THE PREVALENCE AND INCIDENCE of

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1 Podocyte Lesions in Patients With Obesity-Related Glomerulopathy Hui-Mei Chen, PhD, Zhi-Hong Liu, MD, Cai-Hong Zeng, PhD, Shi-Jun Li, PhD, Qing-Wen Wang, MD, and Lei-Shi Li, MD Background: Obesity-related glomerulopathy is an important complication of obesity that shares some pathophysiological factors relevant to renal damage with diabetic nephropathy. It was recognized that podocyte lesions lead to proteinuria and glomerulosclerosis in patients with some proteinuric glomerular diseases, especially diabetic nephropathy. However, podocyte changes in patients with obesity-related glomerulopathy and the underlying mechanisms are unclear. Methods: Glomerular volume, podocyte number, and foot-process width were evaluated in 46 patients with biopsy-proven obesity-related glomerulopathy. Renal tissue from 10 kidney donors served as controls. Results: Glomerular volume was enlarged markedly in patients with obesity-related glomerulopathy (P < 0.001), with a corresponding decrease in podocyte density (P < 0.001) compared with controls. Foot-process width on the peripheral glomerular basement membrane was increased significantly, whereas mesangial volume fraction did not differ between patients with obesity-related glomerulopathy and controls. Degree of proteinuria was associated strongly with decreased podocyte density (P 0.001), increased foot-process width (P 0.001), and decreased podocyte number (P 0.002). In addition, the endogenous creatinine clearance rate correlated with decreased podocyte number (P 0.008). Interestingly, podocyte number and density correlated significantly with abnormalities in fasting glucose (P < 0.001) and insulin levels (P < 0.001) and Homeostasis Model Assessment of Insulin Resistance (P < 0.001). Conclusion: Decreased podocyte density and number were observed in patients with obesity-related glomerulopathy, and changes in podocytes correlated with degree of proteinuria and renal function impairment in these patients. In addition to the enlargement in glomerular volume with consequential mechanical stretch, metabolic disorders also may contribute to the development of podocyte lesions in patients with obesity-related glomerulopathy. Am J Kidney Dis 48: by the National Kidney Foundation, Inc. INDEX WORDS: Obesity-related glomerulopathy; podocyte; proteinuria. From the Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing , PR China. Received April 20, 2006; accepted in revised form July 26, Originally published online as doi: /j.ajkd on October 6, Support: None. Potential conflicts of interest: None. Address reprint requests to Zhi-Hong Liu, MD, Professor of Medicine, Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing , PR China. zhihong@21cn.net 2006 by the National Kidney Foundation, Inc /06/ $32.00/0 doi: /j.ajkd THE PREVALENCE AND INCIDENCE of obesity are increasing rapidly in both developed and developing countries. 1-3 The International Collaborative Study of Cardiovascular Disease in Asia indicated that obesity and overweight have become important public health problems in China. 4 Obesity is an important modifiable risk factor for metabolic diseases, including hypertension, coronary heart disease, type 2 diabetes, and obesity-related glomerulopathy. 5,6 Thus, the epidemic of obesity has led to a progressive increase in number of patients diagnosed with obesity-related glomerulopathy during the last decades. 7,8 Obesity-related glomerulopathy is characterized by increasing proteinuria and then decreasing glomerular function. Renal biopsies showed that glomerulomegaly and focal segmental glomerulosclerosis (FSGS) are the most common histological lesions in patients with obesityrelated glomerulopathy. 7,9 Experimental studies and clinical data showed that hemodynamic changes and metabolic disorders have important roles in the development of obesity-related glomerulopathy. 5 Interestingly, hemodynamic changes and metabolic disorders are the main factors that also contribute to renal damage in patients with diabetes, which has been related to podocyte lesions. 10 Furthermore, podocyte lesions in patients with diabetic nephropathy were shown to precipitate the development of proteinuria and decrease in renal function, as well as glomerulosclerosis. 11,12 To explore the underlying mechanisms of proteinuria and progression of renal disease in patients with obesity-related glomerulopathy, podocyte change was analyzed and its relationship with renal function and coincident metabolic disorders also was evaluated in the present study. 772 American Journal of Kidney Diseases, Vol 48, No 5 (November), 2006: pp

