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Collapsing glomerulopathy is common in the setting of thrombotic microangiopathy of the native kidney

Open ArchivePublished:September 17, 2016DOI:https://doi.org/10.1016/j.kint.2016.07.021
      Thrombotic microangiopathy (TMA) is a poorly recognized cause of collapsing glomerulopathy. The frequency and significance of collapsing glomerulopathy associated with renal TMA have not been specifically studied in native kidney biopsy specimens. Here we retrospectively documented clinicopathologic features of 53 patients with histologically proven TMA in the native kidney, with special emphasis on changes due to focal segmental glomerulosclerosis (FSGS). Histological TMA was related to hypertensive nephropathy in 21 patients, genetic complement abnormalities in 9, drugs in 7, and to other causes in 16 patients. Almost half (26 patients) presented with arteriolar, 6 with glomerular, and 21 with mixed TMA. Using the Columbia classification system for the 53 patients with histological TMA, 33 had concurrent FSGS lesions with collapsing glomerulopathy the dominant variant in 19 patients (58% of the FSGS cases), not otherwise specified in 9 patients, cellular in 3, and perihilar or tip lesions in 1 patient each. The presence of FSGS was associated with a poor renal prognosis, with no prognostic difference between collapsing glomerulopathy and other FSGS variants. Thus, collapsing glomerulopathy is frequently found in native kidney biopsies with TMA, suggesting that endothelial injury may play an important role in the pathophysiology of FSGS.

      Keywords

      Thrombotic microangiopathy (TMA) is a histological pattern describing a constellation of microvascular alterations that particularly affect the kidney. Biologically, TMA is typically characterized by intravascular hemolytic anemia with high lactate dehydrogenase, low haptoglobin, and schistocytes on blood smears, usually in conjunction with impairment of the affected organ(s). TMA exhibits a wide spectrum of features as it occurs in a variety of diseases such as Shiga toxin–associated hemolytic uremic syndrome, “atypical hemolytic uremic syndrome” secondary to genetic alternative complement pathway (ACP) dysregulation, thrombotic thrombocytopenic purpura, malignant hypertension, or antiphospholipid syndrome.
      • George J.N.
      • Nester C.M.
      Syndromes of thrombotic microangiopathy.
      • Barbour T.
      • Johnson S.
      • Cohney S.
      • Hughes P.
      Thrombotic microangiopathy and associated renal disorders.
      The renal pathology associates endothelial injury with various changes, ranging from acute fibrin thrombi of arteries/arterioles and glomeruli to chronic remodeling of vessel walls and glomerular basement membranes.
      • Laszik Z.G.
      • Silva F.G.
      Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and other thrombotic microangiopathies.
      Collapsing glomerulopathy (CG) is a variant of focal segmental glomerulosclerosis (FSGS) characterized by prominent podocyte hyperplasia and collapse of the glomerular capillary tuft.
      • D'Agati V.D.
      • Fogo A.B.
      • Bruijn J.A.
      • Jennette J.C.
      Pathologic classification of focal segmental glomerulosclerosis: a working proposal.
      This pattern of glomerular injury, which is usually associated with heavy proteinuria and a poor renal prognosis,
      • Thomas D.B.
      • Franceschini N.
      • Hogan S.L.
      • et al.
      Clinical and pathologic characteristics of focal segmental glomerulosclerosis pathologic variants.
      • Valeri A.
      • Barisoni L.
      • Appel G.B.
      • et al.
      Idiopathic collapsing focal segmental glomerulosclerosis: a clinicopathologic study.
      • Detwiler R.K.
      • Falk R.J.
      • Hogan S.L.
      • Jennette J.C.
      Collapsing glomerulopathy: a clinically and pathologically distinct variant of focal segmental glomerulosclerosis.
      • Weiss M.A.
      • Daquioag E.
      • Margolin E.G.
      • Pollak V.E.
      Nephrotic syndrome, progressive irreversible renal failure, and glomerular “collapse”: a new clinicopathologic entity?.
      is mainly related to genetic factors, viruses, and drugs.
      • Albaqumi M.
      • Barisoni L.
      Current views on collapsing glomerulopathy.
      TMA and other acute vaso-occlusive processes represent another possible etiology of CG
      • D'Agati V.D.
      • Fogo A.B.
      • Bruijn J.A.
      • Jennette J.C.
      Pathologic classification of focal segmental glomerulosclerosis: a working proposal.
      (referred to as “ischemia-associated”
      • Albaqumi M.
      • Barisoni L.
      Current views on collapsing glomerulopathy.
      ). Knowledge about TMA-associated CG (TMA-CG) is scarce and mostly based on sporadic reports of CG occurring in renal transplant recipients, in association with immunosuppressive drugs’ toxicity.
      • Dogan E.
      • Ghanta M.
      • Tanriover B.
      Collapsing glomerulopathy in a renal transplant recipient: potential molecular mechanisms.
      • Stokes M.B.
      • Davis C.L.
      • Alpers C.E.
      Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations.
      • Meehan S.M.
      • Pascual M.
      • Williams W.W.
      • et al.
      De novo collapsing glomerulopathy in renal allografts.
      In native kidneys, TMA-CG has only been anecdotally described in case reports or small series in conjunction with calcineurin inhibitors-related arteriolopathy,
      • Goes N.B.
      • Colvin R.B.
      Case records of the Massachusetts General Hospital: case 12-2007: a 56-year-old woman with renal failure after heart-lung transplantation.
      sickle-cell disease,
      • Nasr S.H.
      • Markowitz G.S.
      • Sentman R.L.
      • D’Agati V.D.
      Sickle cell disease, nephrotic syndrome, and renal failure.
      antivascular endothelial growth factor (anti-VEGF) therapy-associated TMA,
      • Stokes M.B.
      • Erazo M.C.
      • D'Agati V.D.
      Glomerular disease related to anti-VEGF therapy.
      mixed connective tissue disease-associated TMA,
      • Rifkin S.I.
      • Gutta H.
      • Nair R.
      • et al.
      Collapsing glomerulopathy in a patient with mixed connective tissue disease.
      or malignant hypertension.
      • Fukuda K.
      • Shimizu A.
      • Kaneko T.
      • et al.
      A case of secondary focal segmental glomerulosclerosis associated with malignant hypertension.
      The question of the prevalence and significance of CG changes in the setting of histological TMA has never been addressed in an unselected fashion, using native renal biopsy specimens. The purpose of our study was to explore the prevalence of CG in TMA, its clinicopathologic features, and the outcome of TMA-CG in native kidneys.

