Novel tubular–glomerular interplay in diabetic kidney disease mediated by sirtuin 1, nicotinamide mononucleotide, and nicotinamide adenine dinucleotide Oshima Award Address 2017

Following SIRT1-mediated deacetylation of transcription factors or histones, NAD is converted into nicotinamide, as shown in the NAD-related metabolic maps in Fig. 3, thereby emphasizing that NAD is indispensable for SIRT1 function. In mitochondria, NAD is converted to NADH. Therefore, it is essential that these pathways remain intact to maintain cellular NAD levels, and hence, NAD is supplied via de novo and salvage pathways [6]. In the de novo pathway, the essential amino acid tryptophan is converted to NAD, whereas in the salvage pathway NAD consumed by SIRT1 is recycled via two enzymes—nicotinamide phosphoribosyltransferase (NAMPT) and NMN adenylyltransferase (NMNAT). NMN is a precursor of NAD that was reported to be beneficial in several diseases by restoring NAD levels. However, the reason for NMN but not NAD being more effective in elevating NAD levels remains unknown and requires further investigation. Nicotinamide, a substrate of intracellular NAMPT in the NAD-related salvage pathway, is excluded to the extracellular compartment.

Previous research has revealed the pivotal role of podocyte injury in DKD [7]. Microalbuminuria is considered as an important early diagnostic marker for DKD and indicates functional or morphological damage to the podocytes. However, microalbuminuria is not a specific diagnostic marker for diabetic renal damage in DKD because it is increased in hypertension-induced renal sclerosis as well. Our recent research has revealed that metabolic changes (Fig. 4) in proximal tubules precede the changes in podocytes in DKD [8]. Specifically, proximal tubular SIRT1 was primarily downregulated before evident podocytic injury, thereby leading to a reduction in NMN levels that causes further downregulation of SIRT1 in podocytes. In the NAD salvage pathway, NMN is upstream of NAD and SIRT1. However, the salvage pathway is a circular metabolic pathway and not a linear one. It remains possible that NMN is upstream as well as downstream of SIRT1. In a previous study, we have determined whether NMN was the humoral factor connecting proximal tubules with podocytes using three approaches: experiments utilizing conditioned medium, measurement of endogenous NMN levels, and exogenous treatment with fluorophore-tagged NMN to investigate NMN biodistribution [8, 9]. We termed this cell–cell interplay as the proximal tubule–podocyte communication. As shown in later figures, the two well-known components of the tubuloglomerular communication are tubuloglomerular feedback (TGF) and glomerular–tubular balance (GTB) [10]. Corruption of the TGF and GTB underlies glomerular hyperfiltration observed in DKD. However, our new evidence suggests that the dysfunction in the proximal tubule–podocyte communication occurs prior to a malfunction in TGF and GTB and the podocyte injury.

In podocytes, SIRT1 levels are decreased, which was affected by the proximal tubular SIRT1 downregulation, thereby leading to the ectopic expression of claudin-1 in podocytes, causing albuminuria. Under normal conditions, claudin-1 is expressed in parietal epithelial cells, which creates tight junctions that might prevent leakage from Bowman’s capsule [11]. Furthermore, a recent report in mice overexpressing claudin-1 specifically in podocytes has demonstrated that podocyte damage occurred via reduced nephrin and podocin [12]. Although the authors have not elucidated the exact mechanism for the reduction of nephrin and podocin by claudin-1, they inferred that claudin-1 overexpression competitively inhibited the expression of both nephrin and podocin. These mechanisms should be investigated in future studies.

Our studies assessing the mechanism of SIRT1-mediated control of claudin-1 expression have suggested epigenetic mechanisms are involved in this regulation. Under normal conditions, SIRT1 levels were maintained (Fig. 5). SIRT1 deacetylated histones H3 and H4; this caused histone H3K9 methylation and DNA methylation of the CpG islands around claudin-1 gene by DNA methyltransferase (DNMT) 1, thereby suppressing claudin-1 expression. Conversely, in diabetic conditions, SIRT1 levels are reduced with the consequent elevation of H3 and H4 acetylation (Fig. 6), which in turn suppressed H3K9 methylation and DNMT1-induced CpG methylation of claudin-1 to promote its expression.

