Increasing evidence suggests the heterogeneity of macrophage phenotype and function ultimately determines the outcome of diabetic nephropathy (DN). This study aimed to investigate the effects of vitamin D on macrophage M1/M2 phenotype and its role in preventing podocyte impairment in streptozotocin-induced DN rats. Calcitriol, a bioactive 1,25-dihydroxyvitamin D3, ameliorated proteinuria and renal damage as well as reversed the decline of both nephrin and podocin, crucial structural proteins in podocytes. DN rats showed increased infiltrating macrophages with M1 phenotype characterized by elevated expression of inducible nitric oxide synthase and TNF-α in glomeruli and interstitium, which were inhibited after calcitriol treatment. Interestingly, calcitriol promoted M2 macrophage activation with enhanced expression of CD163, arginase-1, and mannose receptor at week 18 but not at week 8 or 14. The ratio of CD163 to CD68, considered as the proportion of M2 macrophages, was about 2.9-fold higher at week 18 after calcitriol treatment. Furthermore, the protein expression of inducible nitric oxide synthase, a crucial marker of M1 macrophages, was negatively correlated with the expression of either nephrin or podocin, whereas CD163, indicating M2 macrophages, was positively correlated. In vitro, 1,25-dihydroxyvitamin D3 switched high-glucose-induced M1 macrophages toward an M2 phenotype in either U937-derived macrophages or RAW264.7 cells. Our results suggest that vitamin D not only reduces macrophage infiltration and inhibits M1 macrophage activation but also enhances M2 macrophage phenotype to protect against podocyte injury.

Macrophages, especially their activation state, are closely related to the progression of diabetic nephropathy. Classically activated macrophages (M1) are proinflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. 1,25-Dihydroxyvitamin D3 has renoprotective roles that extend beyond the regulation of mineral metabolism, and PPARγ, a nuclear receptor, is essential for macrophage polarization. The present study investigates the effect of 1,25-dihydroxyvitamin D3 on macrophage activation state and its underlying mechanism in RAW264.7 cells. We find that, under high glucose conditions, RAW264.7 macrophages tend to switch to the M1 phenotype, expressing higher iNOS and proinflammatory cytokines, including TNFα and IL-12. While 1,25-dihydroxyvitamin D3 significantly inhibited M1 activation, it enhanced M2 macrophage activation; namely, it upregulated the expression of MR, Arg-1, and the anti-inflammatory cytokine IL-10 but downregulated the M1 markers. However, the above effects of 1,25-dihydroxyvitamin D3 were abolished when the expression of VDR and PPARγ was inhibited by VDR siRNA and a PPARγ antagonist. In addition, PPARγ was also decreased upon treatment with VDR siRNA. The above results demonstrate that active vitamin D promoted M1 phenotype switching to M2 via the VDR-PPARγ pathway.

Podocyte injury plays a critical role in the development and progression of diabetic nephropathy (DN). Over expression of TRPC6 on the podocytes has been revealed to cause podocyte injury in non-diabetic states. Besides, the emerging evidence from clinic revealed that vitamin D could reduce albuminuria and improve renal function, which was associated with podocyte protection. Our study aimed to investigate whether calcitriol ameliorating podocyte impairment is associated with regulation of the expression of TRPC6 in STZ-induced rats. Sprague-Dawley rats were randomly divided into three groups: normal control, DN, and DN treated with calcitriol (DN + VD); VD rats were treated with 0.1 μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozocin. The rats were sacrificed after 18 weeks treatment. DN rats exhibited increased proteinuria accompanied by elevated TRPC6 expression. Treatment with calcitriol not only reduced proteinuria, but also normalized TRPC6 expression. Meanwhile, in DN rats, the expression of podocyte specific markers including nephrin and podocin was significantly decreased, accompanied by increased desmin, a marker of podocyte injury. Treatment with calcitriol reversed above changes. In addition, vitamin D receptor (VDR) was significantly decreased, whereas this reduction was attenuated by the calcitriol treatment. Moreover, TRPC6 was positively correlated with both 24 h urinary protein and desmin. In contrast, TRPC6 was negatively correlated with both VDR and nephrin expression in podocytes. Calcitriol can ameliorate podocyte injury, which is contributed by the inhibition of enhanced TRPC6 expression in the early stages of DN rats.