      Results

       Clinical and histological characteristics of TMA

      We consecutively analyzed 2030 native kidney biopsies in our center between January 1, 2007 and December 31, 2012. Fifty-three of these (2.6%) satisfied the inclusion criteria of histological TMA with no concurrent immune-mediated glomerulonephritis after immunofluorescence (IF) (performed in all 53 cases) or diabetic glomerulopathy. The clinical and biological characteristics at the time of the renal biopsy are summarized in Table 1. The cohort included 31 male (58.5%) and 22 female (41.5%) patients. Forty-eight (90.6%) patients were Caucasians and 5 (9.4%) were black Africans. The mean age was 49.2 years (range, 21–79). The biopsy indications were as follows: chronic renal failure (n = 24), acute renal failure (n = 21), and proteinuria (with or without renal failure) (n = 8). Systemic TMA (defined as the association of ≥3 criteria among the following: schistocytes, thrombocytopenia, anemia, low haptoglobin, and elevated lactate dehydrogenase) was present in a minority of patients (19 of 50). No HIV seropositivity was demonstrated in any patient. After retrospective review of the clinical information, the final diagnoses associated with TMA were malignant hypertension (n = 21), mutations in genes encoding known regulatory proteins of the ACP (n = 9), drugs (n = 7: monoclonal antibodies against VEGF for breast carcinoma [n = 2], calcineurin inhibitors for cardiac transplantation [n = 1] and bone marrow transplantation [n = 1], mammalian target of rapamycin inhibitors for liver transplantation [n = 1], gemcitabine for pancreas carcinoma [n = 1], interferon beta for multiple sclerosis [n = 1]), and miscellaneous causes (n = 9: postpartum nephropathy [n = 2], antiphospholipid syndrome [n = 2], hematopoietic stem cell transplantation [n = 2], sickle cell nephropathy [n = 1], radiotherapy-induced kidney disease [n = 1], Castleman disease [n = 1]); the cause of TMA could not be determined in 7 cases. No Shiga toxin–producing bacterial infection was documented in any patient. Patients with ACP abnormalities had a more severe presentation at the time of initial diagnosis with more frequent systemic TMA (77.8% vs. 29.3%, P = 0.007) and a more frequent need for dialysis (77.8% vs. 9.5%, P < 0.001). No statistical association was found between the initial clinical presentation and TMA etiology (see Supplementary Table S1).
      Table 1Baseline clinical and biological characteristics
      Total (N = 53)
      Age49.2 ± 14.9
      Sex (F/M)22/31
      Ethnicity (C/A)48/5
      Systemic TMA19/50
      Serum creatinine (mg/dl)3.28 ± 2.29
      Dialysis requirement11/53
      Proteinuria (g/d)2.13 ± 1.83
      Nephrotic syndrome4/47
      Hematuria24/49
      SBP (mm Hg)172 ± 39
      DBP (mm Hg)100 ± 29
      Etiologies
       Malignant hypertension21
       ACP abnormalities9
       Drugs7
       Miscellaneous9
       Not determined7
      A, African; ACP, alternative complement pathway; C, Caucasian; DBP, diastolic blood pressure; F, female; M, male; SBP, systolic blood pressure; TMA, thrombotic microangiopathy.
      The pathological features of TMA are summarized in Table 2 and illustrated in Figure 1. The mean number of glomeruli, arteries, and arterioles were 22.0 ± 12.5, 1.7 ± 1.1, and 8.9 ± 5.3, respectively. TMA was exclusively arterial and arteriolar in 26 cases (49.1%), exclusively glomerular in 6 (11.3%), and mixed in 21 (39.6%). Regarding the extent of TMA, changes were focal in 14 of 53 and diffuse in 39 of 53 cases. Active TMA changes, defined as the presence of fibrin thrombi or fibrinoid necrosis or both, were found in 30 of 53 cases. A correlation analysis between clinical and histological data showed that diffuse TMA was associated with more frequent systemic TMA, a higher serum creatinine level, and higher blood pressure (see Supplementary Table S2).
      Table 2Pathological characteristics of TMA
      Glomerular TMAArteriolar TMAGlomerular and arteriolar TMATotal
      Focal (n = 1)Diffuse (n = 5)Focal (n = 8)Diffuse (n = 18)Focal (n = 5)Diffuse (n = 16)
      Glomerular changes
       Endothelial swelling1/15/5NA0/57/1613/27
       Fibrin thrombi0/11/5NA1/59/1611/27
       Congestion0/10/5NA0/56/166/27
       MBG reduplication1/14/5NA2/514/1621/27
       Mesangiolysis0/13/5NA2/510/1615/27
      Arteriolar changes
       Endothelial swellingNA8/818/183/516/1645/47
       Subendothelial edemaNA5/818/184/516/1642/47
       Fibrin thrombiNA6/88/181/511/1626/47
       Fibrinoid necrosisNA0/83/180/53/166/47
       Onion-skin changesNA2/815/183/511/1630/47
       Fibrous occlusionNA2/89/182/54/1617/47
      NA, not applicable; TMA, thrombotic microangiopathy.
      Figure 1
      Figure 1Thrombotic microangiopathy histopathology. (a) Arterial endothelial swelling (Masson trichrome [MT], original magnification ×200; bar = 150 μm). (b) Glomerular endothelial swelling (MT, original magnification ×1000; bar = 30 μm). (c) Arteriolar fibrin thrombi (MT, original magnification ×1000; bar = 30 μm). (d) Glomerular fibrin thrombi (MT, original magnification ×1000; bar = 30 μm). (e) Glomerular congestion (Jones methenamine silver [JMS], original magnification ×400; bar = 75 μm). (f) Arteriolar subendothelial edema (hematoxylin-eosin-saffron, original magnification ×1000; bar = 30 μm). (g) Arteriolar “onion-skin” concentric thickening and fibrin thrombi (MT, original magnification ×1000; bar = 30 μm). (h) Double contours of glomerular capillary walls (JMS, original magnification ×1000; bar = 30 μm). (i) Glomerular ischemic wrinkling (JMS, original magnification ×200; bar = 150 μm). (j) Mesangiolysis (JMS, original magnification ×400; bar = 75 μm). (k) Glomerular platelet thrombi (anti-CD61 immunohistochemistry ×400; bar = 75 μm). (l) Arteriolar platelet thrombi (anti-CD61 immunohistochemistry ×1000; bar = 30 μm).

