Hirokazu Sakan

Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion

Hypercalciuria is one of the early manifestations of diabetic nephropathy. We explored here the role of α-Klotho, a protein expressed predominantly in distal convoluted tubules that has a role in calcium reabsorption. We studied 31 patients with early diabetic nephropathy and compared them with 31 patients with IgA nephropathy and 7 with minimal change disease. Renal α-Klotho expression was significantly lower and urinary calcium excretion (UCa/UCr) significantly higher in diabetic nephropathy than in IgA nephropathy or minimal change disease. Multiple regression analyses indicated that α-Klotho mRNA was inversely correlated with calcium excretion. We next measured these parameters in a mouse model of streptozotocin (STZ)-induced diabetic nephropathy, characterized by glomerular hyperfiltration, as seen in early diabetic nephropathy. We also confirmed a reduction of renal α-Klotho mRNA down to almost 50% and enhanced calcium excretion in mice with STZ-induced diabetic nephropathy in comparison with nondiabetic mice. Hypercalciuria was exacerbated in heterozygous α-Klotho knockout mice in comparison with wild-type mice, each with STZ-induced diabetic nephropathy. Thus, α-Klotho expression was decreased in distal convoluted tubules in diabetic nephropathy in humans and mice. Renal loss of α-Klotho may affect urinary calcium excretion in early diabetic nephropathy.

Reduced renal α-Klotho expression in CKD patients and its effect on renal phosphate handling and vitamin D metabolism.

Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)2 vitamin D3 (1,25VitD3). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (n = 236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD3 levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels.

Fractalkine expression and CD16 monocyte accumulation in glomerular lesions: association with their severity and diversity in lupus models

Fractalkine (Fkn) is expressed on injured endothelial cells and is a membrane-bound chemokine that attracts cells expressing its receptor, CX3CR1, including CD16(+) monocytes (CD16(+) Mos). To clarify the role played by Fkn in the development of glomerular lesions in lupus nephritis, we examined Fkn expression and CD16(+) Mo accumulation induced in experimental C.B-17/Inc-scid/scid (SCID) lupus model mice by injection of IgG(3)-producing hybridoma clones obtained from MRL/lpr mice. Glomerular Fkn expression and accumulation of CD16(+) Mos were semiquantitatively evaluated using laser capture microdissection and real-time PCR. Injection of the 2B11.3 and 7B6.8 clones induced formation of glomerular proliferative and wire-loop lesions, respectively. Immunohistological analysis of the localization of Fkn and CD16(+) Mos revealed that Fkn expression and CD16(+) Mo accumulation were markedly elevated in glomerular lesions induced by 2B11.3, whereas no elevation was detected in those induced by 7B6.8. In addition, to examine the contribution of glomerular Fkn to the development of proliferative lesions, L cells producing an Fkn antagonist (Fkn-AT) were transplanted into SCID mice exhibiting proliferative lupus nephritis (DPLN) induced by 2B11.3. Notably, transplantation of the Fkn-AT-producing cells was functionally and histologically protective against this DPLN. Taken together, our findings suggest that Fkn and CD16(+) Mo accumulation are partially associated with the severity and diversity of histology of lupus nephritis.

Clinical Significance of Fibroblast-Specific Protein-1 Expression on Podocytes in Patients with Focal Segmental Glomerulosclerosis

Backgrounds/Aims: We previously reported that fibroblast-specific protein 1 (FSP1) is a marker of epithelial-mesenchymal transition (EMT) in tubulointerstitial fibrosis. The EMT-like changes observed in podocytes are reportedly associated with podocyte detachment which may cause focal glomerulosclerosis. Methods: In cross-sectional studies, we analyzed podocyte expression of FSP1 immunohistochemically using renal biopsy specimens from 31 patients with focal segmental glomerulosclerosis (FSGS) and 39 patients with minimal change disease (MCD). We also semiquantitatively analyzed glomerular expression of FSP1 mRNA using laser capture microdissection and real-time PCR. Results: We found that FSP1 was localized to podocytes in both FSGS and MCD patients; however, the number of FSP1+ podocytes per glomerular profile was significantly higher in patients with FSGS than in those with MCD, and there was a corresponding difference in the levels of FSP1 mRNA. FSP1+ podocyte counts per glomerular profile in FSGS patients correlated significantly with the prevalence of glomerulosclerosis and the extent of interstitial type-I collagen-positive areas. Conclusion: Taken together, these data suggest that podocyte expression of FSP1 could shed light on the potential linkage between EMT-like changes, detachment of podocytes from the glomerular basal membrane and the pathophysiology underlying FSGS.

Enhanced expression of the soluble form of E-selectin attenuates progression of lupus nephritis and vasculitis in MRL/lpr mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that causes inflammatory tissue damage, including lupus nephritis and vasculitis. Local generation of adhesion molecules and expression of their ligands on inflammatory cells appears to contribute to the progression of SLE. We found significantly increased E‐selectin expression in the glomeruli and renal interstitial microvasculature of MRL/MpJ‐lpr/lpr (MRL/lpr) lupus model mice. This was accompanied with infiltration of inflammatory cells, especially macrophages and CD8+ T cells. Similarly, in 21 patients with proliferative lupus nephritis, there was a significant correlation between renal E‐selectin levels and macrophage and CD8+ T cell infiltration in the affected kidneys. By contrast, in transgenic MRL/lpr mice exhibiting elevated levels of circulating soluble E‐selectin (sE‐selectin) protein, which competitively inhibits E‐ and P‐selectin‐mediated extravasation of inflammatory cells, the progression of lupus nephritis and vasculitis was significantly suppressed and survival was significantly prolonged. This improvement was accompanied by significant reductions in renal infiltration by macrophages and CD8+ T cells. These results suggest that E‐selectin plays a crucial role in lupus nephritis and vasculitis by mediating renal infiltration of inflammatory cells, and that because it inhibits this process, sE‐selectin could potentially serve as an effective treatment for lupus nephritis and vasculitis.

Association between E-selectin expression and histopathological modification of glomerular lesions by non-nephritogenic IgM antibodies in experimental lupus nephritis.

Abstract Objective. To examine the role played by E-selectin in bystander IgM-mediated modification of glomerular lesions in experimental lupus nephritis. Methods. Experimental lupus SCID mice were induced by an intraperitoneal injection of clone 7B6.8, which was derived from a MRL/lpr mouse and shown to induce wire-loop type glomerular lesions. Mice were subsequently administered clone Sp6, a non-nephritogenic IgM antibody- producing hybridoma. E-selectin expression was then evaluated in glomeruli showing histopathological conversion of lesions from wire-loop-like to a cell-proliferative form. We also investigated the effects of a circulating soluble form of E-selectin (sE-selectin) on the modification of glomerular lesions in this lupus model. Results. In experimental lupus mice, glomerular E-selectin expression significantly increased during the conversion from wire-loop-like glomerular lesions to a cell-proliferative type mediated by a non-nephritogenic bystander IgM antibody in presence of a nephritogenic antibody. Intraglomerular infiltration of CD68 + macrophages correlated significantly with the glomerular level of E-selectin expression. In addition, overexpression of circulating sE-selectin significantly suppressed conversion to cell-proliferative glomerular lesions and glomerular macrophage infiltration in these lupus model mice. Conclusions. The histopathological modification of lupus nephritis by non-nephritogenic bystander IgM antibodies is associated in part with glomerular E-selectin expression.