Insig1 deletion in proximal tubular deregulates Aldh1a1 to consume NAD+ and contributes to renal fibrosis

doi:10.21203/rs.3.rs-3140844/v1

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Insig1 deletion in proximal tubular deregulates Aldh1a1 to consume NAD⁺ and contributes to renal fibrosis

https://doi.org/10.21203/rs.3.rs-3140844/v1

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Profibrotic proximal tubules (PTs) were identified as a unique phenotype of PTCs in renal fibrosis. Controlling the process of renal fibrosis requires understanding how to manage the S1 subset's branch to the S3 subset rather than to the profibrotic PT subset. Insig1 is one of the branch-dependent genes involved in controlling this process, although its role in renal fibrosis is unknown. Here, we discovered the pathogenic effect of PTC-specific Insig1 deficiency on renal fibrosis in vivo and in vitro. Overexpression of Insig1 profoundly inhibited renal fibrosis. Insig1 deletion boosted SREBP1 nuclear localization, increasing Aldh1a1 transcriptional activity, causing excessive NAD⁺ consumption and ER enlargement, and accelerating renal fibrosis. We identified nicardipine as a selective inhibitor of Aldh1a1-restored NAD⁺ and ER homeostasis, which attenuated renal fibrosis. Together, our findings support Insig1 as a new therapeutic target for CKD.

Health sciences/Nephrology/Kidney diseases/Chronic kidney disease

Health sciences/Nephrology/Kidney diseases/Renal fibrosis

Insig1

Aldh1a1

NAD+ metabolism

proximal tubule

endoplasmic reticulum expansion

chronic kidney disease

renal fibrosis

Renal fibrosis (RF) is a common outcome of chronic kidney disease (CKD) and leads to the final and irreversible loss of renal function ^{1, 2}. CKD is a leading cause of death, however few effective treatments are available ³. For the identification of new therapeutic strategies and improved management of this chronic condition, a better understanding of the mechanism that mediates renal fibrosis is critical.

Differentially expressed genes (DEGs) have been found to be enriched in metabolic pathways and to be altered in PTCs, as indicated by comprehensive genome-wide human kidney tissue transcriptomics, revealing PTC dysfunction and loss in renal fibrosis ⁴. PTCs constitute the most common type of cell in the kidney and are essential for maintaining healthy renal function and preventing renal fibrosis following injury ⁵. The clustering of PTCs involved in unilateral ureteral obstruction (UUO) has led to the identification of six PT cell types, namely, precursor PT, S1, S2, and S3 subsets; proliferating PT subset, and profibrotic PT subset, as determined by single-cell RNA sequencing (scRNA-seq) of the GSE182256 dataset ⁶. The S1 subset can branch out into the S3 and profibrotic PT subsets. During this crucial phase, branch-dependent genes are crucial, and notably, Insulin-induced gene 1 (Insig1) was included among the branch-specific genes identified.

Insig1, an intrinsic protein in the endoplasmic reticulum (ER), is crucial for lipid and cholesterol synthesis ^{7, 8}. By interacting with the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, Insig1 controls the proteolytic processing and transcriptional activation of SREBP1 ^{9, 10}. SREBP1 transcript levels affect a variety of biological processes, such as tumor development ^{11, 12}, immunity ^{13, 14}, and lipid metabolism ^{15, 16}. Recent evidence indicates that Insig1 expression is downregulated in kidney diseases such diabetic kidney disease (DKD) and renal mass loss, but no study has been done to determine the effect of this reduced Insig1 activity in the pathophysiology of CKD or PTC fibrosis ^{17, 18}.

Before identifying the role of Insig1 in CKD, we performed a bulk RNA-seq analysis to assess the DEGs in PTCs between Insig1^ΔKap and Insig1^flox/flox mice. Among that in the discovered DEGs, aldehyde dehydrogenases 1 family member A1 (Aldh1a1) expression was noticeably greater in the PTCs of Insig1^ΔKap mice than in those of Insig1^flox/flox controls, indicating that Aldh1a1 may be a target gene of Insig1. Aldh1a1 is a cytosolic enzyme that catalyses mainly the oxidation of acetaldehyde into acetic acid. It also functions as a marker for cancer stem-like cells (CSCs) and is essential for retinoid metabolism ¹⁹. Evidence from recent studies has demonstrated that Aldh1a1 is essential for numerous biological systems, including the central nervous system, and is involved in diseases related to inflammation and metabolism. Overexpression of Aldh1a1 has been linked to obesity, diabetes, cancer, and other illnesses ^{20, 21, 22}. Furthermore, Aldh1a1 leverages nicotinamide adenine dinucleotide (NAD⁺) as a coenzyme to oxidize aldehydes into carboxylic acids through reactions that are coupled to the reduction of NAD⁺ to NADH ²³.

