Int Urol Nephrol DOI 10.

1007/s11255-012-0164-6

NEPHROLOGY ORIGINAL PAPER

Evaluation of renal biopsies in type 2 diabetic patients with kidney disease: a clinicopathological study of 216 cases
Li Zhuo Wenwen Ren Wenge Li Guming Zou Jianhua Lu

Received: 8 December 2011 / Accepted: 13 March 2012 Springer Science+Business Media, B.V. 2012

Abstract Objectives The outcome and the therapy of patients with diabetes mellitus (DM), diabetic nephropathy (DN), and non-diabetic renal disease (NDRD) are quite different, so the differential diagnosis is of considerable importance. To evaluate the usefulness of renal biopsy in type 2 diabetic patients, we examined the relationship between the clinical parameters and the histopathological ndings in different age groups. Methods Renal biopsy specimens and clinical and laboratory data from 216 patients with type 2 DM were evaluated. According to their age, three groups were dened: 1735 years (group I), 3659 years (group II), and more than 60 years (group III). Results The study showed that, beside the duration of diabetes, other clinical parameters were not significantly different between the three groups. Chronic nephritic syndrome was the most common clinical manifestation in group I (44.1 %) and in group II (34.0 %). Among patients in group III, we found a high prevalence of chronic renal failure (34.3 %) and nephrotic syndrome (28.6 %). Consistent with the clinical manifestations, IgA nephropathy was the most common pathologic nding in group I (29.4 %) and in

group II (34.7 %), whereas the most frequent abnormalities in group III were membranous nephropathy (25.7 %) and tubulointerstitial lesions (14.3 %). Overall, among these patients, 14 cases were diagnosed with DN (6.5 %), 179 with NDRD (82.9 %), while 23 had concurrent DN and NDRD (10.7 %). Conclusions Our results indicated that the clinical manifestations and pathologic ndings in type 2 diabetic patients in different age groups have different features. This study emphasized the usefulness of renal biopsy for determining the pattern of renal damage and thus for the overall management of type 2 diabetic patients. Keywords Type II diabetes mellitus Diabetic nephropathy (DN) Non-diabetic renal disease (NDRD) Renal biopsy Renal histopathology Age Introduction Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM) and also a major cause of end-stage renal disease (ESRD). The incidences of DM and DN are rising rapidly in the past decades and have emerged as a great burden to the healthcare system in China. However, it has been estimated that up to one-third of diabetic patients who are submitted to renal biopsy may have different nondiabetic renal disease (NDRD). These diseases can be either independent or associated (superimposed) with

L. Zhuo W. Ren W. Li (&) G. Zou J. Lu Department of Nephrology, Chinese-Japanese Friendship Hospital, 2 East Yinghuayuan Street, Hepingli, Beijing 100029, Peoples Republic of China e-mail: wenge_lee2002@yahoo.com

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the DN. The pathogenic mechanism of DN is different to NDRD. And it is usually believed that DN is hard to reverse, but some NDRD are often treatable, even remittable. The therapy and prognosis of DM, DN, and NDRD are quite different, so the differential diagnosis is of considerable importance. Although renal biopsied are not routinely performed in diabetic patients, it is indicated, however, in patients who develop sudden onset proteinuria, hematuria, and/or rapid deterioration of renal function [1, 2]. To our knowledge, there is no such study in the local population where histopathological evaluation of renal biopsy has been performed in diabetic patients. In order to evaluate the usefulness of renal biopsy in patients with type II diabetes mellitus, we examined the relationship between various clinical parameters in patients with type II diabetes mellitus and renal biopsy samples.