       Collapsing glomerulopathy is frequent in the setting of histological TMA

      We found FSGS associated with TMA changes in 33 of 53 cases (62.3%). Among them, CG was the predominant variant, present in 19 of 33 cases (57.6%). As depicted in Figure 2, the other FSGS variants according to the Columbia classification were not otherwise specified (NOS) (n = 9, 27.3%), cellular (n = 3, 9.1%), glomerular tip lesion (n = 1, 3.0%), and perihilar (n = 1, 3.0%).
      Figure 2
      Figure 2Focal segmental glomerulosclerosis (FSGS) variants in thrombotic microangiopathy biopsy specimens. CG, collapsing glomerulopathy; NOS, not otherwise specified.
      The histological characteristics of CG are shown in Table 3 and illustrated in Figure 3. By definition, all CG cases contained ≥1 lesion with glomerular collapse and overlying podocyte hyperplasia. CG lesions were predominantly segmental, that is, with capillary collapse in only some segments of the tuft, whereas other segments maintained a normal architectural appearance (n = 16), as opposed to global, that is, with involvement of >50% of the glomerular tuft (n = 3). We observed podocyte vacuolization, due to intracytoplasmic protein resorption droplets, for all (19 of 19) of the CG cases (Figure 3j). Foam cells were present in capillary loops in the vicinity of glomerular collapse in 8 of 19 cases (42.1%) (Figure 3c). Glomerular foam cells were also present in 2 cases classified as cellular FSGS and in 2 cases classified as NOS. Podocyte mitoses were exceptionally present (Figure 3d). We observed the coexistence of CG and TMA changes in the same glomerulus in some instance (Figure 3m).
      Table 3Comparison of the pathological features of FSGS cases
      No FSGS (n = 20)FSGS (n = 33)PCG

      (n = 19)
      Other FSGS (n = 14)P
      Comparison between the CG group and the “other FSGS” group.
      Cellular (n = 3)Tip (n = 1)Perihilar (n = 1)NOS (n = 9)
      Glomeruli (n)18.5 ± 8.724.1 ± 14.10.1223.5 ± 15.924.6 ± 11.6541822.7 ± 8.40.37
      Global sclerosis (%)14.7 ± 18.235.1 ± 25.00.00336.5 ± 27.228.1 ± 23.525.944.434.4 ± 25.70.78
      Collapsing (%)011.0 ± 13.5NA19.1 ± 12.70000NA
      Cellular (%)00.9 ± 2.6NA0.3 ± 1.36.7 ± 2.9000NA
      Tip lesion (%)01.0 ± 4.6NA0.3 ± 1.402.500NA
      Perihilar (%)00.6 ± 2.6NA03.7 ± 6.4010.00NA
      NOS (%)02.8 ± 8.3NA9.2 ± 12.410.0 ± 17.35.010.016.3 ± 14.10.60
      Interstitial fibrosis (mean score)1.3 ± 0.92.1 ± 1.00.0062.0 ± 1.01.3 ± 1.1312.3 ± 1.00.81
      CG, collapsing glomerulopathy; FSGS, focal segmental glomerulosclerosis; NA, not applicable; NOS, not otherwise specified. Mean percentage (by reference to the total number of nonglobally sclerotic glomeruli) of affected glomeruli per biopsy.
      a Comparison between the CG group and the “other FSGS” group.
      Figure 3
      Figure 3Examples of thrombotic microangiopathy–associated collapsing glomerulopathy. Case 1: (a) arteriolar TMA (Masson trichrome [MT], original magnification ×400; bar = 75 μm), (b) collapsing glomerulopathy with collapsed capillary loops covered by hyperplastic podocytes (Jones methenamine silver [JMS], original magnification ×400; bar = 75 μm), (c) intracapillary foam cells (JMS, original magnification ×1000; bar = 30 μm), (d) podocyte mitosis (JMS, original magnification ×1000; bar = 30 μm). Case 2: (e) arteriolar TMA (MT, original magnification ×400; bar = 50 μm), (f,g) collapsing glomerulopathy (MT, original magnification ×400; bar = 50 μm, and JMS, original magnification ×400; bar = 50 μm). Case 3: (h) arteriolar TMA (periodic acid –Schiff, original magnification ×200; bar = 75 μm), (i) collapsing glomerulopathy (JMS, original magnification ×400; bar = 75 μm), (j) podocyte hyaline droplets (periodic acid–Schiff, original magnification ×1000; bar = 30 μm). Case 4: (k) glomerular TMA with segmental mesangiolysis (MT, original magnification ×400; bar = 75 μm), (l) collapsing glomerulopathy in another glomerulus (JMS, original magnification ×400; bar = 75 μm), (m) double contours indicating coexisting glomerular TMA (JMS, original magnification ×1000; bar = 30 μm), (n) endothelial swelling with severe luminal narrowing (star) without effacement of the visceral epithelial cell foot processes (arrows) (electron microscopy, original magnification ×13,000; bar = 5 μm), (o) luminal obliteration (star) with focal epithelial cell foot processes effacement (arrows) and podocyte vacuolization (asterisk) (electron microscopy, original magnification ×11,000; bar = 6 μm).
      In addition to TMA glomerular ultrastructural changes, electron microscopy, performed in 3 of 19 TMA-CG cases, showed only focal effacement of the visceral epithelial cell foot processes (Figures 3n and o).
      CG changes affected, on average, 19.1% of nonsclerotic glomeruli (Table 3). The 19 CG cases also showed NOS in 9.2% of nonsclerotic glomeruli. Mean global glomerulosclerosis in the CG group was 36.5%, which was not statistically different from the “other FSGS” group. However, CG can occur in cases with few chronic glomerular changes, as the percentage of globally sclerotic glomeruli was below 20% in 5 of 19 CG cases (26.3%). Similarly, 5 of 19 CG cases (26.3%) occurred with only mild or no interstitial fibrosis.
      We did not find any significant statistical associations between the FSGS variant and location, extent, or activity of histological TMA (see Supplementary Table S3).