NAD⁺ is a substrate of key metabolic enzymes involved in energy metabolism, which includes key cellular processes such as glycolysis, the Krebs cycle, DNA repair, and other metabolic reactions in cells (Canto et al., 2015; McReynolds et al., 2020). Many of NAD⁺ cellular functions are realized through its role as a cosubstrate of sirtuins (SIRTs), poly(ADP-ribose) polymerases (PARPs), CD38 ²⁴, and Aldh1a1. Depletion of NAD⁺ leads to numerous inherited and acquired diseases in humans, especially age-related diseases, including neurodegeneration, diabetes, and cancer ^{25, 26, 27}. After acute kidney injury (AKI) and in CKD, substantial decreases in the NAD⁺ level disrupt energy generation. Additionally, NAD⁺ depletion can lead to ER expansion mediated by the deribosylation of the ER stress sensor GRP78/BiP, causing it to be cleaved off from kinase/endoribonuclease inositol-requiring enzyme 1α (IRE1α) ²⁸. Excessive ER stress results in the persistent activation of the unfolded protein response, which is an underlying cause of PTC death and fibrosis. In contrast, increasing evidence indicates that increases in the NAD⁺ level may protect PTCs from a variety of acute stressors, such as ischaemia–reperfusion, toxicity and systemic inflammation ^{27, 29}. NAD⁺-dependent maintenance of PTC metabolic health may also attenuate long-term profibrotic responses that can lead to CKD ³⁰.

Due to the important roles of Aldh1a1 in maintaining NAD⁺ homeostasis in PTCs, compounds that modulate Aldh1a1 activity must be identified. In the present study, nicardipine was identified as a selective Aldh1a1 antagonist based on virtual screening, in vitro enzymatic activity assays ³¹ and BIAcore imaging. Nicardipine is a dihydropyridine-type Ca²⁺ channel blocker (CCB) that exhibits unusual cerebrovascular characteristics and high antihypertensive action ³². However, whether nicardipine affects renal fibrosis or Aldh1a1 deactivation and the related mechanisms are unknown.

In this study, we investigated Insig1 influences on CKD progression and sought to determine whether Aldh1a1 plays a role in mediating the effects of Insig1. We used PTC-specific Insig1-knockout mice and whole-body Aldh1a1-knockout mice subjected to UUO or 5/6 nephrectomy (5/6 Nx) to evaluate the importance of PTC-specific Insig1/Aldh1a1 signalling in conferred protection against renal fibrosis and the maintenance of NAD⁺ homeostasis. We confirmed the therapeutic effect of a selective Aldh1a1 inhibitor by using BIAcore imaging, animal models, PTCs, human PTCs (HPTCs) and human-induced pluripotent stem cell (hiPSC)-derived kidney organoids. The findings suggest potential candidate targets and drugs for treating CKD.

The downregulation of Insig1 is associated with CKD

To identify the co-occurrences with the fibrosis of PTCs, we performed scRNA-seq with dataset GSE182256 from the GEO database. Following a quality control process, the main kidney cell types were clustered (Supplementary Fig. 1A,B), and the PTCs were reclustered to identify PT subsets based on canonical markers from the original publication. Uniform manifold approximation and projection (UMAP) allowed two-dimensional visualization of the main cell types and the PT subset (Fig. 1A and Supplementary Fig. 1C). The pseudo-time trajectory intriguingly revealed that S1 subset were transformed into S3 and profibrotic PT subgroups (Fig. 1B). A heatmap showed that after UUO, the profibrotic PT subgroup exhibited the highest level of ER stress of all the PT subsets (Fig. 1C). Then, in samples from patients with CKD, we observed an apparent ER expansion and vesiculation in PTCs using an electron microscope and immunofluorescence (IF) staining for the ER marker calreticulin (Fig. 1D). Additionally, PTCs from mice with UUO and 5/6 Nx-induced CKD displayed comparable ER alterations and increased ER stress (Fig. 1E and Supplementary Fig. 1D,E). According to these results, the pathophysiological pathways underlying PT fibrosis involved ER stress.

To identify the genes that may prevent the transition of the S1 subset into the profibrotic PT subset, we applied the BEAM technique to branch point 2. The expression of these genes along with the pseudo-time were shown in the heatmap (Fig. 1F). Then, we employed hdWGCNA to cluster branch-dependent genes. Branch point 2 involved three coexpressed gene modules (Fig. 1G). Three gene modules' eigengene levels from four PT subgroups were shown in the boxplot. We chose the blue module after comparing the gene expression variations between the S1 subset transiting into the S3 subset and the S1 subset transiting into profibrotic PT subset. Compared to those in the S2 subset, the eigengene levels gradually increased in the S1 and S3 subsets but decreased in the profibrotic PT subset (Fig. 1H). There were 120 genes in the blue module, and to identify target genes, we compared these genes with genes that were downregulated in both the GSE118341 dataset and our bulk RNA-seq dataset (Fig. 1I). Next, we combined GO analysis of these 17 similar genes with ER stress related genes. Insig1 was the only gene identified in the ER unfolded protein response (Fig. 1J).

Insig1 was widely expressed in PTCs and with its expression was reduced in the UUO group (Fig. 1K,L and Supplementary Fig. 1F-I). Consistent with its alteration in the blue module, Insig1 was downregulated in kidney biopsies of CKD patients compared to controls (Fig. 1M,N). Table S1 presents a list of the clinical details of CKD patients. It was positively correlated with the estimated glomerular filtration rate (eGFR) and negatively correlated with serum creatinine (SCr) level (Fig. 1O). These results demonstrated that the downregulation of Insig1 in CKD may contribute to the progression of renal fibrosis.

