Proximal Tubule Cells Research Articles

Decoding the Intercellular Cross-Talking Between Immune Cells and Renal Innate Cells in Diabetic Kidney Disease by Bioinformatics.

Diabetic kidney disease (DKD) continues to be devastating complication of diabetes mellitus. Immune response and inflammatory reaction play essential roles in the progression of DKD. But the specific mechanism of immune cells, and their act on renal innate cells remains unclear. This article focused on immune cells and their communication with renal innate cells to provide bioinformatic evidence for further understanding the immune mechanism in DKD. Data were analyzed to evaluate the differentially expressed genes (DEGs) and their pathways in DKD patients and mice. Gene set enrichment analysis (GSEA) was used to explore the immune inflammation-related pathways. CIBERSORT was applied to evaluate the distribution of inflammatory cells in different states. Cell-type DEGs and their enrichment pathways were further explored in podocytes, proximal tubule cells and injured tubule cells. Cellchat was used to reveal the cellular communication between immune cells and renal innate cells in DKD. GO and KEGG analysis showed that DEGs were mainly enriched in immune inflammation-related pathways. Monocytes, M2 macrophages and T cells were significantly increased in DKD samples, especially in renal tubule. ScRNA datasets showed that the immune cells number in DKD were significantly increased. Cell-type DEGs were involved in kidney growth and development. In DKD, the interaction numbers and strength between immune cells and innate cells were significantly increased. VISTANT, SPP1 and IGF signal flow were increased in DKD. SPP1-CD44, NRG1-ERBB4, NAMPT-INSR, and Igf1-Igf1r receptor ligand pairs were enhanced in DKD, which mediated the communication between immune-inflammatory cells and innate cells. Our study explored the pathogenesis of renal injury promoted by immunoinflammatory in DKD. VISTANT, SPP1, and IGF signaling pathways and SPP1-CD44, NRG1-ERBB4, NAMPT-INSR, and Igf1- Igf1r receptor ligand pairs might occupy essential place in the occurrence and progress of DKD.

Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.

Fucoidan-proanthocyanidins nanoparticles protect against cisplatin-induced acute kidney injury by activating mitophagy and inhibiting mtDNA-cGAS/STING signaling pathway

Fucoidan (FU) is a natural polymer from marine organisms, which has been widely studied and applied in drug delivery. In this study, FU nanoparticles loaded with proanthocyanidins (PCs) (FU/PCs NPs) were prepared and their effect and mechanism in protecting cisplatin-induced acute kidney injury (AKI) were studied. The in vitro studies confirmed that FU/PCs NPs increased the antioxidant activity of free PCs and protected the death of human kidney proximal tubule (HK-2) cells induced by cisplatin. Further mechanism studies showed that FU/PCs NPs protected the mitochondrial damage induced by cisplatin, activated mitophagy, inhibited the release of mitochondrial DNA (mtDNA), and inhibited the cGAS/STING signal pathway. The in vivo results also indicated that FU/PCs NPs protected cisplatin-induced AKI, including inhibiting the increase of blood urea nitrogen (BUN) and serum creatinine (SCr) levels induced by cisplatin. The mechanism studies confirmed that cisplatin induced an increase in the expression of mitophagy-related protein Pink/Pakrin, mitochondrial mtDNA release and cGAS/STING expression in mice kidney tissues. Pre-administration of FU/PCs NPs further activated mitophagy, as well as inhibiting mtDNA release and cGAS/STING expression. In conclusion, our research proved the role of mitophagy-mtDNA-cGAS/STING signal was involved in cisplatin-induced AKI.