2 PODOCYTES IN OBESITY-RELATED GLOMERULOPATHY 773 Patients and Controls METHODS The World Health Organization s definition of obesity is based on increased body mass index (BMI), whereas it is not appropriate for the Chinese population. 3 Obesity in this study is defined as a BMI of 28 kg/m 2 or greater, according to criteria from the Cooperative Meta-Analysis Group of China obesity task force. 13 Forty-six Chinese patients with obesity, proteinuria, and biopsy-proven obesity-related glomerulopathy were enrolled in the present study. Histological changes in these patients are defined as obesity-associated FSGS with glomerulomegaly or obesity-associated glomerulomegaly alone. 5,7,9 Idiopathic cases of FSGS were ruled out carefully according to clinical and histological characteristics, including variants in glomerular size, diffuse foot-process effacement, and segmental glomerular scarring. Other underlying conditions that could cause secondary FSGS or glomerulomegaly were excluded carefully, such as diabetes and/or diabetic glomerulosclerosis. Ten nonobese kidney donors were used as controls, matched for sex and age. These controls had significantly lower BMIs than our patients with obesity-related glomerulopathy (Table 1). Donors had no history of hypertension, obesity, diabetes, or renal diseases. Approval was obtained from local authorities, and informed signed consent was obtained from each patient and control. Renal Pathological Evaluation Renal biopsies were performed under ultrasound guidance by an experienced investigator, and tissues were processed for light, electron, and immunofluorescence microscopy according to standard techniques. Hematoxylin and eosin, periodic acid Schiff, methenamine silver, and Masson stains were used routinely on formalin-fixed tissue, sectioned at 2 m. Electron microscopic examination was conducted on tissues fixed in 2.5% glutaraldehyde and embedded in epon. Electron Microscopy Photographs obtained at original magnifications 2,000 to 4,000 were assembled for measurement on a single section through the glomerulus using the method of Weibel and Gomez. Although it is not the best choice for podocyte number estimation, the Weibel and Gomez method from electron micrographs can provide estimation similar to the disector/fractionator method, which is considered the theoretic gold standard method. 14 Morphometric analysis was performed on 3 open glomeruli per biopsy specimen. Podocytes are defined as residing within the glomerular tuft, but outside the glomerular basement membrane (GBM). Area density of podocyte nuclei per glomerulus [Na(epinuclei/glom)] was determined by point counting 14 : Na(epinuclei/glom) N/[ Pg (d/mag) 2 ], where N is number of nuclei, Pg is number of points hitting the glomerular profile, d is distance between coarse grid points, and mag is magnification. The volume density of podocyte nuclei per glomerulus [Vv(epi-nuclei/glom)] was calculated: Vv(epinuclei/glom) Pn/( Pg 16), where Pn is number of fine grid points over nuclei and 16 is number of fine points corresponding to each coarse point. Finally, the numerical density of podocytes [Nv(epi/glom)] was calculated: Nv(epi/ glom) (1/1.55) [Na(epi-nuclei/glom) 3 /Vv(nuclei/ glom)] 1/2, where 1.55 is the shape factor for nuclei. The same montages were used to estimate mesangial fractional volume [Vv(mes/glom)] by point counting. 15 Another set of photographs was obtained at higher power micrographs (original magnification 20,000). Podocyte foot-process width on peripheral GBM or filtration surface was measured. The average foot-process width was calculated 16 : Foot-process width ( /4) ( GBM length/ foot process), where GBM length is total length of peripheral Table 1. Clinical Characteristics of Patients With Obesity-Related Glomerulopathy Patients With Obesity-Related Glomerulopathy Controls P Men/women 34/12 7/3 NS Age (y) NS Body mass index (kg/m 2 ) Known duration of obesity (y) Mean blood pressure (mm Hg) Pulse blood pressure (mm Hg) NS ACE-inhibitor/ARB treatment 15 (32.6) Hypertension 17 (37.0) 0 (0) 0.05 Known duration of proteinuria (mo) Proteinuria (g/24 h) Serum creatinine (mg/dl) NS Creatinine clearance (ml/min) Left kidney length (mm) NOTE. Values expressed as mean SD or number (percent). To convert serum creatinine in mg/dl to mol/l, multiply by 88.4; creatinine clearance in ml/min to ml/s, multiply by Abbreviation: NS, not significant; ACE, angiotensin II-converting enzyme; ARB, angiotensin II-receptor blocker.