       Immunohistochemical phenotype of podocytes

      Immunohistochemical staining was not possible for 6 specimens because there was no residual paraffin-embedded renal tissue: hence the final number submitted to immunohistochemistry was 47 cases (classified by light microscopy as CG in 14, NOS in 9, cellular in 3, glomerular tip lesion in 1, perihilar in 1, and no FSGS in 19). We observed the loss of CD10 expression in 7 of 14 CG and 6 of 9 NOS cases, indicating podocyte dedifferentiation. The loss of Wilms tumor 1 was found in 5 of 14 CG and in 3 of 9 NOS cases. The loss of podocyte markers was restricted to collapsed tuft areas with typical crowding of visceral epithelial cells. We did not observe immunohistochemical dedifferentiation of podocytes in unaffected portions of the tuft of segmentally injured glomeruli and glomeruli without visible collapse (Figure 4). Using nuclear staining of Ki67 as a marker, we were unable to find cases associated with podocyte proliferation (whereas a positive staining was occasionally seen in tubular cells).
      Figure 4
      Figure 4Loss of podocyte immunohistochemical markers. (a) Several capillaries show loss of CD10 cytoplasmic staining (arrow) (original magnification ×400; bar = 75 μm). (b) Loss of Wilms tumor 1 nuclear staining in the same glomerulus with a similar segmental distribution (arrow) (original magnification ×400; bar = 75 μm).

       Clinical-pathologic correlation and follow-up

      The demographic characteristics and presenting clinical features for the FSGS variants associated with TMA are summarized in Table 4. At the time of the renal biopsy, there was no significant difference between TMA without FSGS, TMA-CG, and TMA with other FSGS variants with respect to age, sex, and ethnicity. The degree of renal impairment also did not differ among the 3 groups. Proteinuria was significantly higher in cases with FSGS (CG and other FSGS variants) than in cases without FSGS (2.54 ± 2.01 vs. 1.42 ± 1.23 g/d, P = 0.03). Nevertheless, there was no difference of proteinuria between CG and the “other FSGS” category (2.39 ± 1.50 vs. 2.72 ± 2.50 g/d, P = 0.77). The frequency of nephrotic syndrome was low in each group (5.9%, 11.8%, and 7.7% in “no FSGS,” CG, and “other FSGS” groups, respectively [P= 0.79]). We observed systemic TMA more frequently in patients without FSGS than in those with FSGS (CG and others) (68.4% vs. 19.4%, P= 0.001). The comparison between FSGS categories based on the cause of TMA was limited due to the low number of patients in each group and the heterogeneity of the “others” category. However, CG appeared to be not specific to a particular cause because it occurred in each etiological category: 7 cases in malignant hypertension-related TMA, 2 cases in ACP abnormalities-related TMA, 2 cases in drug-related TMA, and 8 cases in TMA related to other causes.
      Table 4Clinical characteristics at baseline according to FSGS variants
      No FSGS (n = 20)CG

      (n = 19)
      Other FSGS (n = 14)P
      Age51.0 ± 14.844.8 ± 14.352.8 ± 15.40.25
      BMI23.0 ± 3.223.0 ± 3.323.5 ± 3.40.89
      Sex (F/M)7/1310/95/90.47
      Ethnicity (C/A)17/318/113/10.58
      Creatinine (mg/dl)3.4 ± 2.73.5 ± 2.02.6 ± 1.90.52
      Proteinuria (g/d)1.42 ± 1.232.39 ± 1.502.72 ± 2.500.1
      No FSGS versus FSGS: 1.42 ± 1.23 versus 2.54 ± 2.01 g/d, P = 0.03; no FSGS versus CG, P = 0.03; no FSGS versus other FSGS, P = 0.14; CG versus other FSGS, P = 0.77.
      Nephrotic syndrome (% [yes/no])5.9 (1/16)11.8 (2/15)7.7 (1/12)0.79
      Systemic TMA (yes/no)13/63/153/100.002
      No FSGS versus CG and other FSGS, P = 0.001.
      Dialysis requirement (yes/no)6/144/151/130.31
      SBP (mm Hg)172 ± 42181 ± 38161 ± 410.39
      DBP (mm Hg)100 ± 31104 ± 2795 ± 290.66
      TMA etiology (n)
       Malignant hypertension (n = 21)1074
       ACP abnormalities (n = 9)720
       Drugs (n = 7)331
       APLS (n = 2)011
       Postpartum (n = 2)011
       HSC transplantation (n = 2)002
       Others (n = 10)055
      A, African; ACP, alternative complement pathway; APLS, antiphospholipid syndrome; BMI, body mass index; C, Caucasian; CG, collapsing glomerulopathy; DBP, diastolic blood pressure; F, female; FSGS, focal segmental glomerulosclerosis; HSC, hematopoietic stem cell; M, male; SBP, systolic blood pressure; TMA, thrombotic microangiopathy.
      a No FSGS versus FSGS: 1.42 ± 1.23 versus 2.54 ± 2.01 g/d, P = 0.03; no FSGS versus CG, P = 0.03; no FSGS versus other FSGS, P = 0.14; CG versus other FSGS, P = 0.77.
      b No FSGS versus CG and other FSGS, P = 0.001.
      Clinical outcomes for the FSGS variants are summarized in Table 5. We performed renal survival analyses for 50 patients because 3 were lost to follow-up after the initial diagnosis. During the follow-up period (32 ± 24 [6–81] months), 12 patients (24.0%) developed end-stage renal disease requiring dialysis. Univariate analysis showed that FSGS occurring in the setting of histological TMA was associated with a poor renal prognosis (11 of 32 patients with FSGS required dialysis vs. 1 of 18 for those without FSGS, P = 0.046) (Figure 5a). The renal prognosis of TMA-CG was not significantly different than for TMA cases associated with other FSGS variants (Figure 5b). Interstitial fibrosis and global glomerular sclerosis were also associated with a poor renal survival (Figure 5c and d). Multivariate analysis was not possible given the limited number of patients and the number of factors affecting renal survival. Proteinuria >0.5 g/d or creatininemia above the median value (>2.65 mg/dl) at the initial diagnosis were not associated with renal survival (data not shown). Proteinuria and estimated glomerular filtration rate were not significantly different in the subgroup of end-stage renal disease–free patients at the end of the follow-up, whether they had FSGS or not.
      Table 5Impact of the FSGS variants on renal outcome (at the last follow-up time point)
      No FSGS (n = 18)CG