404-P: Single-Cell RNA Profiling Changes in Glomerular Cells in Diabetic Nephropathy (DN)

Hyperglycemia, the main risk factor for DN, causes complex pathological damages in glomeruli. To characterize detail changes in cell differentiation and gene expressions induced by diabetes, we performed unbiased single-cell RNA sequencing (scRNA-seq) on glomerular cells from diabetic and nondiabetic mice of long duration. Glomerular cells were isolated from C57 mice after 7-months of STZ-induced diabetes, when clear glomerular pathologies have been documented. Total number of 48,337 high-qualitied glomerular cells were isolated and sequenced from WT and STZ-treated mice. Based on clustering analysis and cell-type specific markers, 12 different cell types were identified, including endothelial cells (EC), podocytes, mesangial cells (MC), proximal tubule cells, parietal epithelial cells (PEC), collecting duct cells, ascending loop of Henle, macrophages, B cells, T cells, NK cells and neutrophils. Over 90% of the cells were from glomeruli (41% EC, 34 % podocytes, 14% MC, 2% of PEC), and 6% immune cells. Interestingly, gene profiling identified 9 clusters of EC, 5 clusters of MC and 7 clusters of podocytes. Diabetes did not change the total cell numbers for EC, MC and podocytes, although macrophage and B cell numbers were increased from 2.2% to 4.8% and 0.35% to 1%, respectively. Cluster subset analysis of EC, MC and podocytes showed that diabetes induced substantial changes. For podocytes, diabetes increased those clusters (5% to 37%) with high expressions of stressed proteins such as Hspa1a, Hspa1b and Hsp90aa1, while decreased those clusters with high expressions of ECM production and CDC42 GTPase cycle, that were involved in cytoskeleton stabilization and foot process formation. Findings from scRNA-seq of glomeruli showed there were multiple subsets of podocytes, EC and MC, which appear to have different functions and their distributions were changed by diabetes. Understanding the mechanism for the distribution changes of the subsets may provide new insight in the pathogenesis of DN. Disclosure J. Fu: None. Q. Li: None. K. Park: None. Q. Huang: None. I. Wu: None. G.L. King: Research Support; Janssen Research & Development, LLC.

12-OR: Urine Lactate Is a Disease Biomarker for Diabetic Kidney Disease

As lactate (LA) elevation is associated with mitochondrial dysfunction, we examined the role of LA in diabetic kidney disease (DKD) progression. Urine LA (ULA) was measured in patients with type 2 diabetes (T2D) in three independent cohort studies (HUNT3, SMART2D, and CRIC). ULA and plasma LA (PLA) were also measured in patients with type 1 diabetes (T1D) following eu- or hyperglycemic clamp, in mouse kidney sections, and sodium-glucose cotransporter-2 (SGLT2)-deficient mice. Mitochondrial functional changes in human kidney proximal tubule (HK2) cells were assayed in response to exogenous LA. Subjects in HUNT3 with DKD (n=39) had elevated ULA levels compared to healthy controls (n=53, p=0.023) (Fig. 1). In subjects with T2D from SMART2D (n=230) and CRIC (n=889), the third tertile of ULA/creatinine ratio was associated with a greater estimated glomerular filtration rate (eGFR) decline [-1.63 mL/min/1.73m2/year, p=0.007, and -0.63 mL/min/1.73m2, p=0.042], relative to the first tertile. Acute hyperglycemia stimulated PLA (p<0.0001) and ULA (p=0.001) in patients with T1D (n=39), and in kidney sections incubated with high glucose. Compared to wild-type, SGLT2-deficient mice had lower ULA levels. LA above 2.5 mM inhibited oxygen consumption rate in HK2 cells. Elevated ULA/creatinine ratio inhibits mitochondrial function in diabetes and identifies subjects at risk of rapid renal decline independent of chronic glycemic control. Disclosure L.Kugathasan: None. L.Natarajan: None. S.Lim: None. S.Waikar: None. Y.M.Osman: None. J.He: None. V.Vallon: Research Support; NIH - National Institutes of Health, Gilead Sciences, Inc., Merck Sharp & Dohme Corp., Novo Nordisk, Maze Therapeutics, Boehringer-Ingelheim, Speaker's Bureau; AstraZeneca. S.Hallan: None. D.Cherney: Other Relationship; Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL-Behring, Otsuka, Novartis, Youngene, Lexicon and Novo-Nordisk, Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL-Behring and Novo-Nordisk. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. Cric study investigators: n/a. M.Darshi: Employee; Janssen Research & Development, LLC. V.Srinivasan sridhar: Other Relationship; Merck & Co., Inc. V.Drel: None. J.Tumova: None. J.Liu: None. J.Wang: None. B.A.Perkins: Advisory Panel; Dexcom, Inc., Insulet Corporation, Novo Nordisk, Sanofi, Vertex Pharmaceuticals Incorporated, Other Relationship; Abbott, Medtronic, Sanofi, Research Support; Novo Nordisk, Bank of Montreal (BMO). Y.Lytvyn: None.