Insig1 deficiency in PTCs aggravated UUO-induced CKD

Next, we produced a PTCs-specific Insig1-knockout (Insig1^ΔKap) mice (Fig. 2A). The protein expression in isolated renal tubule tissue confirmed the reduction in Insig1 expression in Insig1^ΔKap mice compared to Insig1^flox/flox littermates without Cre recombinase activity (Fig. 2B,C). To elucidate the role of Insig1 in CKD renal fibrosis, we subjected Insig1^ΔKap and Insig1^flox/flox mice to UUO for 14 days to generate classical renal fibrosis model mice. The Insig1^ΔKap mice showed worsened collagen and FN deposition in the kidney compared to that in the Insig1^flox/flox mice, as determined by Sirius Red staining and Immunohistochemical (IHC) staining (Fig. 2D). Insig1^ΔKap mice showed significantly higher expressions of the fibrotic indicators (Fn, α-SMA (Acta2), COL-I, and Postn) in the kidneys after UUO (Fig. 2E, F and Supplementary Fig. 1J), as well as higher expression of genes (Pdgfb, Cd74) representative of the profibrotic PT subset and lower expression of genes (Slc6a13, Slc7a13, Cyp4b1) representative of the S3 subset (Fig. 2G and Supplementary Fig. 1K). Additionally, Insig1 deletion in PTCs increased expressions of ER stress markers (Calreticulin and Atf6) and exacerbated UUO-induced ER expansion and vesiculation (Fig. 2H-J), while exerting no effect on the inflammatory response (Supplementary Fig. 1L). These results revealed that Insig1 deficiency in PTCs aggravated UUO-induced renal fibrosis and ER stress.

Insig1 deficiency in PTCs aggravated 5/6 nephrectomy-induced CKD

Then, we investigated the role of Insig1 in another renal fibrosis models prepared with Insig1^ΔKap mice treated with 5/6 Nx for 16 weeks. In accordance with the findings from the UUO-induced CKD model, the kidneys of Insig1^ΔKap mice challenged with 5/6 Nx showed higher levels of urine albumin, SCr, and blood urea nitrogen (BUN) levels than Insig1^flox/flox mice (Fig. 3A,B). Moreover, compared to the Insig1^flox/flox mice, Insig1^ΔKap mice exhibited exacerbated collagen and FN deposition rates and increased expressions of the fibrotic indicators (Fn, Col-I and Col-III) after 5/6 Nx challenge (Fig. 3C, D). Additionally, after 5/6 Nx challenge, Insig1 deficiency in PTCs exacerbated ER expansion and vesiculation and increased expressions of ER stress markers (Calreticulin, Atf6 and Trib3), while decreasing Slc6a13 expression and increasing Cd74 expression (Fig. 3E-H). These results revealed that Insig1 deficiency in PTCs aggravated 5/6 Nx-induced renal fibrosis and ER stress.

Insig1 overexpression alleviated UUO-induced renal fibrosis in mice

In addition to loss-of-function models, gain-of-function model was established by injecting Insig1 and Ctrl plasmids under high pressure into the mouse tail vein. Supplementary Fig. 2A demonstrates that 36 hours after injecting the Insig1 plasmids, the protein expression of Insig1 was significantly increased in the kidneys. These mice underwent UUO surgery 36 hours after receiving the Insig1 plasmids. In contrast to the findings with Insig1^ΔKap mice, injecting the Insig1 plasmids markedly improved renal fibrosis in the UUO-treated mice compared to that in the mice injected with Ctrl plasmids, as evidenced by reduced collagen deposition and fibrotic indicator (COL-I and α-SMA) protein expression, as well as similar changes in the mRNA expression of Fn, Col-I, and Col-III in the kidneys (Supplementary Fig. 2B-D). These results considerably demonstrated Insig1's protective function in renal fibrosis, which was in line with the beneficial role in the blue module.

Silencing Insig1 aggravated TGF-β1-induced fibrotic responses in vitro

To examine the direct effect of Insig1 on renal tubular cells, we transfected HK2 cells with Insig1 siRNA or Ctrl siRNA (Supplementary Fig. 2E) and then treated the cells with TGF-β1 for 24 h. In accordance with our in vivo findings, silencing Insig1 worsened TGF-β1-induced fibrotic responses, as shown by increased expression of Fn and Acta2, as well as worsened ER stress compared to that of the control group (Supplementary Fig. 2F,G). We also found that silencing Insig1 boosted TGF-β1-induced expression of FN, CTGF, Acta2 and Col-I in mouse renal tubular epithelial cells (TKPTS) (Supplementary Fig. 3A-E). Therefore, we hypothesized that silencing Insig1 exacerbated TGF-β1-induced fibrotic responses in HK2 and TKPTS cells.

In contrast, overexpression of Insig1 reduced TGF-1-induced expression of FN, CTGF, Acta2, and Col-I compared to that of the control group (Supplementary Fig. 3F–J). Collectively, these findings showed that Insig1 significantly inhibited the synthesis of extracellular matrix components and ER stress caused by TGF-β1, showing that Insig1 functions as a protective factor against renal fibrogenesis.

