Categorization of the diabetic nephropathy by Tervaert classification in clinical setting Hospital de Santo António – CHP, Portugal Patholo Filipa Moreno*, Ana Pinho**, Renata Dias*, Ramon Vizcaino* *Anatomic Pathology Department – Santo Antonio Hospital, Oporto, Portugal **Nephrology Department – Faro Hospital, Faro, Portugal

Diabetic Nephropathy Diabetic Nephopathy (DN) is the most common cause of end-stage renal disease (ESRD) The percentage of patients with ESRD who have Diabetes Mellitus, specially Type II patients, have increseased over the past decade The incidence of Diabetes Mellitus is rising world wide It is estimated that 20-40% of all diabetic patients will develop diabetic nephropathy

What is the Diabetic Nephropathy? Clinical syndrome Persistente proteinuria Hypertension Progressive decline in renal function Pathologic renal lesions Diabetic microangiopathy –  of basement membrane (BM) material Difuse glomerulosclerosis – difuse  in mesangial matrix and thickening of the capillary walls Nodular glomerulosclerosis – Kimmelstiel-Wilson lesions Insudative lesions – hyalinosis Atubular glomeruli Difuse linear reaction for IgG along the BM

Diabetic Nephropathy Diagnostic histopathologic lesions Glomerular Thickening of glomerular basement membrane (GBM) Mesangial expansion Nodular glomerulosclerosis (Kimmelstiel-Wilson lesions) Interstitial Thickening of tubular basement membrane (TBM) Arteriolar hyalinosis Although the most important structural changes occur in the glomeruli, concomitantly and in proportion to the degree of glomerulopathy, arterioles, tubules and interstitium also develop morphological lesions, the most

Tervaert’s Pathologic Classification of Diabetic Nephropathy “Our aim, commissioned by the Research Committee of the Renal Pathology Society, was to develop a consensus classification combining type I and type II diabetic nephropathies” Jornal of the American Society of Nephrology, 21: 556-563, 2010 The team decided to classify DN due to type I and type II diabetes toguether because there is substantial overlap in histologic lesions and renal complications Similar histologic lesions Similar renal complications Type I DN Type II DN

Tervaert’s Pathologic Classification of Diabetic Nephropathy They created a classification scheme that diveded the Diabetic nephopathy lesions in four progressive classes, based on glomerular lesions

Glomerular Basement Membrane Thickening Class I Glomerular Basement Membrane Thickening Biopsy shows no or only mild, nonspecific changes by light microscopy Changes do not meet the criteria of classes II through IV Absence of mesangial expansion, nodular KW lesions and glomerulosclerosis GBM, measured with EM is, on average Thicker than 430 nm in males Thicker than 395 nm in females Class I – H & E 400x

Class II Mesangial Expansion II a – Mild II b – Severe Class II a – PAS 400x Mild or severe mesangial expansion, not meeting the criteria for class II or IV Mesangial expansion – increase in extracellular material in the mesangium such that the width of the interspace exceeds two mesangial cell nuclei in at least two glomerular lobules Mild – expanded mesangial area < mean area of a capillary lumen Severe - expanded mesangial area > mean area of a capillary lumen Class II b – PAS 400x

Nodular Sclerosis – Kimmelstiel-Wilson lesions. Class III Nodular Sclerosis – Kimmelstiel-Wilson lesions. At least one convincing Kimmelstiel-Wilson lesion is found The biopsy specimen does not have more than 50% global glomerulosclerosis (Class III) Kimmelstiel-Wilson lesion – focal, lobular, round to oval mesangial lesions with an acellular, hyaline/matrix core, rounded peripherally by sparse, crescent-shaped mesangial nuclei Class III – PAS 400x

Advanced Diabetic Glomerulosclerosis Class IV Advanced Diabetic Glomerulosclerosis Class IV – PAS 400x Advanced DN More than 50% global glomerulosclerosis The is clinical or pathological evidence that the sclerosis is attributable to DN

Tervaert’s Pathologic Classification of Diabetic Nephropathy Travaert and his team also sugested a separate evaluation for interstitial and vascular lesions