Methods Renal biopsy specimens from 216 patients with type II diabetes mellitus obtained between January 2003 and December 2010 at the Nephrology Department of Chinese-Japanese Friendship Hospital were evaluated. As sufcient information about the characteristic lesions of DN accumulated, renal biopsy was not performed routinely at this institution. Renal biopsy was performed because urinary abnormalities or renal functions were inconsistent with either the clinical expression or the natural history of DN. The study protocol was approved by the Human Ethics Review Committee of China, and signed consent form was obtained from each patient before biopsy. Type II diabetes mellitus was diagnosed using the criteria of the Chinese Diabetic Society [3]. The age range of patients was 1777 years. In order to elucidate the inuence of aging on the clinical manifestations and histopathological ndings, 3 subgroups were formed: 1735, 3659 years, and more than 60 years. The clinical parameters recorded for each patient at the time of renal biopsy included age, sex, duration of diabetes, blood pressure, electrocardiogram, echocardiogram, kidney ultrasound, and funduscopic ndings. Laboratory studies included fasting blood sugar, sugar tolerance test, HbA1c, urinalysis, urine osmotic pressure, 24-h protein excretion, total protein, albumin, blood urea nitrogen, serum creatinine level, creatinine clearance, serum complement level, serum

immunoglobulin level, and the presence of antinuclear antibodies. All cases were processed routinely for light microscopy, immunouoresence and electron microscopy. Renal tissue submitted for diagnosis was divided into three portions; one piece was xed in buffered formalin and processed into parafn blocks for light microscopy examination. Sections were stained with hematoxylin and eosin, periodic acid Schiff (PAS), silver methanamine, and Masson trichrome. The second piece of tissue was frozen for direct immunouoresence studies with uorescein isothiocyanate (FITC) conjugated antibodies for IgG, IgA, IgM, C3, C4, C1q, and brinogen. The third piece was xed in Trumps EM xative and processed into resin blocks; ultrathin sections stained with uranyl acetate and lead citrate were then examined with the transmission electron microscope. Renal tissue damage was assessed by the mean glomerulosclerosis (GS) score, the score of tubularinterstitial injury and vessel lesions [47]. Each specimen reported included at least ten glomeruli. The considered characteristics were examined, including diffuse mesangial expansion with predominance of increased mesangial matrix, KimmelstielWilson nodular lesions, hyaline exudative lesions, and glomerular basement membrane thickening. GS score was evaluated on a semiquantitative scale of 04 for the degree of glomerulosclerosis (0, no GS; 1, B25 %; 2, 2650 %; 3, 5175 %; and 4, 76100 % GS) per glomerulus and expressed as the mean GS score for the total glomeruli in every tissue section. Tubularinterstitial injury was measured by semiquantitative grading on a 03 scale (score 0, no change; 1,\25 %; 2, 2550 %; and 3, [50 % tubules or interstitium involved). For arterial morphometry, *100 crosssections per patient group of intrarenal arteries with diameter \80 lm (corresponding to arterioles and interlobular arteries) were randomly selected at 409 magnication in PAS-stained sections. The vessel and lumen diameters were measured, and media thickness was obtained by the formula: (vessel diameter-lumen diameter)/2. To avoid bias due to sectioning vessels at various angles, the elliptic proles were measured at the widest part of the shortest diameter. Percent of vessel diameter occupied by media and lumen, media to lumen ratio, and media cross-sectional area were also calculated [5]. Renal lesions were classied according to the Chinese-classication criteria for

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renal diseases. Each biopsy was reviewed by one of the two staff pathologists, both dedicated to renal pathology. Furthermore, the relationship between clinical parameters and histopathological ndings were examined. Statistical analysis was performed using SPSS version 17.0 for windows (SPSS Inc., Chicago, IL, USA). Data are expressed as mean SD. Regression analysis and the Spearman rank correlation test were used to determine the correlation between clinical parameters and histopathological ndings. Differences between groups were analyzed by v2 test or MannWhitney U test. p \ 0.05 was considered signicant.

Results Table 1 lists the biochemical characteristics that were observed in type II diabetic patients at the time of renal biopsy. Of course, the patients in group III were of older age than in groups I and II (p \ 0.05). Moreover, patients in group I had a shorter duration of diabetes (p \ 0.05), but it did not show statistical signicance that between in groups II and III. Compared with group III, the incidence rate of overlapped hypertensive disease was lower in group I and II, although this difference did not reach statistical signicance. Fasting blood sugar, albuminuria, and the incidence rate of retinopathy did not differ between these three groups.