Signaling mechanisms in renal compensatory hypertrophy revealed by multi-omics

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we use a multi-omic approach in a unilateral nephrectomy model in male mice to identify signaling processes associated with renal compensatory hypertrophy, demonstrating that the lipid-activated transcription factor peroxisome proliferator-activated receptor alpha (PPARα) is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

#5592 THE SODIUM PHOSPHATE COTRANSPORTER NAPI-IIB IS UPREGULATED AND EXPRESSED DE NOVO IN KIDNEYS OF PATIENTS WITH CHRONIC KIDNEYS DISEASE

Abstract Background and Aims Renal reabsorption of phosphate is mediated by 3 main transporters located in the luminal brush border membrane of the proximal tubule, namely NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit2 (SLC20A2). The expression of all three proteins has been shown to be reduced in animal models of kidney disease [1]. Recently, we demonstrated that NaPi-IIb, a paralogue of the SLC34 subfamily that is mostly expressed in small intestine and lungs, is also expressed in the loop of Henle. Its mRNA expression is enhanced in rodent models of kidney injury. However, the expression of NaPi-IIb has not been characterized in humans and it is unknown whether NaPi-IIb is also upregulated in chronic kidney disease (CKD). Method In this study, we examined the localization of NaPi-IIa, IIb, and IIc in kidneys of control healthy individuals (kidney donors) (eGFR: 111.6 ± 22.9 ml/min/1.73m2) and patients with diabetic nephropathy (eGFR: 31.9 ± 7.7 ml/min/1.73m2), anti-glomerular basement membrane nephritis (anti-GMB) (eGFR: 27.2 ± 22.6 ml/min/1.73m2), and hypertensive nephropathy (eGFR: 31.5 ± 5.3 ml/min/1.73m2) by immunofluorescence. Moreover, we analyzed data from various transcriptome data bases from healthy individuals and patients with CKD. Results In kidneys from healthy donors, NaPi-IIb staining was restricted to thin and thick limbs of the loop of Henle, while AQP1 was observed in proximal tubules and descending thin limbs. NaPi-IIc was abundantly found in proximal tubule cells. In biopsies from patients, NaPi-IIb signal was also found in addition to thin and thick limbs in cells of the collecting duct and in many proximal tubules. In contrast, the staining of NaPi-IIc was reduced in all groups of patients with CKD. RNA expression data from the European Renal cDNA Bank (ERCB) confirmed our immunofluorescence findings. SLC34A2 (NaPi-IIb) mRNA was progressively more expressed in kidneys of patients with kidney disease of tubulointerstitial origin from CKD stages 1 to 5, while it was strongly upregulated from stage 4 onwards in CKD of glomerular origin. Single cell RNA sequencing data of patients with acute kidney injury and CKD [2] showed elevation of SLC34A2 expression in injured thick ascending limb, degenerative proximal tubule cells (14x higher, adj. p≪0.0001) (de novo expression), and descending thin limb (3.7x higher, adj. p≪0.0001). SLC34A1 (NaPi-IIa) expression levels were reduced in whole kidneys (ERCB data) and SLC34A1 and SLC34A3 expression also decreased in proximal tubule cells of patients with CKD. Conclusion The phosphate transporter NaPi-IIb is highly upregulated in kidneys from patients with various etiologies of CKD and its expression spreads to proximal tubule and collecting duct. In contrast, the expression of the main physiological phosphate transporters NaPi-IIa and NaPi-IIc is reduced in CKD. These findings may have implications for the development and maintenance of hyperphosphatemia in CKD patients and limit the use of specific NaPi-IIa and/or NaPi-IIc inhibitors in patients with CKD.

