetic patients with acute kidney injury ( aki ) , kidney biopsy often reveals non - diabetic kidney pathology . 
 this case describes a patient with known type 1 diabetes who presented with aki , nephrotic syndrome and haematuria . 
 combination pathology of seronegative anti - glomerular basement membrane antibody - mediated glomerulonephritis ( anti - gbm gn ) , membranous nephropathy ( mn ) and diabetic nephropathy ( dn ) was demonstrated . 
 strong linear gbm igg - staining on biopsy with crescentic gn and clinical aki led to a diagnosis of anti - gbm gn , although serum antibodies were not detectable . 
 features of dn , kimmelstiel  wilson nodules and albumin staining were also present , along with features of mn , such as subepithelial deposits on electron microscopy . despite treatment with immunosuppression and plasmapheresis , there was no recovery of kidney function . 
 coexisting anti - gbm gn and mn is well recognized , but the concurrent diagnosis with dn has not been described . 
Background
Case report
Discussion
Conflict of interest statement