3 774 GBM in each picture, foot process is total number of foot processes on peripheral GBM, and the correction factor /4 serves to correct for presumed random variation in the angle of section relative to the long axis of the podocyte. Light Microscopy Periodic acid Schiff stained paraffin-embedded light microscopy slides at original magnification 400 were used to estimate mean glomerular volume by the method of Weibel and Gomez, only when at least 10 glomerular profiles were available. Mean glomerular volumes of controls were estimated by using the same method in paraffin-embedded tissue. Mean glomerular volume was calculated 17 : V(glom) 0.75 [ Pg (d/mag) 2 ] 3/2 0.21, where 0.75 and 0.21 are both factors from line regression. Because mean glomerular volume is greater for plasticembedded tissue than paraffin-embedded tissue, the correction factor 2.09 is used for conversion to allow comparisons with previously published studies. 18 Number of podocytes per glomerulus Epi(N/glom) was determined 14 : Epi(N/ glom) V(glom) Nv(epi/glom). An average of 50 glomerular sections were examined in light microscopy and categorized as global or segmental glomerulosclerosis. Clinical Parameters Patient charts were reviewed for age, sex, and presenting clinical and laboratory data at the time of renal biopsy. Periods of obesity and proteinuria were recorded based on patient statements. Physical examinations, routine biochemical determinations, and 24-hour urine samples for measurement of endogenous creatinine clearance rate and daily proteinuria were obtained. Clearances were adjusted for body surface area by using height and weight according to the following formula: [(height in centimeters)(weight in kilograms)/3,600] 1/2. 7 Hypertension is defined as blood pressure greater than 140/90 mm Hg. Plasma insulin concentrations were measured by means of radioimmunoassay (supplied by the Research Institute of Diabetes Mellitus, Chengdu, PR China). Insulin resistance was quantified by using Homeostasis Model Assessment of Insulin Resistance, a model used successfully for evaluating insulin sensitivity in large studies. 19 Ultrasonographic measurements of kidney length were determined by the same examiner with a 3.75-MHz transducer (Vivid 7; GE Inc, USA). CHEN ET AL Statistical Analysis Data are expressed as mean SD and analyzed using SPSS, version 11.0 (SPSS Inc, Chicago, IL). Comparisons between groups were performed using Student t-test. Relationships between parameters were analyzed using Pearson correlation coefficient. Multivariate analysis for related variables was performed by using stepwise linear regression. Two-tailed P less than 0.05 is considered statistically significant. RESULTS Clinical features of 46 patients with obesityrelated glomerulopathy and 10 controls are listed in Table 1. Patients with obesity-related glomerulopathy had greater proteinuria, creatinine clearance rate, kidney length, and mean blood pressure than controls. Because of retrospective analysis, durations of obesity were available for only 26 subjects according to patient statements. Unfortunately, there was no record of periods and doses of angiotensin II-converting enzyme (ACE)-inhibitor/angiotensin II-receptor blocker (ARB) treatment in this study. Podocyte Lesions and Glomerulopathy Data for podocytes in patients with obesityrelated glomerulopathy are listed in detail (Table 2). Obesity-related glomerulopathy was characterized by glomerulomegaly with a small percentage of glomerulosclerosis. Compared with controls, mean glomerular volume was markedly greater in patients with obesity-related glomerulopathy ( versus m 3 ; P 0.001; Fig 1); whereas podocyte number was numerically lower ( versus ; P 0.05). Because glomerular volume was greater and podocyte number was similar, podocyte density in patients with obesity- Table 2. Comparison of Glomerular and Podocyte Changes Between Patients With Obesity-Related Glomerulopathy and Controls Patients With Obesity-Related Glomerulopathy Controls P Volume of glomerulus ( 10 6 m 3 ) Numerical density of podocytes per glomerulus (/10 6 m 3 ) Podocyte number per glomerulus NS Mean global sclerosis (%) 12.5 (0-81) 2 (0-6.8) 0.05 Mean segmental sclerosis (%) 6.3 (0-20) Foot-process width on peripheral basement membrane (nm) Volume fraction of mesangium in glomerulus NS NOTE. Data expressed as mean SD or median (range). Abbreviation: NS, not significant.