The patient clinical manifestations were summarized in Table 2. The most common clinical manifestation was chronic nephritic syndrome (a syndrome characterized pathologically by diffuse inammatory changes in the glomeruli and clinically by hematuria with RBC casts, mild proteinuria, and, often, hypertension, edema, and azotemia.) which accounted for 44.1 % of all patients in group I. The clinical manifestations were multiformity in group II, chronic nephritic syndrome (34.0 %), nephrotic syndrome (It is a group of symptoms including protein in the urine (more than 3.5 g per day), low blood protein levels, high cholesterol levels, high triglyceride levels, and swelling.) (28.6 %), chronic renal failure (18.4 %), latent glomerulonephritis (It is also known as asymptomatic proteinuria or hematuria, urine routine examination that is abnormal, but no edema, hypertension, or renal dysfunction such as performance.) (14.3 %), and acute renal failure (4.8 %) by turns. It did not show statistical signicance in group II. Among the patients in group III had showed prevalence of chronic renal failure (34.3 %) and nephrotic syndrome (28.6 %). Chronic nephritic syndrome was signicantly greater in groups I and II compared with in group III (p \ 0.05). In group I, the most common pathologic diagnosis found was IgA nephropathy, which accounted for 29.4 % of all patients. Membranous nephropathy and obesity-related glomerulopathy accounted for 11.8 %, respectively, followed by focal segmental glomerular sclerosis (FSGS) (8.8 %) and mesangial proliferative glomerulonephritis (8.8 %). The most common renal

Table 1 The patient demographics and baseline characteristics Clinical indices Patients Mean age, years Fasting blood sugar (mmol/L) Diabetes duration (month) Albuminuria (microalbuminuria and macroalbuminuria) Retinopathy Overlapped hypertension Stage 1 Stage 2 Stage 3 * P \ 0.05 versus Group I
m

Group I 34 29.60 4.4 8.01 4.7 11.99 21.6 33 (97.1 %) 7 (20.7 %) 17 (50.0 %) 5 6 6

Group II 147 48.22 6.6* 7.33 9.4 43.76 57.7* 145 (98.6 %) 27 (18.4 %) 95 (64.6 %) 19 38 38

Group III 35 65.48 4.3*,m 6.77 2.2 63.51 76.9* 33 (94.3 %) 5 (14.3 %) 24 (68.6 %) 3 14 7

P \ 0.05 versus Group II

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lesions found in group II also were IgA nephropathy (34.7 %), followed by membranous nephropathy (15.0 %), mesangial proliferative glomerulonephritis (6.1 %), obesity-related glomerulopathy membranous nephropathy (5.4 %), focal proliferative glomerulonephritis (5.4 %), and tubulointerstitial lesion (5.4 %). In elderly patients (group III), membranous nephropathy was most common, accounting for 25.7 %. Tubulointerstitial lesion ranked second accounting for 14.3 %, followed by mesangial proliferative

glomerulonephritis (11.4 %), crescentic glomerulonephritis (8.6 %) and so on. IgA nephropathy was signicantly greater in groups I and II compared with patients in group III (p \ 0.05). However, the prevalence of membranous nephropathy and tubulointerstitial lesion in group III were signicantly higher than in groups I and II (p \ 0.05). Overall, in the present study, 216 patients were nally included, of which 14 cases were diagnosed as DN, 179 were NDRD, while 23 were concurrent DN and NDRD (Table 3).

Table 2 The patient clinical manifestations Clinical manifestations Chronic nephritic syndrome Nephrotic syndrome Latent glomerulonephritis Acute renal failure Chronic renal failure * P \ 0.05 versus Group I
m j

Group I (n = 34) 15 (44.1 %)j 7 (20.6 %) 6 (17.6 %) 3 (8.8 %) 3 (8.8 %)

Group II (n = 147) 50 (34.0 %)j 42 (28.6 %) 21 (14.3 %) 7 (4.8 %) 27 (18.4)

Group III (n = 35) 6 (17.1 %) 10 (28.6 %) 2 (5.7 %) 5 (14.3 %) 12 (34.3 %)*

P \ 0.05 versus Group II P \ 0.05 versus Group III

Table 3 The patient pathologic diagnoses Pathological types Diabetic nephropathy IgA nephropathy Membranous nephropathy Obesity-related glomerulopathy membranous nephropathy Focal proliferative glomerulonephritis Focal segmental glomerular sclerosis (FSGS) Minimal change glomerulopathy Membranoproliferative glomerulonephritis Mesangial proliferative glomerulonephritis Endocapillary proliferative glomerulonephritis Crescentic glomerulonephritis Thin basement membrane syndrome Amyloid protein Tubulointerstitial lesion Hypertensive renal damage Overlapped DN with NDRD *P \ 0.05 versus Group I
m j