Aldh1a1 is a transcriptional targert of Insig1 in the kidney

As Insig1 regulates lipid metabolism, we first examined whether Insig1 deletion exacerbates UUO-induced renal fibrosis mediated through its effect on lipid metabolism. We observed that UUO-induced lipogenesis was unaffected by Insig1 deletion (Supplementary Fig. 4A). Additionally, TOFA (an inhibitor of acetyl-CoA carboxylase-α) and C75 (an inhibitor of fatty-acid) were applied to an in vitro CKD model. When TGF-β1 was present, silencing Insig1 enhanced the mRNA expression of Fn and Ctgf, and neither TOFA nor C75 reversed this trend (Supplementary Fig. 4B). These findings suggested that lipogenic activity may not be related to the profibrotic effect of Insig1 deletion in PTCs.

Then, utilizing isolated renal tubule tissue, we conducted a bulk RNA-seq analysis to examine differences in gene expression between Insig1^flox/flox and Insig1^ΔKap mouse PTCs to better understand the underlying mechanism by which Insig1 prevents renal fibrosis. Insig1^ΔKap mice showed 179 differentially expressed genes, 109 downregulated genes and 70 upregulated genes, in comparison to Insig1^flox/flox mice (Fig. 4A). The retinol metabolism pathway was one of the most significantly altered pathways, according to KEGG pathway enrichment results (Fig. 4B). Aldh1a1, a crucial enzyme in retinol metabolism, was markedly increased in the kidneys of Insig1^ΔKap mice in contrast to Insig1flox/flox mice (Fig. 4C). Aldh1a1 was also noticeably elevated in Insig1-silenced TKPTS cells (Fig. 4D). Additionally, the luciferase reporter gene data showed that Insig1 negatively modulated Aldh1a1 transcriptional activity (Fig. 4E,F).

According to the literature, after deletion, Insig1 was unavailable to interact with SCAP, causing the SCAP-SREBP1 complex to translocate to the Golgi apparatus and increasing the amount of SREBP1 entering the nucleus ⁸. Moreover, SREBP1 protein expression was markedly decreased in the cytoplasm but increased in the nucleus of Insig1-silenced TKPTS cells (Fig. 4G). Aldh1a1 transcriptional activity was significantly elevated when SREBP1 was overexpressed (Fig. 4H). A chromatin immunoprecipitation (ChIP) assay revealed that SREBP1 bound to the Aldh1a1 promoter region (Fig. 4I). Additionally, fatostatin (an SREBP1 inhibitor) markedly reversed the increase in transcriptional activity of Aldh1a1 in Insig1-silenced TKPTS cells (Fig. 4J). Although the active form of SREBP1 (SREBP1 1-480 bp) increased Aldh1a1 transcriptional activity, fatostatin was failed to reverse this trend (Fig. 4K). These findings demonstrated that Insig1 prevented SREBP1 protein entry into the nucleus, thereby lowering Aldh1a1 transcriptional activity (Fig. 4L).

Inhibiting Aldh1a1 alleviated renal fibrosis in vivo and in vitro

The Aldh1a1 level was found to be significantly higher in the UUO model mice (Fig. 5A and Supplementary Fig. 4C–E), and it positively correlated with the SCr level and negatively correlated with the eGFR (Fig. 5B). To verify the role of Aldh1a1 in CKD, we subsequently constructed Aldh1a1-KO mice (Supplementary Fig. 4F,G). In contrast to wild-type (WT) mice, Aldh1a1 deletion markedly decreased collagen deposition and FN expression in the kidneys after UUO (Fig. 5C). The fibrotic index (α-SMA, COL-I, Col-III, Postn, and Ctgf) and the profibrotic PT maker (Pdgfb) level were also significantly lower in the Aldh1a1-KO animals after UUO, while the S3 marker (Slc6a13, Slc7a13 and Cyp4b1) levels were significantly higher (Fig. 5D–F and Supplementary Fig. 4H). Moreover, Aldh1a1 deletion significantly decreased ER dilation and ER stress makers (Calreticulin, Atf6 and Perk) (Fig. 5G–I).

In TKPTS cells, overexpression of Aldh1a1 (Supplementary Fig. 4I) worsened TGF-β1-induced fibrotic responses, as shown by increased expression of FN, Acta2 and Ctgf (Fig. 5J,K). In contrast, Aldh1a1-silencing reduced ER stress and the TGF-β1-induced expression of FN, α-SMA and Col-I in TKPTS cells compare to the levels in the control group (Fig. 5L and Supplementary Fig. 4J–L). Additionally, modifying the protein's active site may be able to block Aldh1a1's profibrotic effects (Fig. 5M).

In line with these findings, pharmacological intervention using the Aldh1a1 inhibitor NCT501 reduced collagen deposition and the expression of FN, Acta2, and COL-I in kidneys after UUO (Supplementary Fig. 5A–C). NCT501 also reduced the veracity of TGF-β1-induced fibrotic responses in TKPTS and HK2 cells (Supplementary Fig. 5D-F). These results together demonstrated that Aldh1a1 overexpression exacerbated renal fibrosis.