      (n = 18)
      Other FSGS (n = 14)P
      ESRD (requiring dialysis)1/187/184/140.06
      No FSGS versus CG, P = 0.046; CG versus other FSGS, P = 0.7.
      Patients without ESRD(n = 17)(n = 11)(n = 10)
       eGFR (ml/min/1.73 m2)57 ± 3451 ± 3060 ± 480.85
       Proteinuria (g/d)0.28 ± 0.490.87 ± 1.130.69 ± 1.020.22
      No FSGS versus CG, P = 0.057; CG versus other FSGS, P = 0.57.
       Nephrotic syndrome0/170/111/10NA
       Systemic TMA0/170/110/10NA
       SBP (mm Hg)130 ± 13124 ± 16123 ± 170.45
       DBP (mm Hg)81 ± 877 ± 1073 ± 130.21
      CG, collapsing glomerulopathy; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ESRD, end-stage renal disease; FSGS, focal segmental glomerulosclerosis; NA, not applicable; SBP, systolic blood pressure; TMA, thrombotic microangiopathy.
      a No FSGS versus CG, P = 0.046; CG versus other FSGS, P = 0.7.
      b No FSGS versus CG, P = 0.057; CG versus other FSGS, P = 0.57.
      Figure 5
      Figure 5Renal survival according to pathological features. (a) Kaplan-Meier survival curves comparing renal survival for cases with focal segmental glomerulosclerosis (FSGS) with those without FSGS (No FSGS). (b) Kaplan-Meier survival curves comparing renal survival for cases with collapsing glomerulopathy (CG), cases with non-CG FSGS, and cases without FSGS (No FSGS). (c) Kaplan-Meier survival curves comparing renal survival for cases with slight interstitial fibrosis (IF grade ≤1) with those with at least moderate interstitial fibrosis (IF grade ≥2). (d) Kaplan-Meier survival curves comparing renal survival for cases with slight global glomerular sclerosis (<20% sclerotic glomeruli) with those with more advanced global glomerular sclerosis (≥20% sclerotic glomeruli).