Group I (n = 34) 1 (2.9 %) 10 (29.4 %)j 4 (11.8 %) 4 (11.8 %) 0 3 (8.8 %) 0 0 3 (8.8 %) 2 (5.9 %) 2 (5.9 %) 0 0 0 1(2.9 %) 4 (11.8 %)

Group II (n = 147) 11 (7.5 %) 51 (34.7 %)j 22 (15.0 %) 8 (5.4 %) 8 (5.4 %) 2 (1.4 %) 3 (2.0 %) 0 9 (6.1 %) 1 (0.7 %) 2 (1.4 %) 1(0.7 %) 1 (0.7 %) 8 (5.4 %) 3(2.0 %) 17(11.6 %)

Group III (n = 35) 2 (5.7 %) 1 (2.9 %) 9 (25.7 %)*,m 1 (2.9 %) 2 (5.7 %) 1 (2.9 %) 1 (2.9 %) 0 4 (11.4 %) 0 3 (8.6 %) 0 2 (5.7 %) 5 (14.3 %)*,m 2(5.7 %) 2(5.7 %)

P \ 0.05 versus Group II P \ 0.05 versus Group III

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Discussion Diabetic nephropathy is also known as diabetic GS, and diabetic kidney disease. It is a complication of diabetes associated with the kidney. DN is the leading cause of kidney disease in patients starting renal replacement therapy and affects *40 % of diabetic patients. It increases the risk of death, mainly from cardiovascular causes. However, it takes a long time for the symptoms to become evident and thus generally cannot be diagnosed in the early stages. DN is inferred if a patient had diabetes at least 710 years, exhibited demonstrable diabetic retinopathy, and had previous microalbuminuria without sudden onset of heavy proteinuria, no hematuria and non-small-sized kidney in the absence of other renal diseases [8]. DN is categorized into stages: microalbuminuria (urinary albumin excretion, UAE [ 20 lg/min and B199 lg/ min) and macroalbuminuria (UAE C 200 lg/min) [9]. Renal biopsy in this setting will not be diagnostically useful. Actually, a variety of renal lesions other than DN can occur in diabetic patients, just be dened as NDRD, such as IgA nephropathy, membranous nephropathy, mesangial proliferative glomerulonephritis, hypertensive renal disease, and FSGS. Although some studies suggest that the incidence of NDRD is relatively rare, the possible involvement of NDRD needs to be considered. The challenges still exist to differentiate NDRD from DN in DM patients. Nevertheless, the low prevalence of NDRD may be partly due to unsuitable criteria for renal biopsy in DM patients. In the present study, 93.5 % of patients with DM had NDRD. There are no standardized criteria for renal biopsy in DM; therefore, obtaining a renal biopsy from patients with DM is currently a matter of judgment by the primary physician. In our center, renal biopsy is commonly performed in patients with DM who show the following features: (1) clinical renal lesions (microalbuminuria) occurring within 5 years of onset of DM; (2) microalbuminuria without diabetic retinopathy; (3) macroscopic hematuria and microscopic hematuria; (4) rapidly progressive renal failure; (5) proteinuria [3.5 grams per day and eGFR C 60 ml/min/1.73 m2; (6) suspected other renal diseases secondary to systemic disease. Our experiences demonstrated that high detection rate of NDRD could be achieved, if patients performed renal biopsy according to the six characters.