Insig1/Aldh1a1 activation mitigated renal fibrosis by increasing NAD⁺ levels

An untargeted metabolomics analysis was carried out to determine the mechanism of Aldh1a1 in renal fibrosis. As expected, we discovered a considerable decrease in the levels of NAD⁺ and its precursors (nicotinamide riboside and nicotinamide ribotide) in kidneys after UUO (Supplementary Fig. 5G–I), and this decline was even more pronounced following the loss of Insig1 in PTCs (Supplementary Fig. 5J, K and Fig. 6A,B). Consistent with the metabolomic analysis results, after UUO or 5/6 Nx challenge, the NAD⁺ concentration was considerably lower in the kidneys of the Insig1^ΔKap mice than in the kidneys of the Insig1^flox/flox mice (Fig. 6C). Additionally, compared to WT mice, Aldh1a1-KO mice showed significantly higher levels of NAD⁺ in the kidneys after UUO (Fig. 6C). NCT501 treatment increased the NAD⁺/NADH ratio in TKPTS cells despite Insig1 silencing or Aldh1a1 overexpression decreasing it. Additionally, Aldh1a1 overexpression worsened TGF-β1-induced NAD⁺ consumption (Fig. 6D,E). In hiPSC-derived kidney organoids, we also found that NAD⁺ reduced TGF-β1-induced extracellular matrix component production (Supplementary Fig. 5L). These findings suggested that the antifibrotic activity of the Insig1/Aldh1a1 pathway may depend on NAD⁺ homeostasis.

Recent research suggested that NAD⁺ controls ER membrane expansion by deribosylating the ER sensor GRP78/BIP ²⁸ (Fig. 6F). In TKPTS cells, NCT501 treatment or Insig1 overexpression significantly increased ADP-ribosylated BIP levels (Fig. 6G,H). Insig1 silencing also resulted in an expansion of the ER membrane and reduced IRE1α and BIP binding. NAD⁺ or Aldh1a1 silencing, however, increased BIP and IRE1α binding (Fig. 6I,J and Supplementary Fig. 5M). Together, Insig1 overexpression or Aldh1a1 silencing restored the ADP ribosylation of BIP, enhanced the binding of IRE1α and BIP, and maintained ER homeostasis by increasing NAD⁺ levels.

Identification of a novel human Aldh1a1 inhibitor that alleviated UUO-induced CKD in a Aldh1a1-dependent manner

The TargetMol-Approved Drug Screening Library (a total of 1600 compounds) and TargetMol-Anti Cancer Compound Library (a total of 3145 compounds) were used in our study for virtual screening (Fig. 7A). On the basis of the virtual screening results, nineteen compounds were chosen for additional examination by in vitro enzymatic activity assays. The human recombinant Aldh1a1 (hAldh1a1) protein was profoundly inhibited by nicardipine (13-F9), and its docking score was − 9.4128 (Fig. 7B,C). Then the BIAcore system was used to directly evaluate the binding of nicardipine and Aldh1a1. The equilibrium dissociation constant (KD) value of Aldh1a1 with nicardipine was 1.98E-06 M, indicating strong binding between nicardipine and Aldh1a1, while the KD value of Aldh1a1 with NCT501 was 3.31E-05 M (Fig. 7D and Supplementary Fig. 5N). In vivo, nicardipine treatment decreased Aldh1a1 protein expression in the kidney but exerted no effect on Aldh1a1 mRNA expression (Fig. 7E and Supplementary Fig. 6A). Consistent with in vivo results, nicardipine lowered Aldh1a1 protein expression in TKPTS cells in a dose-dependent manner but exerted no effect on Aldh1a1 mRNA expression or transcriptional activity (Fig. 7F and Supplementary Fig. 6B,C). Additionally, the protease inhibitor MG132 profoundly reversed the nicardipine-induced reduction in Aldh1a1 protein expression (Supplementary Fig. 6D). Therefore, nicardipine inhibited Aldh1a1 expression via a proteasomal degradation mechanism. Thus, nicardipine may function as a novel Aldh1a1 inhibitor.

For further analysis of the effects of nicardipine on UUO-induced CKD, mice were given nicardipine (1 mg/kg/day intragastrically (i.g.)) for 2 days before UUO surgery. Nicardipine administration decreased renal fibrosis in the mice after UUO, which was consistent with the outcomes observed in mice treated with NCT501. Following nicardipine treatment, collagen deposition and FN expression were reduced in the mice after UUO (Fig. 7G), and profibrotic marker expression (FN, CTGF, Col-I, and Col-III), ER stress index (Atf6, Trib3), and ER expansion, were decreased in the kidneys compared to UUO-only exposed animals (Fig. 7H–K). Similarly, nicardipine markedly reduced the fibrotic reactions caused by TGF-β1 in TKPTS cells, HPTCs, and hiPSC-derived kidney organoids (Supplementary Fig. 7A–F). Mechanistically, nicardipine greatly elevated the NAD⁺ concentration and the NAD⁺/NADH ratio, which had been decreased by TGF-β1, significantly boosting ADP-ribosylated BIP levels and increasing IRE1α and BIP binding, which prevented ER expansion (Fig. 7L–N).