4 PODOCYTES IN OBESITY-RELATED GLOMERULOPATHY versus ; P 0.05). Footprocess width on the peripheral GBM clearly was greater in patients with obesity-related glomerulopathy than controls ( versus nm; P 0.001), whereas a majority of foot processes presented no effacement or fusion. Fig 1. Glomerular hypertrophy in patients with obesityrelated glomerulopathy compared with healthy controls: (A) a normal glomerular section in controls, and (B) enlarged glomerulus in patients with obesityrelated glomerulopathy. (Periodic acid Schiff; original magnification 400.) related glomerulopathy thus was significantly decreased compared with controls ( versus /10 6 m 3 ; P 0.001). However, mesangial volume fraction of glomerulus was similar between patients with obesityrelated glomerulopathy and controls (0.21 Podocyte Changes and Renal Function The relationship between podocyte changes and renal function parameters was analyzed (Table 3). Proteinuria was related inversely to podocyte number and density (r 0.587; P and r 0.565; P 0.002, respectively) and correlated directly with foot-process width on the peripheral GBM (r 0.641; P 0.001). However, proteinuria had no obvious relationship with glomerular volume or mesangial volume fraction. Conversely, creatinine clearance directly related to only podocyte number (r 0.618; P 0.008). There was no correlation between creatinine clearance and podocyte density, glomerular volume, mesangial volume fraction, or foot-process width. Stepwise linear regression showed that both podocyte density and foot-process width were the strongest predictors of proteinuria (r 0.746; P 0.001), whereas podocyte number did not add to the relationship. Podocyte Changes and Metabolic Disturbances Table 4 lists univariate correlations between podocyte lesions and metabolic data. Both podocyte number and density were related inversely to fasting glucose level (P 0.001), insulin level (P 0.001), and HOMA-IR (P 0.001), whereas foot-process width on peripheral GBM did not correlate with metabolic variables. Multiple regression analysis showed that podocyte density correlated positively with fasting glucose Table 3. Correlations Between Podocyte Changes and Proteinuria and Renal Function in Patients With Obesity-Related Glomerulopathy Proteinuria Creatinine Clearance Pearson Correlation P Pearson Correlation P Volume of glomerulus 0.15 NS NS Numerical density of podocytes per glomerulus NS Podocyte number per glomerulus Foot-process width on peripheral basement membrane (nm) NS Volume fraction of mesangium in glomerulus NS NS Abbreviation: NS, not significant.

5 776 CHEN ET AL Table 4. Correlations Between Podocyte Lesions and Metabolic Disturbances in Patients With Obesity-Related Glomerulopathy Podocyte Number Podocyte Density Foot-Process Width on Peripheral Basement Membrane Pearson Correlation P Pearson Correlation P Pearson Correlation P Age (y) NS NS NS Body mass index (kg/m 2 ) NS NS NS Waist circumference (cm) NS NS NS Waist-hip ratio NS NS NS Mean blood pressure (mm Hg) NS NS NS Total cholesterol (mmol/l) NS NS NS Triglycerides (mmol/l) NS NS NS High-density lipoprotein cholesterol (mmol/l) NS NS NS Low-density lipoprotein cholesterol (mmol/l) NS NS NS Glucose (mmol/l) NS Insulin (miu/l) NS HOMA-IR NS Uric acid (mmol/l) NS NS NS Abbreviations: NS, not significant; HOMA-IR, Homeostasis Model Assessment of Insulin Resistance. level (r 0.826; P 0.006) and negatively with the other metabolic variables. Meanwhile, podocyte number was related to both fasting glucose and insulin levels (r 0.953; P 0.001), and age, sex, BMI, and levels of lipid protein and blood pressure did not contribute further. When patients with ACE-inhibitor/ARB treatment were analyzed as a subgroup, podocyte number was greater in patients with than without ACEinhibitor/ARB therapy ( versus ; P 0.022). DISCUSSION Increasing evidence shows that podocyte injury is the primary driving force in the development of proteinuria and glomerulosclerosis. 20 Podocytes are vulnerable to many forms of injury, including both immune-mediated and nonimmune processes. 21,22 Glomerular enlargement with consequential challenge of mechanical stretch and activation of the renin-angiotensin system is considered an important contributor to the development of podocyte injury. 12,21,23 Metabolic disturbances also are involved in the aggravation of podocyte injury, such as lipid dysmetabolism. 