We have determined that among the three DNMT isoforms, DNMT1 plays an important role in CpG methylation of claudin-1 (Fig. 7). We have specifically demonstrated that DNMT1 is involved in the methylation of the CpG islands located in the first exon and not of those in the promoter region of claudin-1. The CpG islands are typically localized in the promoter regions, and our findings regarding DNMT1-mediated claudin-1 methylation is unique. However, a recent report [13] has stated that first exon CpG islands played an important role in gene transcription in addition to those in promoter regions [14]. Therefore, future studies will be ciritical to further elucidate the role of exon CpGs.

The detailed molecular mechanisms of claudin-1 damage in podocytes involving the β-catenin/snail pathway is shown in Fig. 8. We used DKD models, such as the streptozotocin-induced diabetes mellitus model and db/db mice, to demonstrate the corruption of proximal tubule–podocyte interaction including a reduction in proximal tubular SIRT1 levels concomitant with decreased SIRT1 and increased claudin-1 in podocytes. Additional experiments using proximal tubule-specific Sirt1 conditional knockout mice showed phenotypes that were similar to that observed in diabetic animal models [8]. These data indicated that proximal tubular SIRT1 was a critical molecule in proximal tubule–podocyte communication. In contrast, proximal tubule-specific Sirt1 transgenic mice rescued streptozotocin- and db/db-induced albuminuria, thereby validating the pivotal roles of SIRT1 in proximal tubules. Although we have previously demonstrated that proximal SIRT1 protected against reactive oxygen species-mediated kidney injury [15‐17], we recently uncovered that SIRT1 was specifically protective against hyperglycemia and diabetes-induced kidney damage [8]. Another report by Inagi and colleagues [18] has demonstrated that podocyte-specific Sirt1 knockout provoked podocyte damage in mice, further supporting the ciritical role of SIRT1 in protection against kidney disease. A recent review on the pivotal roles of surtuins in kidney disease [19] raise the possibility of sirtuins as novel diagnostic and/or therapeutic targets of DKD.

Finally, we summarize the relationship between SGLT2 and SIRT1 (Fig. 9). In our recent study, we have uncovered that SGLT2 is elevated during early stages of DKD, which could upregulate intracellular glucose levels in proximal tubules and subsequently decrease SIRT1 expression. Conversely, we demonstrated that SGLT2 inhibitors preserved SIRT1 expression. Our current studies [20] focus on elucidating whether SGLT2 inhibitors can maintain the proximal tubule–podocyte communication.

Glucose reabsorption in proximal tubular cells is depicted in Fig. 10. Briefly, Na⁺ and glucose transports into proximal tubules via SGLT2, whereas Na⁺/K⁺ ATPase-coupled channels pump out Na⁺. Glucose is transported via glucose transporter 2 (GLUT2). Contrary to the physiological regulation of glucose, we hypothesized that GLUT2-mediated regurgitation occurs, particularly in very early stages of DKD. Further, this could occur in the stages of the disease with increased urinary glucose when hyperglycemia might not be observed because of compensatory hyperinsulinemia.

Using a two-chamber model [20], we found that high glucose levels in the basolateral side of proximal tubules (Fig. 11) might trigger intracellular signal transduction, leading to SGLT2 upregulation. We determined that GLUT2 was coupled with importin-α1, which was bound to HNF1α. We further elucidated that after detecting glucose flow, the importin-α1/HNF1α complex was then unbound from GLUT2, and HNF1α transported into the nucleus by importin-α1 to increase the activity of the SGLT2 promoter.

In DKD, disruption of the GTB and TGF leads to the hyperfiltration and increase in Na⁺ reabsorption. The mechanisms we uncovered imply that SGLT2 inhibitors might be utilized as a novel therapeutic target in DKD (Fig. 12) to block the disruption of GTB and TGF. Blocking the GLUT2/importin-α1/HNF1α interaction might reduce SGLT2 expression and prevent the reduction in SIRT1 expression, which may further contribute to the protection of the proximal tubule–podocyte communication in DKD.