      Discussion

      A secondary “reactive” form of CG related to acute vasoocclusive processes is mentioned in the seminal paper of the Columbia classification of FSGS.
      • D'Agati V.D.
      • Fogo A.B.
      • Bruijn J.A.
      • Jennette J.C.
      Pathologic classification of focal segmental glomerulosclerosis: a working proposal.
      However, although reference textbooks indicate that “segmental collapse of the glomerular capillary lumina” and “visceral epithelial cell hyperplasia” can be found during glomerular TMA,
      • Laszik Z.G.
      • Silva F.G.
      Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and other thrombotic microangiopathies.
      CG potentially driven by ischemia or endothelial injury is very poorly documented, almost exclusively in conjunction with renal transplantation
      • Canaud G.
      • Bruneval P.
      • Noël L.H.
      • et al.
      Glomerular collapse associated with subtotal renal infarction in kidney transplant recipients with multiple renal arteries.
      • Nadasdy T.
      • Allen C.
      • Zand M.S.
      Zonal distribution of glomerular collapse in renal allografts: possible role of vascular changes.
      and the use of calcineurin inhibitors.
      • Stokes M.B.
      • Davis C.L.
      • Alpers C.E.
      Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations.
      • Meehan S.M.
      • Pascual M.
      • Williams W.W.
      • et al.
      De novo collapsing glomerulopathy in renal allografts.
      • Goes N.B.
      • Colvin R.B.
      Case records of the Massachusetts General Hospital: case 12-2007: a 56-year-old woman with renal failure after heart-lung transplantation.
      Other vaso-occlusive diseases such as cholesterol embolism have also occasionally been reported to cause CG.
      • Greenberg A.
      • Bastacky S.I.
      • Iqbal A.
      • et al.
      Focal segmental glomerulosclerosis associated with nephrotic syndrome in cholesterol atheroembolism: clinicopathological correlations.
      Ours is the first study to specifically focus on the prevalence of the different FSGS variants occurring in the setting of histological TMA in native kidneys. The primary result of our study is the striking overrepresentation of CG. Indeed, we found CG in 35.8% (19 of 53) of all TMA patients; CG was by far the most frequent FSGS variant among cases with FSGS, representing 57.6% (19 of 33) of all FSGS cases occurring in association with histological TMA. In contrast, the frequency of CG has been reported to be 5% among a Dutch population with FSGS,
      • Deegens J.K.
      • Steenbergen E.J.
      • Borm G.F.
      • et al.
      Pathological variants of focal segmental glomerulosclerosis in an adult Dutch population—epidemiology and outcome.
      demographically similar to that in our study (notably for the high prevalence of Caucasian patients). Another important finding is that TMA-CG was not associated with a specific cause of TMA and can occur also in malignant hypertension, ACP genetic abnormalities–related TMA, or drug-related TMA.
      A small number of clinicopathologic studies have highlighted the occurrence of CG associated with either TMA or severe arteriolar occlusion in the setting of specific glomerular diseases, that is, IgA nephropathy
      • El Karoui K.
      • Hill G.S.
      • Karras A.
      • et al.
      Focal segmental glomerulosclerosis plays a major role in the progression of IgA nephropathy. II. Light microscopic and clinical studies.
      and diabetic nephropathy.
      • Salvatore S.P.
      • Reddi A.S.
      • Chandran C.B.
      • et al.
      Collapsing glomerulopathy superimposed on diabetic nephropathy: insights into etiology of an under-recognized, severe pattern of glomerular injury.
      Indeed, 11 IgA nephropathy–related CG cases were described by El Karoui et al.
      • El Karoui K.
      • Hill G.S.
      • Karras A.
      • et al.
      Focal segmental glomerulosclerosis plays a major role in the progression of IgA nephropathy. II. Light microscopic and clinical studies.
      in their series of 128 cases of IgA nephropathy; strikingly, 10 of 11 cases had evidence of concomitant TMA. Similarly, Salvatore et al.
      • Salvatore S.P.
      • Reddi A.S.
      • Chandran C.B.
      • et al.
      Collapsing glomerulopathy superimposed on diabetic nephropathy: insights into etiology of an under-recognized, severe pattern of glomerular injury.
      found CG superimposed in 5% of their 534 diabetic nephropathy cases; they hypothesized that CG could be the consequence of glomerular ischemia induced by severe arteriolar hyalinosis. Our study, in which cases with either immune deposits glomerulonephritis or diabetic nephropathy were excluded, suggests, along with those previous works, a major role for ischemia or endothelial injury or both in the pathophysiology of CG. In addition, the relationship between TMA and CG is underlined by the ability of different therapeutic drugs to induce either TMA or CG, such as calcineurin inhibitors,
      • Stokes M.B.
      • Davis C.L.
      • Alpers C.E.
      Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations.
      • Nizze H.
      • Mihatsch M.J.
      • Zollinger H.U.
      • et al.
      Cyclosporine-associated nephropathy in patients with heart and bone marrow transplants.
      interferons,
      • Hunt D.
      • Kavanagh D.
      • Drummond I.
      • et al.
      Thrombotic microangiopathy associated with interferon beta.
      • Markowitz G.S.
      • Nasr S.H.
      • Stokes M.B.
      • D’Agati V.D.
      Treatment with IFN-α, -β, or -γ is associated with collapsing focal segmental glomerulosclerosis.
      • Zuber J.
      • Martinez F.
      • Droz D.
      • et al.
      Alpha-interferon-associated thrombotic microangiopathy: a clinicopathologic study of 8 patients and review of the literature.
      sirolimus,
      • Izzedine H.
      • Brochériou I.
      • Frances C.
      Post-transplantation proteinuria and sirolimus.
      • Crew R.J.
      • Radhakrishnan J.
      • Cohen D.J.
      • et al.
      De novo thrombotic microangiopathy following treatment with sirolimus: report of two cases.
      • Barone G.W.
      • Gurley B.J.
      • Abul-Ezz S.R.
      • Gokden N.
      Sirolimus-induced thrombotic microangiopathy in a renal transplant recipient.
      and anti-VEGF molecules.
      • Stokes M.B.
      • Erazo M.C.
      • D'Agati V.D.
      Glomerular disease related to anti-VEGF therapy.
      • Izzedine H.
      • Escudier B.
      • Lhomme C.
      • et al.
      Kidney diseases associated with anti-vascular endothelial growth factor (VEGF): an 8-year observational study at a single center.
      • Eremina V.
      • Jefferson J.A.
      • Kowalewska J.
      • et al.
      VEGF inhibition and renal thrombotic microangiopathy.
      • Frangié C.
      • Lefaucheur C.
      • Medioni J.
      • et al.
      Renal thrombotic microangiopathy caused by anti-VEGF-antibody treatment for metastatic renal-cell carcinoma.
      In line with the fact that CG is not considered a postadaptative FSGS variant,
      • Kambham N.
      • Markowitz G.S.
      • Valeri A.M.
      • et al.
      Obesity-related glomerulopathy: an emerging epidemic.
      the presence of CG was not related to the percentage of glomerulosclerosis or the degree of interstitial fibrosis because 26.3% of CG cases occurred in patients with <20% sclerotic glomeruli and only mild interstitial fibrosis.
      In our study, a TMA-CG diagnosis was based on the strict application of the Columbia classification criteria, that is, all CG cases showed glomerular capillary wall collapse with wrinkling of basement membranes causing obliteration of the capillary lumen associated with overlying podocyte hypertrophy and hyperplasia. Our results suggest that TMA-CG and “classical” CG (i.e., CG related to ethnicity, viruses, or drugs, or a combination of these) differ on many points although they are indistinguishable by light microscopy. First, in opposition to “classical” CG, which is more common in black patients,
      • D'Agati V.D.
      • Alster J.M.
      • Jennette J.C.
      • et al.
      Association of histologic variants in FSGS clinical trial with presenting features and outcomes.
      • Stokes M.B.
      • D'Agati V.D.
      Morphologic variants of focal segmental glomerulosclerosis and their significance.
      almost all TMA-CG patients of our series (18 of 19, 94.7%) were Caucasian. However, this should be viewed with caution given the small number of black patients included in the present study. Second, nephrotic syndrome was infrequent in TMA-CG (2 of 17, 11.8%) and the level of proteinuria was equivalent in CG and other FSGS variants; this is consistent with the previously reported characteristics of TMA-CG, which, in contrast to “classical” CG, is often not associated with heavy proteinuria.
      • Stokes M.B.
      • Davis C.L.
      • Alpers C.E.
      Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations.
      • Goes N.B.
      • Colvin R.B.
      Case records of the Massachusetts General Hospital: case 12-2007: a 56-year-old woman with renal failure after heart-lung transplantation.
      Third, we found that dysregulation of the immunohistochemical phenotype of podocytes was less marked in our TMA-CG cases than in “classical” CG: although we observed podocyte dedifferentiation in one-half of the tested cases, proliferation of podocytes was not detected. This result is in accordance with the fact that the degree of podocyte dysregulation is less prominent in the reactive forms of CG.
      • Albaqumi M.
      • Barisoni L.
      Current views on collapsing glomerulopathy.
      Lastly, although we could perform electron microscopy in only 3 of 19 TMA-CG cases, there appeared to be fewer ultrastructural changes of podocytes than in “classical” CG as illustrated by the low degree of foot process effacement in our cases. Altogether, these findings suggest that TMA-CG is associated with attenuated podocyte changes relative to “classical” CG and may be insufficient to trigger a full-blown clinical, immunohistochemical, and ultrastructural phenotype. However, the similar prognosis of TMA-CG and other FSGS variants in our study could indicate that CG is an early form of FSGS that subsequently evolves toward NOS, in accordance with previous studies.
      • Zhong Y.
      • Xu F.
      • Li X.
      • et al.
      The evolution of morphological variants of focal segmental glomerulosclerosis: a repeat biopsy-based observation.
      • Schwartz M.M.
      • Lewis E.J.
      Focal segmental glomerular sclerosis: the cellular lesion.
      A purely mechanical effect of ischemia on glomeruli is unlikely to be sufficient to account for the pathophysiology of TMA-CG. Indeed, ischemic kidneys frequently show wrinkling of the glomerular basement membrane but without prominent podocyte damage. It is thus almost certain that factors other than ischemia may play a significant pathogenic role. Endothelial injury, which is common to TMA, may be pivotal for the pathogenesis of CG. This is supported by the frequent observation of intraglomerular foam cells in 8 of 19 TMA-CG cases of our series, as the presence of foam cells is a sign of severe endothelial injury.
      • Schwartz M.M.
      Focal segmental glomerulosclerosis.
      The concept of “endothelial-podocyte crosstalk” relies mainly on signaling by podocyte-derived mediators, such as VEGF-A,
      • Eremina V.
      • Jefferson J.A.
      • Kowalewska J.
      • et al.
      VEGF inhibition and renal thrombotic microangiopathy.
      to glomerular endothelial cells. However, endothelial-derived factors that signal to podocytes are probably also crucial for glomerular physiology.
      • Haraldsson B.S.
      The endothelium as part of the integrative glomerular barrier complex.
      Indeed, during preeclampsia, a disease in which glomerular endothelial injury (“endotheliosis”) is central, the sera of patients can induce in vitro podocyte damage through the production of endothelin-1 by endothelial cells.
      • Collino F.
      • Bussolati B.
      • Gerbaudo E.
      • et al.
      Preeclamptic sera induce nephrin shedding from podocytes through endothelin-1 release by endothelial glomerular cells.
      This mechanism could explain the frequent occurrence of preeclampsia-associated podocyturia (reviewed in Craici et al.
      • Craici I.M.
      • Wagner S.J.
      • Weissgerber T.L.
      • et al.
      Advances in the pathophysiology of pre-eclampsia and related podocyte injury.
      ) as well as the dysregulation of the podocyte phenotype in preeclamptic kidney sections.
      • Zhao S.
      • Gu X.
      • Groome L.J.
      • Wang Y.
      Decreased nephrin and GLEPP-1, but increased VEGF, Flt-1, and nitrotyrosine, expressions in kidney tissue sections from women with preeclampsia.
      • Garovic V.D.
      • Wagner S.J.
      • Petrovic L.M.
      • et al.
      Glomerular expression of nephrin and synaptopodin, but not podocin, is decreased in kidney sections from women with preeclampsia.
      It is also possible that TMA-CG is a downstream consequence of endothelial injury, mediated by alteration of the paracrine relationship between injured glomerular endothelial cells and podocytes.
      This work has important limitations related to its retrospective design, the relatively small population studied, and the low percentage of cases with electron microscopy. However, this study provides the first evidence that CG is a frequent histological finding in the setting of native kidney TMA, independent of the etiology of TMA. Our results strongly suggest that endothelial injury plays an important role in the development of FSGS and, particularly, CG.

