In the current study, GBM anionic sites were characterized as heparan sulfate proteoglycan and their densities were similar in BB/DPh and age-matched controls. These results varied from those of several investigators, including Chakrabarti and Sima (1989), who demonstrated significantly reduced site density using the cationic dye cuprolinic blue in 6-month diabetic BB rats. Likewise, significant reductions in PEI or cuprolinic blue labeling have been demonstrated in streptozotocin-induced diabetic rats at up to 8 months of diabetes (Moriya et al., 1993; van den Born et al., 1995a).
Reductions in GBM AS have been tightly linked with increased filtration unit permeability and significant proteinuria (Kanwar, 1984). In this regard, reduced GBM-heparan sulfate proteoglycan is associated with increased capillary permeability and passage of proteins into the ultrafiltrate (Kanwar et al., 1980; Rosenzweig and Kanwar, 1983). Also, Goode et al. (1995) concluded that a reduction in the density of cationic gold-labeled GBM-associated AS in patients with chronic diabetes (greater than 15 years) correlated closely with increased urine protein excretion. Moreover, van den Born et al. (1995b) showed that at 8 months postinduction of diabetes, streptozotocin diabetic rats demonstrated increased capillary permeability to albumin and IgG that correlated with relative decreases in GBM-heparan sulfate proteoglycan. Accordingly, reduction of AS density in GBMs is widely regarded as contributory to diabetic proteinuria. However, since only modest levels of urine protein have been reported for moderately hyperglycemic BB rats (Cohen et al., 1987; Velasquez et al., 1990; Zamlauski-Tucker et al., 1992; Feld et al., 1995), it seems possible that maintenance of normal complements of AS as shown in the current investigation may be related functionally to known relatively low levels of proteinuria in diabetic BB rats.
It is difficult to speculate on the reasons why AS density in the current study conflicts with those of other investigators. However, our PEI staining procedures differ from those utilizing cuprolinic blue (Chakrabarti and Sima, 1989; van den Born et al., 1995a) and the cationicity of these two probes may not be identical. Moreover, the studies were carried out at disparate times postonset of hyperglycemia. Future combined AS/functional studies including creatinine clearance and assessment of proteinuria in BB/DR, BB/DPh, and BB/DPn rats may shed some light on this apparent disparity.
Acellular renal tissues.
Although our LM observations of acellular glomeruli from BB/DR, BB/DPh, and BB/DPn rats at 1 year postonset of diabetes showed few structural differences, increased resolution at the level of SEM and TEM showed several important distinctions. Simple TEM inspection showed that at 3 months, MM was only slightly increased in BB/DPh kidneys over BB/DR controls. However, GBMs in the diabetics appeared markedly thickened, and surprisingly GBMs in BB/DPn animals also appeared wider than those in BB/DR controls. Similar though exaggerated differences were observed in 1-year animals, where modestly increased MM was more extensively distributed in BB/DPh animals and GBMs were substantially widened.
However, by far the most unexpected result in the current investigation related to substantially increased GBM widths in 1-year BB/DPn rats relative to age-matched BB/DR controls. Although BB/DPn rats never became diabetic and remained normoglycemic until death, their GBM thickness appeared similar or even greater than those seen in age-matched hyperglycemics.
Morphometric analyses of all animal types showed substantial increases in GBM thickness with age, and these data were consistent with several studies that indicated this parameter was a reliable biomarker of aging in rats (Yagahashi et al., 1978; Cohen et al., 1987; Feld et al., 1995). Our morphometric data also showed that GBMs in BB/DPh animals were significantly thicker than age-matched BB/DR controls at 3 months, 6 months, and 1 year postonset of hyperglycemia. This was also not unexpected as other investigators showed that as early as 6 months postonset of hyperglycemia, GBMs in BB/DP rats were thicker than age-matched BB/DR rats (Brown et al., 1983; Cohen et al., 1987; Chakrabarti and Sima, 1989; Feld et al., 1995). Also, Feld et al. (1995) showed that at 1 year, GBMs were 25% wider in BB/DP rats than in age-matched BB/DR controls.