24 Our study shows decreased podocyte density and increased glomerular volume in patients with obesity-related glomerulopathy, whereas podocyte numbers were similar between patients and healthy controls. Interestingly, univariate correlation analysis showed that proteinuria was related positively to podocyte density, foot-process width, and podocyte number in patients with obesityrelated glomerulopathy. White et al 25 observed similar numbers of podocytes in healthy individuals and normotensive patients with type 1 diabetes. They also showed a correlation between podocyte number and albuminuria excretion rate, but only in proteinuric patients. A decrease in podocyte density was observed to be related to microalbuminuria in patients with diabetes. That study suggested that podocyte density may be functionally more relevant than absolute number in microalbuminuric patients with diabetes. 18 Meyer et al 26 showed that podocyte number was related to progression of proteinuria in a longitudinal study, but information for density was not reported. Thus, on the basis of published data, podocyte density seems to be relevant to patients with microalbuminuria, whereas podocyte number is described more in patients with proteinuria or microalbuminuria for a long time. 18,25,26 Therefore, correlation of proteinuria and podocytes may vary in patients with different stages of obesity-related glomerulopathy. 27 At early stages, a decrease in podocyte density with correspond-

6 PODOCYTES IN OBESITY-RELATED GLOMERULOPATHY 777 Fig 2. Glomerular capillary loop enlargement and mesangial expansion in patients with obesity-related glomerulopathy compared with controls: (A) normal capillary loops (C) and mesangial area (M) in controls, and (B) expansion of both C and M in patients with obesity-related glomerulopathy. (Electron microscopy; original magnification 3,000.) ing widening of foot processes results in the magnitude of the permselectivity defect and then proteinuria. At later stages, podocyte loss aggravates the decrease in podocyte density and progression of proteinuria. Therefore, a decrease in podocyte number in patients with obesity-related glomerulopathy might not be an independent predictor of the development of proteinuria. The result of multiple regression analysis supports this interpretation, showing that podocyte number does not add to the relationship between proteinuria and podocyte density. However, podocyte number is described as the only variable associated with decreased creatinine clearance, suggesting that podocyte loss represents relatively severe impairment of renal function in patients with obesity-related glomerulopathy. Obviously, podocyte density and podocyte number are unequally relevant to obesity-related glomerulopathy. Podocyte density is markedly decreased, whereas podocyte number is only numerically decreased. Because density is calculated as number of cells divided by glomerular volume, even if podocyte number remains stable, increased glomerular volume will result in decreased podocyte density. Glomerular volume is increased greatly in patients with obesity-related glomerulopathy compared with controls. Previous studies also showed that the consistent presence of glomerulomegaly was the most common histological characteristic of patients with obesityrelated glomerulopathy. 1,6 An increase in glomerular volume can be consequent to: (1) expansion in capillary loops; (2) increase in solid components, ie, mesangial cells and matrix; or (3) a combination of both factors. Our results are in keeping with the third possibility. Solid components increase with glomerular volume proportionally, and the percentage of mesangium in patients with obesityrelated glomerulopathy is similar to that in controls ( versus ; P 0.05). Expansion of capillary loops will contribute further to glomerular hypertrophy. Because podocytes are thought to be incapable of replication, an increase in both mesangial area and capillary loops (Fig 2), especially the latter, will make the remaining podocytes grow and extend their foot processes to maintain the area covered. Previous studies showed that podocytes have progressive injury when obliged to cover increased surface area. They also indicated that sustained mechanical stress is associated with the podocyte injury caused by glomerular hypertrophy. 23,28 Eventually, the decrease in podocyte number will cause foot-process widening and decrease the ability of podocytes to remain attached to GBM, with consequent areas of bare GBM, which are potential starting points for glomerulosclerosis.