Objective To assess the reliability and prognostic value of the Tervaert’s pathologic classification of diabetic nephropathy in renal biopsies performed on type 2 diabetes mellitus patients with an atypical clinical presentation of renal disease

Intermediate Pathologist Methods Study design Population Single-center study in a tertiary referral hospital for renal pathology Retrospective evaluation of 710 consecutive renal biopsies Selection of Diabetic Nephropathy (DM) positive biopsies In our study, a senior pathologist, na intermediate and a junior pathologist retrospectively evaluated consecutive renal biopsies, selecting the DN positive ones and aplying the Travaert classification on them, blinded for inter-observer and clinical outcome. Population Represented? Single-center study Tertiary referral center for renal pathology Exposure & Comparison Allocated well – Measurements accurate? Blind or Objective? Well Maintained? Participants in each group Follow-up Completed follow-up? Drop-outs / lost during follow-up? Exposure & Comparison Senior Pathologist >10 years experience (A – Gold standard) Intermediate Pathologist 3 years experience (B) Junior Pathologist 1st year of practice (C) Blinded inter-observer and blinded for clinical outcome categorization of DM biopsies

Methods Categorization with Tervaert Classification To aplly the classification, we followed the flow chart sugested by Travaert to evaluate the glomaruli in each biopsy. We also classifed the interstitial lesions with the previously shown table.

Methods Study analysis Outcomes Time Results Primary: Start dialysis Secondary: Death (censored death not related with diabetic disease) Time Between biopsy data and outcome (Cohort retrospective) Results Testing of the different baseline characteristics among classes Evaluation of Tervaert’s classification reproducibility Survival analysis by classes - Kaplan Meier

Characterization of the Population Results Characterization of the Population Table 1 Baseline data at the moment of biopsy** Among 710 RN revised, there were 22 pureDN and 8 DN coexisting with other types of pathology (mixedDN). The mixedDN forms were associated to IgA nephropathy (3), transplant glomerulopathy (2), light chain disease (1), amyloidosis (1) or HIV-Associated Nephropathy (1). ** All patients had Type II diabetes mellitus

Results Reproducibility Table 2 Senior Pathologist (A – Gold standard) vs Intermediate Pathologist experience (B) (A) Classes II III IV (B) 9 1 14 - -  1  3 kappa= 0.85 As it was previously reported, the inter-observer reproducibility for Terveaert classification was good (K = 0.82) Table 3 Senior Pathologist (A – Gold standard) vs Junior Pathologist experience (C) (A) Classes II III IV (C) 8 1 2 13 -  - 2  3  kappa=0.79 Global Kappa – 0.82

Results Renal survival Figure 1 Kaplain Meier curves by Tervaert Classification DN The estimated 5-year renal survival rate was 98.4% in Classes II, 54.3% in Class III, and 36.2% in Class IV (p=0.04). At 5 years follow-up Renal survival II= 98.4% III= 54.3% IV= 36.2%

Discussion Study validity Recruitment Allocation Maintenance Blind or objective assessment of outcomes Results analysis Recruitment: was the setting / EP appropriate given the study goals &/or the reviewer’s interests? If relevant, were participants representative of the EP? Could the results be generalised to relevant populations? This should be able to be determined from risk factor/prognostic profile of participants. In prognostic studies – were participants at similar stage in progression of their disease or condition? Allocation: how well were participants allocated to E&C? If a trial were they randomised to E&C? ! If randomised, was allocation concealed (i.e. knowledge of group (EG or CG) participants allocated to concealed from staff & participants until after allocation documented)? Was randomization successful (i.e. EG & CG similar after randomisation – were baseline characteristics similar in each group)? !If not randomised (observational study) were measurements of E&C accurate & similarly for EG & CG? Were differences between EG & CG documented Maintenance: did participants remain in the groups (EG or CG) they were initially allocated to? Blinding: were participants / investigators blind to whether participants were exposed to E or C? Compliance: % participants allocated to EG (or CG) who remained exposed to E (or C) during study? Contamination: % participants allocated to CG who crossover to EG (& visa versa if CG an exposure)? Co- intervention: other significant interventions received unequally by EG&CG during follow-up? Completeness of follow-up: was it high & similar in EG & CG? Blind assessment of outcomes: were outcome assessors unaware if participants in EG or CG? or Objective assessment of outcomes. eg. biopsies; x-rays, validated questionnaires?