Age-related GBM thickness data in normoglycemic BB/DPn animals have not been reported previously. This may be related to several factors, including the relative scarcity of the animals (∼ 10% of BB/DP rats), and a belief that diabetic-resistant (BB/DR) rats served as the most appropriate normoglycemic control for diabetics. As expected, morphometric analyses confirmed our early impressions that GBMs in BB/DPn rats were wider than predicted for a normoglycemic animal group. Remarkably, they not only significantly exceeded those of age-matched normoglycemic BB/DR rats, but were substantially thicker than diabetic BB/DPh rats at 3 months, 6 months, or 1 year.
The precise mechanisms by which basement membranes increase in width are not clear, but most investigators agree that GBM widths increase with age and in several diseases, most notably diabetes mellitus. A number of pathogenetic mechanisms for diabetic basement membrane disease were reviewed recently by Tsilibary (2003). They include hyperglycemia-induced increased type IV collagen synthesis, decreased expression of matrix metalloproteinases (MMP-2 and -3), and increased tissue inhibitors of metalloproteinase (TIMP). Vascular endothelial growth factor (VEGF) may also be involved, as anti-VEGF antibody treatment reduces GBM thickening. Moreover, oxygen radicals/oxidative stress and advanced glycation end products (AGEs) may play a role since AGE inhibitors (e.g., aminoguanidine, which also has antioxidant properties) attenuate diabetic nephropathy. In addition, the polyol enzymatic pathway is stimulated by hyperglycemia and has been implicated in the pathogenesis of chronic diabetic complications. In this regard, diabetic hyperpermeability, an early feature of diabetic microangiopathy, is reduced by aldose reductase inhibitors.
A unifying hypothesis for these mechanisms has been proposed (Nishikawa et al., 2000) and suggests that since increased blood glucose apparently increases the generation of reactive oxygen species (ROS), activates aldose reductase, and induces AGE formation, hyperglycemia-driven ROS production may represent a final common pathway leading to diabetic microvascular damage, including increased basement membrane thickness.
It is clear from a plethora of animal and clinical studies (Skyler, 1996; Feener and King, 2001; Tsilibary, 2003) that most hypotheses on the etiology of diabetic GBM thickening begin with hyperglycemic microvascular damage. A direct causal relationship between chronic hyperglycemia and microvascular complications of diabetes has been established by data from the Diabetes Control and Complications Trial (1993), a randomized multicenter prospective control study. However, data in the current study strongly suggest that microvascular alterations may also occur in genetically diabetic animals in the absence of hyperglycemia.
It should be pointed out that although a large number of GBM measurements were made, the current study focused on relatively few animals, and our data should be confirmed in future studies using larger populations. Furthermore, recent studies have shown that in BB/Wistar rats, females are more resistant than males to activation of protein kinase C and decreased Na+-K+ adenosine triphosphatase activity, which generally are associated with diabetic complications (Sieber et al., 2001). Since the animals used in the current study were of mixed gender, it may be possible that gender-specific differences in GBM thickness were masked in our data set. This also must be considered in future experiments.
In summary, it is difficult to reconcile our GBM thickness data with numerous morphometric studies, including our own (Carlson et al., 1997, 2003), that indicate increased microvascular basement membrane thickness is a concomitant of hyperglycemia. It is also difficult to escape the conclusion that high blood glucose may be sufficient but not required for excessive GBM thickening to occur. Although this clearly is contrary to widely held theories on the mechanisms by which microvessel basement membranes increase in width, it must be pointed out that such mechanisms are not completely understood and likely are multifactorial. Accordingly, resolution of conflicting correlative data on GBM thickness and hyperglycemia requires further study, including careful analyses of prediabetic basement membrane thickening in other spontaneous models of the disease (e.g., NOD mice). Additionally, a reconsideration of early TEM investigations that demonstrate increased capillary basement membrane thickness in genetically diabetic but normoglycemic patients may be appropriate (Barbosa and Saner, 1984).