#3022 REDUCED TUBULAR MIR-190A-5P: A NOVEL BIOMARKER FOR STRATIFICATION OF PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Circulating microRNA (miRNAs) have been proposed as potential diagnostic and prognostic biomarkers and are functionally important in disease pathogenesis. In this study, we used an unbiased measurement of circulating miRNAs in patients with type 2 diabetes to identify a miRNA differentially expressed in kidney disease before moving to a larger, unselected cohort of patients with CKD to assess its potential to predict progression in CKD. Method MiRNA next generation sequencing (NGS) studies were undertaken to measure differential expression of plasma miRNAs in a discovery cohort of 3 groups; individuals with type 2 diabetic kidney disease (T2DKD, n = 9), age and sex matched patients with type 2 diabetes mellitus and normal renal function (T2DNRF, n = 13) and patients without diabetes and with normal renal function (NDNRF, n = 11). Differentially expressed miRs were validated in a separate cohort of the same groups (each n = 10). The prognostic value of miR-190a-5p in a general CKD population was assessed using the seNSOR cohort (n = 395, excluding patients on RRT, with an eGFR of < = 20 ml/min/1.73 m2 or AKI at recruitment). The primary outcome of CKD progression was defined as reaching ESKD (starting RRT or maintaining an eGFR <15mls/min) or >30% reduction in renal function from eGFR at baseline. Reaching ESKD alone was used as a secondary outcome. Results MiR-190a-5p was the only miRNA differentially expressed between T2DKD and both control groups in both the discovery and validation cohorts. In the seNSOR cohort, miR-190a-5p levels correlated positively with eGFR (rho = 0.12, p = 0.04) and inversely with age (rho = -0.12, p = 0.04). MiR-190a-5p levels below the median predicted CKD progression in individuals with minimal and moderate albuminuria (ACR < 3mg/mmol and 3-300mg/mmol respectively) but not in those with severe albuminuria (ACR>300mg/mmol, see figure). In those without severe albuminuria, miR-190a-5p levels predicted CKD progression in multivariate Cox proportional hazards models (HR 0.8, 95% CI: 0.66-0.96, p = 0.015), independently of baseline eGFR, ACR, age, SBP, DBP and sex. When participants with an ACR > 300mg/mmol were included, miR-190a-5p was not predictive of the composite CKD progression endpoint (multivariate HR 0.86, p = 0.064) but was predictive for reaching ESKD alone (multivariate HR 0.68, 95% CI: 0.5-0.93, p = 0.015). Analysis of miR-190 expression in individual renal cell types in the reversible unilateral ureter obstruction mouse model revealed that it is enriched in proximal tubule cells and falls significantly following injury before increasing again the repair phase. Conclusion miR-190a-5p is expressed by healthy proximal renal tubular cells and serum miR-190a-5p levels correlate positively with eGFR. Low serum miR-190a-5p levels may predict progression of CKD in patients with low or moderate proteinuria independently of existing risk factors.