Macrophage infiltration has been linked to the pathogenesis of diabetic nephropathy (DN). However, how infiltrating macrophages affect the progression of DN is unknown. Although infiltrating macrophages produce pro-inflammatory mediators and induce apoptosis in a variety of target cells, there are no studies in podocytes. Therefore, we tested the contribution of macrophages to podocytes apoptosis in DN. in vivo experiments showed that apoptosis in podocytes was increased in streptozocin (STZ)-induced diabetic rats compared with control rats and that this apoptosis was accompanied by increased macrophages infiltration in the kidney. Then, we established a co-culture system to study the interaction between macrophages and podocytes in the absence or presence of high glucose. Macrophages did not trigger podocytes apoptosis when they were co-cultured in the absence of high glucose in a transwell co-culture system. Additionally, although podocyte apoptosis was increased after high glucose stimulation, there was a further enhancement of podocyte apoptosis when podocytes were co-cultured with macrophages in the presence of high glucose compared with podocytes cultured alone in high glucose. Mechanistically, we found that macrophages were activated when they were exposed to high glucose, displaying pro-inflammatory M1 polarization. Furthermore, conditioned media (CM) from such high glucose-activated M1 macrophages (HG-CM) trigged podocytes apoptosis in a reactive oxygen species (ROS)-p38mitogen-activated protein kinases (p38MAPK) dependent manner, which was abolished by either a ROS inhibitor (Tempo) or a p38MAPK inhibitor (SB203580). Finally, we identified tumor necrosis factor (TNF-α) as a key mediator of high glucose-activated macrophages to induce podocytes apoptosis because an anti-TNF-α neutralizing antibody blunted the apoptotic response, excess ROS generation and p38MPAK activation in podocytes induced by HG-CM. Moreover, addition of recombinant TNF-α similarly resulted in podocytes apoptosis. In summary, the TNF-α that was released by high glucose-activated macrophages promoted podocytes apoptosis via ROS-p38MAPK pathway. Blockade of TNF-α secretion from high glucose activated macrophages and ROS-p38MAPK pathway might be effective therapeutic options to limit podocytes apoptosis and delay the progression of diabetic nephropathy.

BackgroundKidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown.MethodsTo investigate PKCδ’s potential role in injury during cold storage–associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice.ResultsWe observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage–associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage–associated transplantation injury.ConclusionsThese results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage–associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

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Objective The present study is to investigate the effect of vitamin D/Vitamin D Receptor (VDR) signaling on podocyte autophagy in diabetic nephropathy. Methods Kidney tissue sections from patients with diabetic nephropathy and nontumor kidney were checked under electronic microscope and VDR immunohistochemistry. Diabetic rat models were induced by intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). Calcitriol treatment was achieved by gavage at dose of 0.1μg/kg/d. Blood, urine and kidney tissue specimens were used for serum, urine biochemistry, histopathology and molecular biology testing. Podocyte cell line MPC-5 was cultured under hyperglycaemic conditions in the absence or presence of 100 nmol/L calcitriol to investigate podocyte injury and autophagy. Results VDR and autophagosomes in podocytes were significantly decreased in renal biopsy from patients with diabetic nephropathy, compared to healthy kidney tissue. Rats with STZ treatment developed typical diabetic kidney disease with low VDR expression. Calcitriol, the active form of vitamin D, could activate VDR and attenuate diabetic nephropathy including proteinuria and glomerular sclerosis. Calcitriol treatment also alleviated the podocyte foot process fusion, reduced podocyte injury marker desmin and preserved slit diaphragms proteins in diabetic nephropathy. Reduced LC3II/I, Beclin-1 and elevated p62 in renal homogenate and reduced autophagosomes and LC3II in podocytes indicated podocytes autophagy impairment in diabetic nephropathy. Whereas calcitriol treatment restored podocyte autophagy activities. In cultured podocytes, the protective effect of calcitriol against high glucose induced podocyte injury could be abated by autophagy inhibitor chloroquine. Conclusion Our study delivered the evidence that calcitriol/VDR signaling attenuated diabetic nephropathy and podocytes injury by restoring podocytes autophagy. This finding may have potential implication for exploring protective mechanisms of calcitriol/VDR in diabetic nephropathy.