      Materials and Methods

       Patient selection

      Computerized records of the Department of Pathology, Lille University Hospitals, were searched to identify all native kidney biopsy specimens with histological TMA from January 1, 2007 to December 31, 2012. These biopsies came from 5 different nephrology centers of northern France.
      We excluded cases with significant Ig or complement glomerular deposits by IF, as immune-mediated glomerulonephritides are confounding factors that can cause FSGS and particularly CG. We also excluded cases with no adequate frozen tissue for IF. Patients with diabetes mellitus were also excluded from the study because severe diabetic arteriolopathy may be difficult to distinguish from arteriolar TMA.
      The referring physician obtained informed consent from each patient for biopsy as well as for the use of clinical data and leftover histological material for research.

       Clinical and laboratory data

      We reviewed the clinical presentation of renal disease including clinical symptoms, laboratory evidence of renal dysfunction, and exploration of the complement pathway. We also systematically recorded the evolution of clinical and biological data obtained 1 year after the initial diagnosis or during the last follow-up time point or both.
      Systemic TMA was defined as the association of ≥3 of the following criteria: presence of >2% schistocytes on the blood smear, thrombocytopenia (<150.000), anemia, low haptoglobin level, and elevated lactate dehydrogenase level. Hypertension was defined as systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg, or receiving antihypertensive medications. Malignant hypertension was defined as a marked elevation of blood pressure, associated with central nervous system symptoms, such as papilledema. To assess renal function, acute kidney injury was defined according to the most recent Kidney Disease: Improving Global Outcomes (KDIGO) criteria.
      Section 2: AKI definition.
      Nephrotic syndrome was defined as the association of nephrotic range proteinuria (>3.5 g/d) and hypoalbuminemia (<30 g/l).

