In this report, we extended our early study on KIM-1 expression in damaged tubules of diabetic animals [19
], and revealed that KIM-1 was also expressed in glomerular epithelial cells in diabetic kidney disease. Our results show that glomerular KIM-1-positive cells were initially localized in the hyperplastic parietal epithelium of Bowman's capsule in the early stages of diabetes. There was a significant increase in cells positive for KIM-1 staining in the glomerular tuft with disease progression and massive proteinuria. Multiple labeling for KIM-1 and nephrin indicated a strong correlation between glomerular KIM-1 induction and podocyte loss in damaged glomeruli. Furthermore, antiproteinuric treatment with AT1 receptor antagonist attenuated diabetes-related KIM-1 expression, in association with a reduction of pro-inflammatory markers and an improvement of podocytopenia and renal function in the diabetic animals.
Tubular KIM-1 expression in both acute and chronic kidney diseases has been well established [8
]. Consistent with previous findings, a progressive increase in tubular expression and urinary excretion of KIM-1 was observed in the Fa/Fa diabetic rats. Additionally, the present study demonstrates that glomerular epithelial cells also express KIM-1. Fa/Fa animals had increased PEC proliferation and hyperplasia, morphologically similar to proximal tubular epithelia, from 7 to 20 weeks of age. The lack of lotus tetragonolobus lectin staining in hyperplastic PECs, however, suggests that they are not proximal tubule cells encroaching into Bowman's capsule in rat kidneys (data not shown). In the absence of microalbuminuria, only few hyperplastic PECs expressed KIM-1 in Fa/+ lean controls. In contrast, the cells double-positive for KIM-1 and claudin-1 were robustly increased in the hyperplastic parietal epithelium of Bowman's capsule in the Fa/Fa animals. The increase in KIM-1 expression prior to the development of hyperglycemia suggests that KIM-1 is a sensitive marker for early PEC activation. As expected, albuminuria occurs early in 7-week-old Fa/Fa rats. It is conceivable that hyperplastic (or proliferative) PECs are extremely sensitive to an elevation of albumin in the Bowman's space and express KIM-1 upon activation. This concept is supported by our in vivo and in vitro findings showing an attenuation of parietal hyperplasia and glomerular KIM-1 expression after antiproteinuric treatment in the diabetic animals and a striking induction of KIM-1 in primary cultured glomerular epithelial cells in response to albumin overload. Although KIM-1 in the primary cultures of rat glomerular epithelial cells was increased slightly after high glucose treatment, the fact that losartan administration reduced proteinuria but did not change blood glucose suggests that urinary glucose may play a minor role in activation and impairment of renal epithelial cells in this rat model of type 2 diabetes mellitus.
While KIM-1 protein was primarily present along the parietal epithelium of Bowman's capsule in 7-week-old Fa/Fa rats, there was a significant increase in the number of cells positive for KIM-1 staining in the glomerular tuft in the diabetic rats at the age of 20–28 weeks. Since podocytopenia has been reported in both diabetic and non-diabetic proteinuric kidney diseases [2
], we next asked whether KIM-1 induction was associated with podocyte loss in the damaged glomeruli. To this end, we performed triple immunostaining for KIM-1, nephrin and WT-1, to evaluate their colocalization in the glomeruli of diabetic animals. Initially, the reduction of nephrin protein was detected in the podocytes next to the hyperplastic PECs expressing KIM-1 in 12-week-old diabetic animals. Thereafter, both WT-1 and nephrin were further decreased in the glomerular tuft of diabetic animals. Interestingly, within a single glomerulus, we often observed strong KIM-1 expression in the tuft area with less nephrin and WT-1 staining. Together, our results demonstrate a tight correlation between the expression level of KIM-1 in the glomerular tuft and the extent of loss of podocyte differentiation markers in the damaged glomeruli.