#4942 GENOTYPE-PHENOTYPE ANALYSIS IN FAMILIAL RENAL GLUCOSURIA PATIENTS: THE CRUCIAL ROLE OF CONSERVED RESIDUES

Abstract Background and Aims Familial renal glucosuria (FRG) is a hereditary disorder caused by mutations in SLC5A2, the gene encoding sodium-glucose cotransporter 2 (SGLT2) in proximal tubule cells. In this study, we aimed to characterize the genotype-phenotype relationship in FRG patients using data from our cohort and literature review. Method We sequenced SLC5A2 in a cohort of 21 FRG patients and measured the renal threshold of glucose (RTG) in 15 patients using urinary glucose excretion in a 4-hour oral glucose tolerance test (OGTT). We built an open-source online calculator to facilitate the calculation of RTG. Besides, we reviewed published literature and obtained information on SLC5A2 variants and 24-hour urinary glucose (24hUG) in FRG patients. Results We identified 27 rare SLC5A2 variants, including 13 novel variants (G484D, R564W, A212S, c.574+1G>C, W649*, S592Cfs*6, Q579*, A89T, Y339*, V39F, G491E, A464E, and G360D), in our cohort and yielded 107 SLC5A2 variants from literature review. RTG in our cohort ranged from 1.0 to 9.2mmol/L. Patients carrying two SLC5A2 variants had lower RTG (3.9 vs. 6.2 mmol/L, p = 0.057) and larger amounts of 24-hour urinary glucose excretion (24hUG) (52.39 vs. 7.20 g/1.73m2, p = 2.7*10−8) than those carrying a single variant. Patients with homozygous missense or in-frame indels had mean 24hUG of 82.30g/1.73m2, comparable to those with homozygous truncating variants (81.66g/1.73m2) and significantly more than those with homozygous splicing variants (35.38g/1.73m2, p = 0.026). Patients with homozygous missense variants involving conservative residues had more 24hUG (104.76g/1.73m2) than those with variants at non-conservative residues (46.37g/1.73m2, p = 0.0031). Conclusion We built a comprehensive map showing the impact of SLC5A2 variant type, variant location, and zygosity on glucosuria severity. Our results highlighted that conserved residues play an essential role in maintaining the transport function of SGLT2.

Chloroquine and Hydroxychloroquine, as Proteasome Inhibitors, Upregulate the Expression and Activity of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3), at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous widely used drugs. Earlier investigation from our laboratory revealed that ubiquitin conjugation to OAT3 leads to OAT3 internalization from the cell surface, followed by degradation in the proteasome. In the current study, we examined the roles of chloroquine (CQ) and hydroxychloroquine (HCQ), two well-known anti-malarial drugs, in their action as proteasome inhibitors and their effects on OAT3 ubiquitination, expression, and function. We showed that in cells treated with CQ and HCQ, the ubiquitinated OAT3 was considerably enhanced, which correlated well with a decrease in 20S proteasome activity. Furthermore, in CQ- and HCQ-treated cells, OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate, were significantly increased. Such increases in OAT3 expression and transport activity were accompanied by an increase in the maximum transport velocity and a decrease in the degradation rate of the transporter. In conclusion, this study unveiled a novel role of CQ and HCQ in enhancing OAT3 expression and transport activity by preventing the degradation of ubiquitinated OAT3 in proteasomes.

