PD and reduced glomerular ECF are quantifiable structural features of nephropathy in T2DM that correlate positively with ACR and worsen in parallel with the classic structural features of diabetic kidney disease. Reduced glomerular ECF also correlates negatively with GFR. This is the first study to demonstrate an association between loss of normal ECF and declining GFR. We confirm that increasing PD correlates positively with increasing albuminuria, as shown in T1DM [9
PD correlated negatively with podocyte number per glomerulus, a finding consistent with previous reports of reduced podocyte number and/or podocyte density in T1DM and T2DM [4
] and with the presence of podocyturia in patients with T2DM and diabetic nephropathy [14
]. PD also correlated positively with albuminuria, whereas podocyte number did not, providing further evidence to implicate podocyte injury rather than podocyte loss as the primary structural factor responsible for decreased permselectivity of the glomerulus in diabetic nephropathy. Studies in Pima Indians with T2DM found that subjects with macroalbuminuria had increased filtration of high molecular weight dextrans through an excess of large pores that serve as macromolecular shunts, whereas microalbuminuric subjects had a shunt size no different from controls [15
], suggesting that low-grade albuminuria may result from a different mechanism than nephrotic-range proteinuria. The significant increase in PD in the macroalbuminuric subjects suggests that these areas of detachment may represent the physical substrate of the excess shunt magnitude, and complete detachment of foot processes may occur along some portions of the GBM without loss of the entire podocyte. PD also correlated modestly, but not significantly, with declining GFR. As we suggested previously, when foot processes become detached, the remaining podocytes may spread their foot processes more broadly to cover what would otherwise be denuded basement membrane [10
]. This broadening of the foot processes reduces filtration slit frequency, which in turn should lower hydraulic permeability and probably GFR [15
]. The negative correlation we found between PD and filtration slit frequency is consistent with this scenario.
ECF was reduced significantly in normoalbuminuric subjects compared with healthy kidney donors, and among those with T2DM, loss of ECF correlated significantly with albuminuria and GFR (). Diminution of the endothelial glycocalyx occurs with exposure to high glucose concentrations. We speculate that the loss of this negatively charged portion of the filtration barrier could then result in the enhanced filtration of polyanionic albumin that has been reported both in vitro
and in vivo
]. Loss of ECF may represent another pathological response of endothelial cells to hyperglycemia, but our fixation methods do not allow investigation of whether the degree of ECF correlates with the thickness of the glycocalyx. Lowered glomerular hydraulic permeability due, in part, to a reduction in the density and size of endothelial fenestrations is believed to be responsible for the decline in GFR in pre-eclampsia [19
], and may also contribute to the decline in GFR in diabetes. The only other morphometric variable to correlate significantly with both albuminuria and GFR was fractional mesangial area, a finding reported previously in T1DM [20
] and T2DM [10
Participants with diabetes had significantly fewer endothelial fenestrae than nondiabetic kidney donors, yet the GFR in those with normo- or microalbuminuria was higher (). This seeming paradox is explained, in part, by diabetes-related glomerulomegaly increasing the total filtration surface area, thereby offsetting declines in glomerular hydraulic permeability caused by the reduced fenestration and the diminished filtration surface area density ().
The percentage of PD was determined by measuring the fraction of the entire peripheral capillary GBM length completely devoid of podocyte foot processes. Although we did not measure PD along the peri-mesangial GBM, PD was observed there as well. We defined PD as complete absence of foot processes because we wanted to avoid characterizing electron-lucent foot processes (which may represent sublethal cell injury) as areas of detachment, as filtration slit diaphragms between foot processes are still potentially intact in these areas, and the functional characteristics of the glomerular membrane potentially remain unchanged [21
]. Moreover, we did not want to include areas with a mixture of intact and detached foot processes in our definition of detachment, because of uncertainties about their effect on permselectivity. In comparison, Toyoda et al.
] counted areas including a mixture of intact and detached foot processes (“mixed” foot processes) in their measurements of detachment. These authors did not mention how electron-lucent foot processes were characterized according to their definitions of ‘attached,’ ‘detached,’ and ‘mixed.’ Accordingly, they reported 22% PD, whereas our different definition results in just over 1% PD. Regardless of these quantitative differences, the two studies demonstrate a process common to diabetic glomerulopathy in both T1DM and T2DM, but absent in normal kidneys [22
]. Moreover, the two studies demonstrate strong similarities in the relationship of PD with the classic lesions of diabetic nephropathy and its associated functional abnormalities.
Podocyte production of vascular endothelial growth factor (VEGF) is important for the maintenance of normal ECF. Blocking production of VEGF or its effector pathways decreases ECF, capillary permeability, and GFR, and increases proteinuria [19
]. However, when we specifically examined endothelium abutting sections of basement membrane that were devoid of overlying foot processes we found no decrease in the mean proportion of normal ECF compared with the overall proportion of normal ECF, suggesting that loss of foot processes does not affect ECF locally and that loss of ECF does not affect local foot process attachment. One potential explanation is that impairment in production and release of VEGF by podocytes may be independent of foot process detachment. Indeed, PD was localized and limited, whereas reduction in ECF was relatively widespread, especially in macroalbuminuric subjects. Despite demonstrated podocyte-endothelial cross-talk in some circumstances, these cellular compartments appear to be differentially regulated (and perturbed).
The present cross-sectional study was designed to assess the functional and structural correlates of PD and loss of ECF at various stages of diabetic kidney disease defined by level of albuminuria. This comparison took place within an ongoing clinical trial, and treatment with angiotensin blockade may have affected the level of albuminuria. Moreover, although albuminuria and GFR strongly predict progressive kidney disease in diabetic Pima Indians [26
] and the combination of these functional variables is a better predictor of ESRD and death than either measure alone [27
], some investigators suggest that microalbuminuria is an unreliable predictor of diabetic nephropathy because it may regress spontaneously [28
], and albuminuria may never increase in some patients despite worsening GFR [29
]. Accordingly, some caution should be exercised in interpreting the relationship between these structural and functional measures.
Pima kidney transplant donors were not available to serve as controls. Instead, morphometric measurements from healthy non-diabetic Caucasian, African American, and Asian kidney donors were used for comparison. There were striking structural differences between the normal kidney donors and noromalbuminuric diabetic subjects in many stereological and morphometric variables. However, in the absence of information from non-diabetic Pima controls, conclusions regarding genetic, environmental, or disease-based differences cannot be inferred with certainty.
The results of this study are likely to be applicable to other populations. Previous studies in Pima Indians have provided valuable, generalizable insights into the pathophysiology of diabetic nephropathy. Aside from the larger size of glomeruli in both diabetic and non-diabetic Pima Indians [30
], the observed structural changes of diabetic nephropathy are indistinguishable from those in other populations. Moreover, because details of the onset and duration of diabetes are known with much greater precision than in other populations, and because nearly all kidney disease is attributable to diabetes [31
], findings in the Pima Indians may in fact offer a clearer picture of the effects of T2DM on the kidney than can be found in other populations.
In conclusion, we document PD and loss of ECF in humans with T2DM even prior to the development of overt nephropathy, and correlate these quantitative structural variables with other glomerular structural and functional characteristics of diabetic nephropathy. Our findings are consistent with those reported previously in T1DM [9
], and confirm the important role that these distinct cellular injuries may play in the development and progression of diabetic glomerular disease in both T1DM and T2DM.