Recruitment The setting was appropriate, given the study goals. The participants were at a similar stage in terms of progression of their disease. The participants were not representative of all Tervaert’s Pathologic Classification of Diabetic Nephropathy with pure nephropathy diabetic

Problems Recruitment Biopsies are performed only when clinical course is not typical for diabetic nephropathy The biopsies do not represent the pathologic range of DN in type II diabetic patients The biopsies only represent patients with atypical clinical course A wide range of non-diabetic renal disease may be present

Allocation The participants were consistently allocated to Tervaert’s classes The measurements of baseline data were accurate and similar for the different classes The differences between classes were documented at the biopsy date Recruitment: was the setting / EP appropriate given the study goals &/or the reviewer’s interests? If relevant, were participants representative of the EP? Could the results be generalised to relevant populations? This should be able to be determined from risk factor/prognostic profile of participants. In prognostic studies – were participants at similar stage in progression of their disease or condition? Allocation: how well were participants allocated to E&C? If a trial were they randomised to E&C? ! If randomised, was allocation concealed (i.e. knowledge of group (EG or CG) participants allocated to concealed from staff & participants until after allocation documented)? Was randomization successful (i.e. EG & CG similar after randomisation – were baseline characteristics similar in each group)? !If not randomised (observational study) were measurements of E&C accurate & similarly for EG & CG? Were differences between EG & CG documented Maintenance: did participants remain in the groups (EG or CG) they were initially allocated to? Blinding: were participants / investigators blind to whether participants were exposed to E or C? Compliance: % participants allocated to EG (or CG) who remained exposed to E (or C) during study? Contamination: % participants allocated to CG who crossover to EG (& visa versa if CG an exposure)? Co- intervention: other significant interventions received unequally by EG&CG during follow-up? Completeness of follow-up: was it high & similar in EG & CG? Blind assessment of outcomes: were outcome assessors unaware if participants in EG or CG? or Objective assessment of outcomes. eg. biopsies; x-rays, validated questionnaires?

Problems Allocation The pathologic findings in diabetic nephropathy differ substantially between type I and type II diabetic patients Kidney lesions underlying renal dysfunction are more heterogeneous in type II patients In Type I patients, the most important renal structure changes occur in the glomeruli Type II patients are more complex and only a minority have histopathological patterns similar to the typical DN of Type I patients

Problems Allocation The mechanisms underlying the associations between cause, natural history and histopathological pattern in the DN of Type II patients are inadequately defined Only a percentage of samples from type II diabetic patients with proteinuria have typical diabetic glomerulopathy The association between the clinic and pathologic findings is not always linear in Type II patients Differences found between class IIb and III may only be due to different pathogenic processes and not progression of disease

Maintenance The participants remained in the groups they were initially allocated to The participants / investigators were blind to participants categorization with Tervaert Classification /clinical baseline data, respectively Clinical co-intervention during the follow-up period was unknown Completeness of follow-up was high and similar in allocated groups Compliance and contamination problems were probably absent Recruitment: was the setting / EP appropriate given the study goals &/or the reviewer’s interests? If relevant, were participants representative of the EP? Could the results be generalised to relevant populations? This should be able to be determined from risk factor/prognostic profile of participants. In prognostic studies – were participants at similar stage in progression of their disease or condition? Allocation: how well were participants allocated to E&C? If a trial were they randomised to E&C? ! If randomised, was allocation concealed (i.e. knowledge of group (EG or CG) participants allocated to concealed from staff & participants until after allocation documented)? Was randomization successful (i.e. EG & CG similar after randomisation – were baseline characteristics similar in each group)? !If not randomised (observational study) were measurements of E&C accurate & similarly for EG & CG? Were differences between EG & CG documented Maintenance: did participants remain in the groups (EG or CG) they were initially allocated to? Blinding: were participants / investigators blind to whether participants were exposed to E or C? Compliance: % participants allocated to EG (or CG) who remained exposed to E (or C) during study? Contamination: % participants allocated to CG who crossover to EG (& visa versa if CG an exposure)? Co- intervention: other significant interventions received unequally by EG&CG during follow-up? Completeness of follow-up: was it high & similar in EG & CG? Blind assessment of outcomes: were outcome assessors unaware if participants in EG or CG? or Objective assessment of outcomes. eg. biopsies; x-rays, validated questionnaires?