The occurrence of NDRD in diabetic patient is well recognized. Different frequencies for NDRD have been reported from different parts of the world [2, 10 13]. In fact, the prevalence of NDRD from these researches would be unreliable in small samples. And less researchers focus on the clinical manifestations and histopathological types in NDRD patients in different age. In the present study, 93.5 % of diabetic patients had NDRD. Our results showed the prevalence of diabetic retinopathy was rarely in the three groups, while it was similar to other literatures. Diabetic retinopathy is one of the microvascular complications of DM, which might have the same pathogenetic pathways as DN. Retinopathy, when it coexists with nephropathy, is thought to be a window of renal complication. Just in this study, 39 cases were diagnosed as diabetic retinopathy and 37 cases were diagnosed as DN, which accounted for 18.1 and 17.1 % of all patients. So, retinopathy may serve as an indicator of DN. However, the duration of diabetes was short, and the mean duration of diabetes was less than 5 years in each group, which was consistent with the frequency of diabetic retinopathy. Durations of diabetes in middle- and older-age patients were signicantly longer than young. The duration of diabetes may affect not only the incidence and extent of retinopathy, but also the extent of renal lesions in these patients. Therefore, a number of studies have conrmed that duration of diabetes was associated with DN lesions [14, 15]. So, the short duration of diabetes and absence of retinopathy could be a useful predictor for NDRD in diabetic patients, as other researchers mentioned [16, 17]. A surprising nding in our study was the poor correlation between hypertension on age and frequency of NDRD. In this study, 133 cases were diagnosed as hypertension, which accounted for 60.9 % of all NDRD patients and 61.6 % of all DM patients. Meanwhile, the incidence rate of hypertension did not differ between different ages. It is worth noting that absence of hypertension in DM was one of the differential diagnoses of NDRD in some literature [18]. However, our ndings are indicated that blood pressure control is of primary importance in the prevention of progression of renal disease in both diabetic disease and NDRD. So, emphasis is given on antihypertensive therapy in patients with DM to slow down or prevent the progression of DN.

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The most common clinical manifestation was chronic nephritic syndrome in young patients, and nephrotic syndrome and chronic renal failure in elders. However, in middle-age patients, the clinical manifestations were multiformity. It might be the direct cause of different pathologic diagnosis in the population. IgA nephropathy is the most common NDRD in diabetics and also the most common glomerulonephritis in adult biopsies of non-diabetics in Asian reports [13, 17, 19]. Similarly, the current study also showed that IgA nephropathy was the common pathologic type in young and middle-age patients. IgA nephropathy is considered to be multifactorial disease in which pathogenesis involves genetic and environmental factors [20]. It is the most common glomerular disease in young adults [21], so the frequency of IgA nephropathy was low in elderly patients, which merely accounted for 2.9 % in our study. The most common pathologic types in elders were membranous nephropathy and tubulointerstitial lesion. It was consistent with the common clinical manifestations in older patients, which were nephrotic syndrome and chronic renal failure, respectively. Although membranous nephropathy and tubulointerstitial lesion were described more frequently in elders, we are unable to infer whether any of the NDRD is more common among diabetic patients when compared with the general population. Taken together, these data suggest that the clinical manifestation and pathologic ndings in different age groups have different features. Anyway, our results are no different with the prevalence of glomerular disease in nondiabetic patients in previous studies. Thus, there seems a need for further multicenter randomized prospective studies to conrm it. This study has emphasized that renal complications might be due to heterogeneous non-diabetic lesions in diabetic patients. These non-diabetic lesions may occur alone or may be superimposed on underlined DN, which are associated with different renal outcome and probably beneted with treatment modalities. And the clinical manifestation and pathologic types in different age groups have different features: The most common clinical manifestation was chronic nephritic syndrome in young patients, and nephrotic syndrome and chronic renal failure in elders. However, in middle-age patients, the clinical manifestations were multiformity. As well as the common pathologic type in young and middle-age patients was IgA

nephropathy, while in elder patients was membranous nephropathy and tubulointerstitial lesion. Though many indicators have been found to be important distinguishing DN from NDRD in the literature, renal biopsy is still the most useful in the overall management of patients with type II diabetes mellitus. It should be noted that the effective renal biopsy criteria could increase the detection rate and diagnostic accuracy of NDRD.
Acknowledgments This work was supported by the National Natural Science Foundation of China (81170675), the Science Foundation of Chinese-Japanese Friendship Hospital (2010ZD-05) and Postdoctoral Science Foundation of China (20090450200). Conict of interest We have had no involvements that might raise the question of bias in the work reported or in the conclusions, implications, or opinions stated. The results presented in this paper have not been published previously in whole or part.

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