To determine whether the protective effect of nicardipine on UUO-induced CKD involved an Alhd1a1-dependent mechanism, we administered nicardipine to WT and Aldh1a1-KO mice for 14 days. As expected, the protective effects of nicardipine on UUO-induced CKD were abolished in Aldh1a1-KO mice (Fig. 7O–Q), showing that Aldh1a1 is involved in the effect of nicardipine on UUO–induced CKD. According to these findings, nicardipine might be a new highly targeted Aldh1a1 inhibitor.

Based on the results of genetic, in vivo, in vitro, and pharmacological investigations, this study revealed that PTC-specific Insig1 plays a significant role in the progression of renal fibrosis in CKD. Here, we found an apparent reduction in Insig1 in the kidney under CKD conditions. Furthermore, we reported the pathogenic effect of PTC-specific Insig1 deficiency on renal fibrosis in vivo and in vitro. Overexpression of Insig1 potently inhibited renal fibrosis and attenuated CKD progression. All these in vivo and in vitro experimental data strongly suggested that the overexpression of Insig1 exerted protective effects against renal fibrosis in CKD.

Re-analysing the scRNA-seq data that had been uploaded ⁶, we discovered that Insig1 was a branch-specific gene that was crucial for controlling how the S1 subset transitioned into the S3 and profibrotic PT subsets. Furthermore, by using hdWGCNA to cluster branch-dependent genes, we discovered that Insig1 was in the blue gene module. Notably, the eigengene levels in the S1 and S3 subsets steadily increased compared to those in the S2 subset, while the were decreased in the profibrotic PT subset; these expression changes were consistent with our experimental results. These findings suggested that increased Insig1 expression might prevent the S1 subset from branching into the profibrotic PT subset.

As SREBP1 is a well-characterized lipogenic transcription factor ³³, Insig1 controls lipogenesis and cholesterol metabolism by controlling SREBP1 in the nucleus ⁸. We investigated whether the profibrotic effect of Insig1 deficiency may be mediated by lipogenic activity. However, we found that the lipogenesis caused by UUO was unaffected by Insig1 deletion in PTCs. In line with this finding, when Insig1 was knocked down, TOFA and C75 were unable to block the profibrotic effect of TGF-β1. Thus, the profibrotic effect of Insig1 deletion in PTCs did not appear to be linked to lipogenic activity.

In our research, we discovered for the first time that Insig1 may control the transcription of Aldh1a1 via the action of SREBP1. When Insig1 was deleted, SREBP1 nuclear translocation was enhanced, and Aldh1a1 transcription was subsequently activated. The transcriptional inhibition of Aldh1a1 by Insig1 was reversed when SREBP1 was prevented from moving to the Golgi apparatus from the ER by inhibitor (fatostatin) treatment. By mutating the Aldh1a1 active site, we prevented the protein from reducing the NAD⁺ level and reversing the profibrotic effects of Aldha1. Together, these data suggested that Insig1 altered NAD⁺ homeostasis through the transcriptional control of Aldh1a1, which might explain how Insig1 influences ER stress. Additionally, Insig1 might play an important role in ageing-related disorders.

NAD⁺ preservation is increasingly being recognized as a way of delaying the effects of ageing on health and thus potentially extend lifetime ^{34, 35, 36}. The kidney is a crucial organ in de novo NAD⁺ biosynthesis ³⁷. In AKI and CKD, de novo NAD⁺ biosynthesis is impaired, and substantial decreases in the levels of NAD⁺ can reduce energy generation ³⁴. More recently, NAD⁺ augmentation through supplementation with nicotinamide mononucleotide (NMN) or nicotinamide (NAM), an NAD⁺ precursor, has been proposed as for the prevention of numerous kidney illnesses, including AKI, CKD, the AKI-to-CKD transition, and diabetic nephropathy ^{27, 29, 38, 39, 40, 41, 42}. Our experimental results showed that NAD⁺ lowered TGF-β1-induced extracellular matrix deposition in kidney organoids produced from hiPSCs, confirming its therapeutic efficacy in CKD.

The function of the ER stress sensor GRP78/BIP depends on NAD⁺-dependent ADP ribosylation. By deribosylating BIP and forcing it to separate from IRE1α, NAD⁺ deficiency resulted in ER expansion ²⁸. Excessive ER stress results in the persistent activation of the unfolded protein response, which is an underlying cause of PTC death and fibrosis. Additionally, de novo NAD + biosynthesis in the kidney may be impaired by the ER stress response ⁴³, which suggests interplay between NAD⁺ and ER stress. Here, we discovered that therapy with NCT501 or overexpression of Insig1 might restore BIP ADP ribosylation. Additionally, NAD⁺ treatment or Insig1 overexpression increased the binding of IRE1α and BIP, but Aldh1a1 overexpression reversed this trend, suggesting that decreased Insig1 might boost Aldh1a1 transcription-regulating activity, which in turn may results in excessive NAD⁺ consumption and ER expansion. The communication between the ER and mitochondria is important to a cell ⁴⁴. Lack of mitochondrial aspartate attenuated NAD⁺ regeneration and led to BIP ADP deribosylation ²⁸. As a result, further exploration into how changes in CKD-related mitochondrial function are caused by Insig1 deletion in PTCs is warranted.