Assessment of outcomes The outcome assessors were unaware of participants categorization with Tervaert Classification (Blind assessment of outcomes) The assessment of outcomes was objective (start dialysis date) The death not related with diabetic disease was censored Recruitment: was the setting / EP appropriate given the study goals &/or the reviewer’s interests? If relevant, were participants representative of the EP? Could the results be generalised to relevant populations? This should be able to be determined from risk factor/prognostic profile of participants. In prognostic studies – were participants at similar stage in progression of their disease or condition? Allocation: how well were participants allocated to E&C? If a trial were they randomised to E&C? ! If randomised, was allocation concealed (i.e. knowledge of group (EG or CG) participants allocated to concealed from staff & participants until after allocation documented)? Was randomization successful (i.e. EG & CG similar after randomisation – were baseline characteristics similar in each group)? !If not randomised (observational study) were measurements of E&C accurate & similarly for EG & CG? Were differences between EG & CG documented Maintenance: did participants remain in the groups (EG or CG) they were initially allocated to? Blinding: were participants / investigators blind to whether participants were exposed to E or C? Compliance: % participants allocated to EG (or CG) who remained exposed to E (or C) during study? Contamination: % participants allocated to CG who crossover to EG (& visa versa if CG an exposure)? Co- intervention: other significant interventions received unequally by EG&CG during follow-up? Completeness of follow-up: was it high & similar in EG & CG? Blind assessment of outcomes: were outcome assessors unaware if participants in EG or CG? or Objective assessment of outcomes. eg. biopsies; x-rays, validated questionnaires?

Results Survival analyses include all participants allocated by exposure factor Reasonable precision (low confidence limits) There was consistency with other studies No adjusted analyses was needed for confounders Recruitment: was the setting / EP appropriate given the study goals &/or the reviewer’s interests? If relevant, were participants representative of the EP? Could the results be generalised to relevant populations? This should be able to be determined from risk factor/prognostic profile of participants. In prognostic studies – were participants at similar stage in progression of their disease or condition? Allocation: how well were participants allocated to E&C? If a trial were they randomised to E&C? ! If randomised, was allocation concealed (i.e. knowledge of group (EG or CG) participants allocated to concealed from staff & participants until after allocation documented)? Was randomization successful (i.e. EG & CG similar after randomisation – were baseline characteristics similar in each group)? !If not randomised (observational study) were measurements of E&C accurate & similarly for EG & CG? Were differences between EG & CG documented Maintenance: did participants remain in the groups (EG or CG) they were initially allocated to? Blinding: were participants / investigators blind to whether participants were exposed to E or C? Compliance: % participants allocated to EG (or CG) who remained exposed to E (or C) during study? Contamination: % participants allocated to CG who crossover to EG (& visa versa if CG an exposure)? Co- intervention: other significant interventions received unequally by EG&CG during follow-up? Completeness of follow-up: was it high & similar in EG & CG? Blind assessment of outcomes: were outcome assessors unaware if participants in EG or CG? or Objective assessment of outcomes. eg. biopsies; x-rays, validated questionnaires? Mazzucco, G. et al. Different patterns of renal damage in type 2 diabetes melitus: a multicentric study on 393 biopsies. AJKD 39 (4), 713-720 (2002) Se Won Oh et al. Clinical implications of pathologic diagnosis and classification for diabetic nephropathy. Diabetes research and clinical practice 97 418–424 (2012)

Conclusions In our study, Tervaert classification proved to be user friendly, accurate and clinically useful There was a good inter-observer reproducibility A uniform and consistent classification of DN will improve the communication between renal pathologists and clinicians, allowing better clinical management. Our findings corroborate the results from experimental centers Larger and more significant trials are therefore recommended

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