#4708 RENAL ORGANOID FROM URINE-DERIVED HUMAN ADULT RENAL PROGENITOR CELLS CAN SPONTANEOUSLY GENERATE LONG RENAL TUBULES

Abstract Background and Aims Organoids are self-organizing 3D aggregations of cells that represent the structure and function of organs. Kidney organoids have the potential to advance the field of nephrology by providing a tool for the study of human kidney development and disease, by providing a tool for in vitro drug screening, and ultimately, for regenerative therapy. They can be derived from embryonic stem cells or induced pluripotent stem cells. We aimed to explore for the first time, the potentiality of human adult renal progenitor cells (ARPCs), isolated from urine of healthy subjects and patients, to generate spheroids and organoids for regenerative purpose. Method ARPCs were isolated from urine by immunolabeling. Optical microscopy, immunofluorescence experiments and cytofluorimetric analysis were used to characterize spheroids and organoids. CD133, NANOG, SSA4, OCT 3-4 and GATA-3, SOX2 stemness marker antibodies were used to study spheroids and CD13, Lotus tetragonolobus lectin, ZO-1, uromodulin, and aminopeptidase antibodies were used to investigate renal tubular markers. PKH26 was used to study cell migration from organoids to tubules. Real-time PCR was used to generate gene expression data of aquaporin profile in renal tubules. Chick embryo chorioallantoic membrane (CAM) assays were used as in vivo model for angiogenesis. Results We isolated ARPCs from urine of healthy subjects or CKD patients. Usually, organoids were generated using complicate induction protocols with several chemical factors. However, for the first time we generated renal spheroids and organoids under 3D culture conditions without any stimulation, such as chemokines or growth factors, starting from ARPCs isolated from subject urine. We showed that the spheroids express high levels of stem cell markers as CD133 and NANOG, functional and constitutional marker of ARPCs, and high levels of stem cell markers that normally are low or not at all expressed in ARPCs and are typical of embryonic stem cells: GATA-3, SSEA4, and Sox2. Moreover, the spheroids were able to induce angiogenesis when implanted in chick embryo chorioallantoic membrane. The urine-derived CD133+ organoids can spontaneously generate long renal tubules in 6–12 days, as showed by PKH26 tagging. The renal tubules were positive for the proximal tubule cell markers CD13 (aminopeptidase N), Lotus tetragonolobus lectin and ZO-1. Moreover, renal tubules expressed the aminopeptidase A (CD249) that is normally present in proximal tubules and glomeruli of the kidneys and that catalyzed the conversion of the Angiotensin II in Angiotensin III witch is important in the local regulation of blood pressure. Also uromodulin was expressed by tubules. It is normally present in epithelial cells of the thick ascending limb of Henle's loop, and after proteolytical cleavage, is secreted into urine. In addition, the renal tubules express several genes typical of renal tubules as AQP1 channel protein that facilitates the flow of water molecules into the cells of the proximal tubule and descending limb of the loop of Henle; γGlut enzyme used for the transport of amino acids across cell membranes; Na/H exchanger 1 (SLC9A1), transporter involved in the regulation of pH homeostasis, cell migration and cell volume; Na/Gluc-1 co-transporter SGLUT1, which promotes the passage of glucose across the membrane in renal tubular cells. In some cases, tubule structures were similar to that found in nephrons, including proximal convoluted tubule, Loop of Henle, Distal Convoluted tubule and collecting duct, as shown by optical microscopy. Conclusion For the first time we demonstrated that kidney organoids can be generated starting by urine-derived human adult renal progenitor cells from patients and for the first time we showed that these organoids can generate, spontaneously, without specific stimulation, long portions of renal tubules. These results open new perspectives in the field of the regenerative medicine as ARPCs can be isolated from urine of patients and used to generate spheroids for the study of human kidney development and disease, for in vitro drug screening, and ultimately, for regenerative therapy.

#4234 ADAM17 MODULATES IFN-MEDIATED INFLAMMATION IN RENAL TUBULAR CELLS

Abstract Background and Aims In diabetic patients, hyperglycemia has been associated with a hypoxic state that contributes to an increased rate of morbidity due to Acute Kidney Failure (AKI). For this reason, we are interested in studying the molecular mechanisms involved in the development of AKI to contribute to the search for effective therapies that can help prevent these episodes. We focused on ADAM17, a disintegrin and metalloproteinase that sheds the ectodomains of cell surface proteins, among which inflammatory cytokines stand out. In an animal model, we showed that the absence of ADAM17 in the renal tubule reduces protein expression related to fibrosis and inflammation induced by diabetes. In this context, we aimed to study the role of ADAM17 in modifying the expression of inflammatory molecules regulated by interferon (IFN) in renal proximal tubule cells subjected to hyperglycemic stress. In this study, we analyzed the effect of ADAM17 deletion in human renal tubular cells (HKC-8) incubated in high-glucose medium and subjected to hypoxia. Method ADAM17 was depleted using CRISPR/Cas9 (ADAM17-KO). Cells were incubated for 6 h or 24 h with high (35 mM HG) or normal (5.5 mM NG) glucose concentrations and were subjected to hypoxia plus 2h re-oxygenation (HIPX). Gene expression of several IFN-regulated inflammatory markers (IL6, IL8, CXCL10, ISG15, IL1a, IL1b, IL12A, CCL5, and CCL2) was determined in wild-type (WT) and ADAM17-KO cells. Results The absence of ADAM17 in HKC-8 cells induced changes in the mRNA expression of genes controlled by IFN, which is accentuated by the hyperglycemic stimulus. Under hypoxic and re-oxygenation conditions, these changes were in the same line, with more discrete values compared to control cells. TNFa, CXCL10 and LNC2 were the most relevant genes reduced in injury conditions in the ADAM17-KO cell line. Conclusion ADAM17 deletion modulates the expression of most IFN-regulated genes that could undergo post-transcriptional changes and subsequently act as an acute mechanism of protection from injury. The results show that ADAM17 plays a major role in the underlying inflammatory response to acute kidney injury, indicating its potential as a therapeutic target. The analysis of circulating proteins will provide information about this process to better understand the mechanism of action of ADAM17 in the protection of renal tubular damage.