       Kidney biopsies

      All biopsies were processed for light microscopy and direct IF. Tissue for histology was fixed in acetic acid-formalin-absolute alcohol. For light examination, at least 24 serial sections for each biopsy specimen were stained with Masson trichrome, periodic acid–Schiff, Jones methenamine silver, or hematoxylin-eosin-saffron. Six-micrometer frozen sections were stained for IF with fluorescein isothiocyanate–conjugated antibodies specific for human IgG, IgM, IgA, C1q, C3, kappa and lambda light chains, and fibrinogen (DAKO, Carpinteria, CA, USA). Kidney biopsies were recalled from the tissue registry and reviewed by a senior renal pathologist (DB) without knowledge of the clinical information.
      Regarding TMA changes, the following lesions have been registered: endothelial swelling (arteriolar and glomerular), fibrin thrombi (arteriolar and glomerular), glomerular congestion, arteriolar subendothelial edema, fibrinoid necrosis of arteriolar walls (defined as a deeply eosinophilic material containing apoptotic debris of endothelial cells and myocytes present, unlike fibrin thrombi, within arteriolar walls), “onion-skin” concentric thickening of arteriolar walls, fibrous arteriolar occlusion, remodeling of glomerular capillary walls with double contours, ischemic wrinkling of the glomerular tuft, and mesangiolysis. The changes were classified as either focal (changes in <50% of glomeruli and arterioles) or diffuse (changes in ≥50% of glomeruli or arterioles or both). The presence of fibrin thrombi or fibrinoid necrosis of arteriolar walls defined “active” TMA.
      The Columbia classification criteria
      • D'Agati V.D.
      • Fogo A.B.
      • Bruijn J.A.
      • Jennette J.C.
      Pathologic classification of focal segmental glomerulosclerosis: a working proposal.
      were strictly applied when FSGS changes were observed to distinguish between the different variants. At least 1 glomerulus with global or segmental collapse of the capillary tuft, wrinkling of the glomerular basement membrane, and prominent hyperplasia and hypertrophy of the overlying epithelial cells was mandatory for diagnosing the collapsing variant.
      • D'Agati V.D.
      • Fogo A.B.
      • Bruijn J.A.
      • Jennette J.C.
      Pathologic classification of focal segmental glomerulosclerosis: a working proposal.
      Podocyte hyperplasia was distinguished from cellular crescent formation by the identification of the Bowman space external to the cell accumulation, absence of intercellular matrix, and lack of spindled-cell morphological characteristics and glomerular necrosis.
      The severity of interstitial fibrosis, tubular atrophy, and arterial intimal fibrosis was semiquantitatively scored on a scale of 0 to 3+ (0%–5%: none, 1: 6%–25%, 2: 26%–50%, 3: >50%).

       Transmission electron microscopy

      Kidney tissue specimens were fixed in Carson fixative and postfixed in 1% osmium tetroxide, dehydrated with acetone, and embedded in Epon 812. The ultrathin sections were contrasted with uranyl acetate and lead citrate and studied using a LEO EM 906 electron microscope (Zeiss, Oberkochen, Germany).

       Immunohistochemistry

      The podocyte phenotype was assessed using antibodies directed against 2 markers of podocyte differentiation: CD10 (56C6; dilution 1:100; Novocastra, Newcastle Upon Tyne, UK), and Wilms tumor 1 (sc-192; dilution 1:100; Santa Cruz Biotechnology, Heidelberg, Germany). Podocyte proliferation was assessed using Ki67 (MIB1; 1:50; Dako, Trappes, France) antibody. CD61 (2f2; 1:50; Novocastra) antibody was used for the detection of platelets thrombi. Immunohistochemistry was performed on paraffin-embedded biopsy tissue for every case with available histological material. All immunohistochemical studies were performed according to the standard automated immunohistochemical procedure (Ventana XT autostainer; Ventana Medical Systems, Benchmark XT, Strasbourg, France).

       Statistical analysis

      Baseline characteristics and histological data were compared using chi-square tests of Fisher exact test for categorical variables and analyses of variance. T-tests (when applicable) or nonparametric tests (Mann-Whitney or Kruskal-Wallis) were used for continuous variables. The end-stage renal disease–free survival time was calculated using the method of Kaplan and Meier and statistical comparisons were performed using the log-rank test.
      Statistical testing was performed at the 2-tailed α level of 0.05. Statistical analyses were performed using the SPSS package (version 15.0 for Windows; Chicago, IL, USA).

      Disclosure

      All the authors declared no competing interests.

      Acknowledgments

      The authors thank A. Hertig for his comments on the manuscript.
      The authors also thank A. Rodenas-Osorio (Pathology Department, Tenon Hospital, Paris, France), M.-C. Verpont (electron microscopy platform, INSERM, UMR S 1155, Paris, France), D. Langui (UMR S1127, and INSERM U1127, and CNRS UMR7225, and ICM, Sorbonne Université, Université Pierre et Marie Curie, Paris, France), R.-M. Siminski and M.-H. Gevaert (Histology Department, Centre Hospitalier Régional Universitaire de Lille, Lille, France), and all the technical staff of the Pathology Department of the Centre Hospitalier Régional Universitaire de Lille for their technical assistance.

      Supplementary Material

      • Table S2

        Correlation between clinics, biology, and pathology of TMA. A, African; C, Caucasian; DBP, diastolic blood pressure; SBP, systolic blood pressure; TMA, thrombotic microangiopathy. aP < 0.05 for mixed versus glomerular TMA. bP < 0.05 for arteriolar versus glomerular TMA.

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      1. Section 2: AKI definition.
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