7 778 Another interesting finding of the current study is that patients with obesity-related glomerulopathy showed a tight correlation between insulin resistance and decreased podocyte number and density. Glucose concentration is shown as an independent risk factor for podocyte loss in the present study. Insulin resistance was proved to have an essential role in obesity-related proteinuria, 29 but how to explain the links between glucose dysmetabolism and podocyte loss is still a question. One possible explanation is that insulin resistance leads to podocyte damage, mediated by glomerular hypertrophy. Insulin resistance has important roles in glomerular hyperfiltration, and hyperinsulinemia can stimulate the synthesis of growth factors. 5,30 Such growth factors and glomerular hyperfiltration were indicated to promote glomerular hypertrophy, which results in podocyte lesions. Another possibility is that a cluster of insulin resistance, including greater glucose and insulin levels, might damage podocytes directly. Study of cell culture showed that high glucose levels could affect regulation of integrin-linked kinase synthesis in podocytes. 31 Our previous study 32 using microdissection techniques combined with Affymetrix (Santa Clara, CA) microarray analysis showed that insulin resistance had important roles in the development of obesity-related glomerulopathy. All these studies support the hypothesis that insulin resistance is one of the major events in the development of podocyte lesions and proteinuria in patients with obesity-related glomerulopathy. However, in addition to hyperglycemia, hypertension was found to induce podocyte loss and progressive nephropathy in patients with diabetes. 25 Patients with obesity-related glomerulopathy showed greater blood pressure compared with controls, but the correlation between blood pressure and podocyte number was not found in this study. Interestingly, we found that patients on ACE-inhibitor/ARB therapy had a greater number of podocytes than those without therapy. It indicates that ACE-inhibitor/ARB therapy might protect podocytes from damage in patients with obesity-related glomerulopathy. Our previous study showed that lipid dysmetabolism also participated in the pathogenesis of obesityrelated glomerulopathy. 32 Blanco et al 24 reported that cholesterol was an important determinant of early podocyte damage in the Zucker rat. They CHEN ET AL also mentioned that atorvastatin could ameliorate those lesions through diminution of lipids. Conversely, a correlation between lipid levels and podocyte changes was not found in patients with obesity-related glomerulopathy. Nevertheless, we could not rule out the interference on results from previous therapeutic interventions. Similarly, it is too early to draw a conclusion about the protective effects of ACE-inhibitor/ ARB treatment on podocytes from our crosssectional data. Method limitations of the present study should be noted. First, the Weibel and Gomez method we used to estimate podocyte number has its inherent bias. This method makes assumptions about the shape and size of cells. 33 If the size or shape of podocytes changes and varies, underestimation or overestimation of podocyte density will happen. Second, the Weibel and Gomez method is not the gold standard for glomerular volume assessment. Moreover, paraffin embedding causes marked shrinkage in glomeruli compared with plastic-embedded tissues. To limit the shortcomings, a converted value of glomerular volume is used in this study according to the previous report. 18 In addition, because all samples including controls are handled in the same way, we believe these estimations are useful for comparisons. In conclusion, this study illustrates a decrease in podocyte density and podocyte number observed in patients with obesity-related glomerulopathy, which contributes to degree of proteinuria and renal function impairment in these patients. In addition to enlargement of glomerular volume with consequential mechanical stretch, metabolic disorders also might have a role in the development of podocyte injury in patients with obesity-related glomerulopathy. REFERENCES 1. Flegal KM, Carroll MD, Kuczmarski RJ, Johnson CL: Overweight and obesity in the United States: Prevalence and trends, Int J Obes Relat Metab Disord 22:39-47, Saw SM, Rajan U: The epidemiology of obesity: A review. Ann Acad Med Singapore 26: , World Health Organization Western Pacific Region, International Association for the Study of Obesity, and International Obesity Task Force: The Asia-Pacific Perspective: Redefining Obesity and Its Treatment. Melbourne, Australia, Health Communications Australia, 2000

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