#4158 URINE DERIVED STEM CELL AMELIORATES RENAL FIBROSIS VIA KLOTHO ACTIVATION

Abstract Background and Aims After renal IRI, regeneration and recovery of the renal tubular cell occurs. However, if the renal repair process is maladaptive, it progresses to renal fibrosis. The role of stem cells in kidney regeneration or fibrosis has not been fully elucidated. we evaluated the urine drived stem cells (UDSC) for renal inflammation and fibrosis after renal ischemia reperfusion (IR). Method 10 week old balb/c nude male mice were used. sham, sham with UDSC, IR, IR with UDSC. UDSC were infused 3 times via tail vain at 6, 7, 8th day after renal IR. Urine NGAL/creatinine (Cr) were checked. The kidneys tissue were harvested at day 14 day. In vitro, TGF-β treated HK2 cell were co-cultured with UDSC. Klotho siRNA silencing was performed in UDSC. Results Urinary NGAL/Cr were significantly increased in IR mice after 14 day IR, compared to sham mice. Urinary NGAL/Cr significantly decreased in UDSC treated IR mice, compared to IR mice. In H&E stain, renal tubulo-interstitial injury were significantly decreased in UDSC treated IR mice, compared to IR mice. In masson trichrom stain, renal fibrosis area were were significantly decreased in UDSC treated IR mice, compared to IR mice. The renal expression of TGF-β, α-SMA, and collagne IV were significantly decreased in UDSC treated IR mice, compared to IR mice. The renal expression of Klotho were increased in UDSC treated IR mice, compared to IR mice. in vitro, UDSCs were stem cells that expressed Klotho protein more strongly than other mesenchymal stem cells (MSCs). UDSCs also suppressed fibrosis by inhibiting transforming growth factor (TGF)-β in HK-2 human renal proximal tubule cells in an in vitro model. Klotho siRNA silencing reduced the TGF-β-inhibiting ability of UDSCs. Conclusion UDSC attenuate renal fibrosis after renal IR. Klotho-secretion of UDSC play a role in these anti- fibrotic effects.

#6014 SINGLE-CELL SPATIAL TRANSCRIPTOMICS OF THE KIDNEY IN HEALTH AND DISEASE DEFINES INJURY-SPECIFIC DOMAINS AND IDENTIFIES NOVEL CELL-CELL INTERACTIONS