In the current study, we first established that nicardipine is a selective Aldh1a1 antagonist. A Ca²⁺ channel blocker, nicardipine, exerted few harmful effects on mice (Supplementary Fig. 6E-H). Consistent with the protective effect of NCT501 on mice after UUO, pretreatment with nicardipine mitigated renal fibrosis in vivo and in vitro, and this protective effect was abolished in Aldh1a1-KO mice, suggesting the involvement of Aldh1a1 in the activity of nicardipine against renal fibrosis, but we cannot rule out a potential off-target effect. Mechanistically, nicardipine might restore BIP ADP ribosylation and increase the binding of IRE1α and BIP, which results in an increase in NAD⁺ level and the maintenance of ER homeostasis. Additionally, we discovered that nicardipine strongly binds to Aldh1a1, facilitating Aldh1a1 protein degradation and thus lowering Aldh1a1 activity. Thus, Aldh1a1 may be a novel nicardipine target. Insig1 expression was low and Aldh1a1 expression was high in CKD patients with hypertension (Supplementary Fig. 7G,H). Nicardipine is effective in the treatment of blood pressure, but here, we found that it effectively attenuated CKD, expanding its clinical indications to treatment of CKD patients with hypertension.

A limitation of our study is that the roles of Insig1 and Nicardipine in ameliorating CKD were observed mainly in models of mouse, PTCs, HPTCs and hiPSCs-derived kidney organoids but not in humans. Although we detected the downregulation of Insig1 in patients with CKD, the effectiveness and importance of Insig1 and Nicardipine in protecting against human CKD remain to be further explored in the future.

In conclusion, activation of Insig1 in PTCs markedly attenuated CKD, possibly by maintaining NAD⁺ homeostasis and controlling ER expansion via the transcriptional repression of Aldh1a1 in PTCs. These findings from the present study not only increase our understanding of the pathogenesis of CKD but also highlight the therapeutic potential of activating the Insig1/Aldh1a1 pathway. Determination of the efficacy and safety of selective Aldh1a1 antagonists and future clinical trials for patients with CKD might reveal a viable approach to CKD therapy.

Human kidney biopsy samples

The tissue samples of 15 diagnosed patients were provided by the Department of Nephrology, Children’s Hospital of Nanjing Medical University. Paracarcinoma kidney tissues, which were obtained from 6 renal cell cancer patients who underwent nephrectomy, were used as normal controls. Clinical data, such as age, sex, and causes of disease, were recorded (Table S1). All studies involving patient kidney specimens were approved by the Local Committee on Human Subjects at the Children’s Hospital of Nanjing Medical University.

Animals

All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (registration number: IACUC-1809017). All mice in the study were housed under controlled conditions (temperature 20–26°C, humidity 40–70%)and a 12-h light-dark cycle). Insig1^flox/flox mice were purchased from GemPharmatech (T020103, Nanjing, China), and Aldh1a1-KO mice were purchased from Cyagen (S-KO-00984, Suzhou, China). Insig1^ΔKap was produced by bleeding Insig1^flox/flox mice and Kap-cre transgenic mice. Eight-week-old male Insig1^flox/flox and Insig1^ΔKap mice were chosen to establish classical CKD models (UUO, 2 weeks; 5/6 Nx, 4 months). For the drug treatment experiments, 8-week-old male C57BL/6 mice were pretreated with NCT501 (HY-18768, MCE, 1.3 mg/kg/day, i.g.) or nicardipine (HY-12515A, MCE, 1 mg/kg/day, i.g.) for 2 days, followed by UUO surgery. NCT501 or nicardipine was continuously administered for the next 14 days. The mice were sacrificed with isoflurane anaesthesia, and blood and kidney tissue were immediately collected for further study.

Cell culture and treatment

All cells were obtained from the American Type Culture Collection (ATCC) and cultured in a humidified incubator (with 5% CO₂ and 37°C). The cells were specifically grown in DMEM or RPMI 1640 medium plus 10% foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were transfected with Insig1 or Aldh1a1 plasmids or siRNA using PolyJet reagent or Lipo3000 and then stimulated with TGF-β1 (10 ng/ml) for 24 h. For the pharmaceutical intervention combined with TGF-β1, cells were pretreated with NCT501 (60 µM) or nicardipine (3 µM) for 2 h before stimulation with TGF-β1 (10 ng/ml) or vehicle for 24 h. The information of plasmids or siRNA are listed as Table S2.

Dual luciferase reporter assay

First, pGL4.19-mAldh1a1 promoter constructs were cotransfected into HEK-293T cells with Insig1, SREBP1 plasmids or si-Insig1. Second, the pGL4.19-mAldh1a1 promoter constructs were transfected into HEK-293T cells with si-Insig1 or SREBP1 (1-480 bp), followed by treatment with either vehicle or fatostatin. Cells were assayed using a Dual-Luciferase Reporter Assay System (Promega, E1910). Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity.

ChIP assay

ChIP assays were conducted using a Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9006) according to the manufacturer’s protocol. After the ChIP DNA sequences were obtained, qPCR was performed using primers (Table S3) to amplify genomic regions containing Aldh1a1-/SREBP1-binding motifs. Enrichment for gene loci was reported as the fold difference in + enrichment of the Aldh1a1/SREBP1 antibody relative to that of the control IgG antibody.