One might ask the cell origin of visceral KIM-1-positive cells. One possibility is that damaged podocytes loss their normal markers and begin to express cell injury markers, such as KIM-1. Another is that KIM-1-expressing PECs move to the glomerular tuft. These two notions are supported by a recent study showing that cells in the glomerular tuft coexpress both podocyte and PEC proteins in several different experimental nephropathy [5
]. In this study, cells positive for both KIM-1 and claudin-1 (PEC marker) were present in the parietal epithelium of the Fa/Fa rats. The great majority of KIM-1-positive cells were initially restricted within the parietal epithelium of Bowman's capsule with subsequent increase in the glomerular tuft. Sometimes, a clear connection was seen between visceral and parietal KIM-1-expressing cells. These data suggest that the visceral KIM-1-positive cells are mobile PECs. Indeed, Appel et al. [27
] provided evidence that PECs possess the capability to migrate onto the glomerular tuft via the vascular stalk, where they differentiate into podocytes under normal conditions. Additionally, PEC migration and hyperplasia combined with progressive podocytopenia have been shown in cellular/collapsing FSGS [7
]. However, not all KIM-1-expressing cells in the glomerular tuft were positive for claudin-1 staining (data not shown). Thus we cannot exclude the possibility that podocytes undergo trans-differentiation and express KIM-1 protein, though no significant overlap between KIM-1 and desmin was detected in the glomeruli of Fa/Fa animals. In fact, previous reports have indicated that dedifferentiated (dysregulated) podocytes characterized by loss of podocyte markers are no longer growth restricted and regain the ability to proliferate in FSGS and HIV-associated nephropathy [28
Our in vivo data indicate a strong positive correlation between glomerular KIM-1 mRNA and the level of urine protein in the diabetic animals. Furthermore, antiproteinuric treatment with AT1 receptor antagonist ameliorated the diabetes-related increase in renal expression and urinary excretion of KIM-1. It is possible that microalbuminuria (or mild proteinuria) could trigger the expression of KIM-1 in activated renal epithelial cells. Therefore, we further performed an in vitro study to examine the action of albumin overload on KIM-1 expression in cultured glomerular epithelial cells. We observed that KIM-1 protein was robustly induced in response to albumin stimulation in primary cultured glomerular epithelial cells. Based on our in vivo and in vitro observations, we speculate that initial mild microalbuminuria resulting from increased glomerular sieving caused by changes in transglomerular pressure and/or glomerular basement membrane activates glomerular and tubular epithelial cells leading to KIM-1 expression, podocyte loss, overt proteinuria and renal scarring in diabetic metabolic syndrome. This notion is supported by a reduction of blood pressure in association with an improvement of renal structure and function in losartan-treated Fa/Fa diabetic rats. However, our results do not exclude a direct regulatory action of AT1 receptor blockade on renal epithelial cells.
The selective expression by injured epithelial cells provides the foundation for KIM-1's use as a biomarker. More recently, studies by Ichimura et al. [10
] have raised the exciting possibility that KIM-1 might be important in renal pathogenesis. They demonstrated that KIM-1 is a phosphatidylserine receptor and plays a predominant role in the uptake of apoptotic cells by renal tubular epithelium [10
]. Interestingly, our preliminary experiments show that KIM-1 promotes the uptake of FITC-labeled bovine serum albumin by renal epithelial cells in primary culture (data not shown). Therefore, it is possible that KIM-1 may work as a scavenger of filtered proteins by endocytotic process in proteinuric kidney disease. Further studies are warranted to evaluate the functional consequences of KIM-1 expression in proteinuric nephropathy.
Taken together, the current study demonstrates the expression of KIM-1 in parietal and visceral epithelial cells in the glomeruli of diabetic animals. The increase in KIM-1 was associated with proteinuria and podocytopenia, which was attenuated by AT1 receptor antagonist. Our results support that glomerular KIM-1 expression, especially in the tuft area, could be used to predict glomerular epithelial cell activation and injury in proteinuric kidney disease.