RNA sequencing analysis

Total RNA samples were obtained from isolated renal tubule tissues (Insig1^ΔKap and Insig1^flox/flox mice). Transcriptome sequencing was carried out by Shanghai Applied Protein Technology, which performed RNA preparation, library creation, sequence analysis, and identification. Differential expression and KEGG analyses were performed using the DESeq2 R package (1.16.1). A P value < 0.05 and | log2fold change | >1 were considered to be significant.

Untargeted metabolomics analysis

Kidney tissues were harvested and processed following the manufacturer’s instructions ⁴⁵. The metabolomics analysis was carried out by Suzhou PANOMIX Biomedical Tech, which included sample preparation, UPLC/Q-TOFMS conditions, and metabolite identification. Significantly altered metabolites were identified based on Student’s t test with a P value < 0.05 and |FC (fold change)|>2 and then labelled with KEGG ID using Thermo Scientific™ Compound Discoverer™ 3.1 software for further metabolic pathway enrichment analysis in KEGG databases.

NAD⁺ measurement

The concentration of NAD⁺ in kidney tissues and cells were measured with an ELISA kit (Abcam, ab65348) following the manufacturer’s instructions. The absorbance of each sample was analysed with a Synergy-H1 fluorimeter from Bio-Tek (Winooski, VT, USA).

Statistical analysis

All experimental data are presented as the means ± SDs. Statistical analyses were performed with GraphPad Prism (version 9.1.0). Statistically significant differences were determined via ANOVAs followed by a Bonferroni multiple comparisons test or Student’s t test. A value of P < 0.05 was considered to be significant.

Data availability

Source Data underlying the display items in the manuscrip related to Figures 1–7 and S1–S7 are provided as Data S1. Raw data file of RNA-Seq have been submitted to the database of NCBI and can be viewed at https://dataview.ncbi.nlm.nih.gov/object/PRJNA989392?reviewer=4labdiajk06p93slpitofp980. All other data supporting the findings of this study are included in the supplementary information or available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (no. 82170753).

Author contributions

S.L. performed experiments, collected and analyzed data, and wrote the manuscript. S.L. and X.Y. screened Nicardipine. S.L. gathered human kidney biopsy samples. Y.Z. helped with in vitro experiments. J.Q. helped with animal experiments and drew the figures. J.W. analyzed the scRNA-seq dataset. X.Y. oversaw the design of experiments and interpretation of results, the writing and organization of the manuscript and did final editing.

Competing interests

The authors declare no competing interests.

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There is NO Competing Interest.

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Insig1 deletion in proximal tubular deregulates Aldh1a1 to consume NAD⁺ and contributes to renal fibrosis

Status:

Version 1

Abstract

Figures

Introduction

Results

The downregulation of Insig1 is associated with CKD

Insig1 deficiency in PTCs aggravated UUO-induced CKD

Insig1 deficiency in PTCs aggravated 5/6 nephrectomy-induced CKD

Insig1 overexpression alleviated UUO-induced renal fibrosis in mice

Silencing Insig1 aggravated TGF-β1-induced fibrotic responses in vitro

Aldh1a1 is a transcriptional targert of Insig1 in the kidney

Inhibiting Aldh1a1 alleviated renal fibrosis in vivo and in vitro

Insig1/Aldh1a1 activation mitigated renal fibrosis by increasing NAD⁺ levels

Identification of a novel human Aldh1a1 inhibitor that alleviated UUO-induced CKD in a Aldh1a1-dependent manner

Discussion

Methods

Human kidney biopsy samples

Animals

Cell culture and treatment

Dual luciferase reporter assay

ChIP assay

RNA sequencing analysis

Untargeted metabolomics analysis

NAD⁺ measurement

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Insig1 deletion in proximal tubular deregulates Aldh1a1 to consume NAD+ and contributes to renal fibrosis

Status:

Version 1

Abstract

Figures

Introduction

Results

The downregulation of Insig1 is associated with CKD

Insig1 deficiency in PTCs aggravated UUO-induced CKD

Insig1 deficiency in PTCs aggravated 5/6 nephrectomy-induced CKD

Insig1 overexpression alleviated UUO-induced renal fibrosis in mice

Silencing Insig1 aggravated TGF-β1-induced fibrotic responses in vitro

Aldh1a1 is a transcriptional targert of Insig1 in the kidney

Inhibiting Aldh1a1 alleviated renal fibrosis in vivo and in vitro

Insig1/Aldh1a1 activation mitigated renal fibrosis by increasing NAD+ levels

Identification of a novel human Aldh1a1 inhibitor that alleviated UUO-induced CKD in a Aldh1a1-dependent manner

Discussion

Methods

Human kidney biopsy samples

Animals

Cell culture and treatment

Dual luciferase reporter assay

ChIP assay

RNA sequencing analysis

Untargeted metabolomics analysis

NAD+ measurement

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Insig1 deletion in proximal tubular deregulates Aldh1a1 to consume NAD⁺ and contributes to renal fibrosis

Insig1/Aldh1a1 activation mitigated renal fibrosis by increasing NAD⁺ levels

NAD⁺ measurement