Abstract Background and Aims Single-cell sequencing has revealed an unexpected diversity of cell types throughout the body. However, the loss of spatial context in many single-cell sequencing techniques hampers our understanding of cell-cell interactions, which are central to almost all (patho-)physiological processes, particularly in highly complex organs such as the kidney. In this study, we aim to profile the spatial organization of, and cell-cell interactions within, the healthy and diseased mouse kidney with single-cell resolution. We focus on changes induced by acute kidney injury (AKI), since AKI is highly prevalent and can progress to chronic kidney disease (CKD), with no targeted treatment strategy to prevent this AKI-to-CKD transition existing to date. Method To model ischemic AKI in the mouse, we performed bilateral ischemia-reperfusion injury on C57BL/6J mice. Kidneys were collected in the transition phase from AKI to CKD at 4 weeks after AKI (n = 3). Kidneys from non-surgery mice were used as controls (n = 3). To characterize the spatial complexity of the kidney with single-cell resolution we used seqFISH+, a sequential fluorescence in situ hybridization approach that allows the multiplexing of thousands of genes by sequential hybridization and confocal imaging, thus enabling RNA-quantification at the single-transcript level. Results Quantifying and clustering 1300 genes in 230,422 cells identified all major cell types of the kidney and revealed changes in the kidneys’ cellular composition at 4 weeks post AKI: While the cortical vasculature and cells of the proximal tubule segment 3 were reduced, the abundance of injured proximal tubule cells (identified by Havcr1 and Vcam1 expression), immune cells and fibroblasts increased. Clustering the identified cell types on their neighbors within a 30um radius revealed distinct spatial domains comprising different combinations of cell types. Certain domains were consistently present across samples and corresponded to the known regional organization of the kidney, thus providing an internal validation. Other domains developed de novo after AKI, illustrating the structural changes induced by AKI. One of these injury-specific domains was comprised of injured proximal tubule cells, macrophages and fibroblasts, while another mainly comprised macrophages, dendritic cells and T cells. Ligand-receptor analysis within domains revealed novel cell-cell interactions, for example highlighting a Crlf1-expressing fibroblast population in close proximity to injured proximal tubule cells, which express the interleukin-6 cytokine Clcf1, a usually co-secreted binding partner of Crlf1. Zooming in on one injury-specific domain, we found that the fraction of fibroblasts and macrophages in the vicinity of injured proximal tubule cells increases with the degree of injury-related gene expression in these cells, suggesting a causal role of injured proximal tubule cells in defining a “pathogenic niche” associated with kidney disease progression. Conclusion This study provides a spatial characterization of the kidney with unprecedented resolution, highlights AKI-induced structural changes in the kidney, defines injury-specific spatial domains and reveals cell-cell-interactions relevant to disease progression from AKI to CKD.

Intrinsic TGF-β signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease

Excessive TGF-β signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-β failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-β signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-β signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-β receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-β signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression.

Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT1 (AT1a) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways.

The current prevailing paradigm in the renin-angiotensin system dictates that most, if not all, biological, physiological, and pathological responses to its most potent peptide, angiotensin II (Ang II), are mediated by extracellular Ang II activating its cell surface receptors. Whether intracellular (or intracrine) Ang II and its receptors are involved remains incompletely understood. The present study tested the hypothesis that extracellular Ang II is taken up by the proximal tubules of the kidney by an AT1 (AT1a) receptor-dependent mechanism and that overexpression of an intracellular Ang II fusion protein (ECFP/Ang II) in mouse proximal tubule cells (mPTC) stimulates the expression of Na+/H+ exchanger 3 (NHE3), Na+/HCO3- cotransporter, and sodium and glucose cotransporter 2 (Sglt2) by AT1a/MAPK/ERK1/2/NF-kB signaling pathways. mPCT cells derived from male wild-type and type 1a Ang II receptor-deficient mice (Agtr1a-/-) were transfected with an intracellular enhanced cyan fluorescent protein-tagged Ang II fusion protein, ECFP/Ang II, and treated without or with AT1 receptor blocker losartan, AT2 receptor blocker PD123319, MEK1/MEK2 inhibitor U0126, NF-кB inhibitor RO 106-9920, or p38 MAP kinase inhibitor SB202196, respectively. In wild-type mPCT cells, the expression of ECFP/Ang II significantly increased NHE3, Na+/HCO3-, and Sglt2 expression (p < 0.01). These responses were accompanied by >3-fold increases in the expression of phospho-ERK1/2 and the p65 subunit of NF-кB (p < 0.01). Losartan, U0126, or RO 106-9920 all significantly attenuated ECFP/Ang II-induced NHE3 and Na+/HCO3- expression (p < 0.01). Deletion of AT1 (AT1a) receptors in mPCT cells attenuated ECFP/Ang II-induced NHE3 and Na+/HCO3- expression (p < 0.01). Interestingly, the AT2 receptor blocker PD123319 also attenuated ECFP/Ang II-induced NHE3 and Na+/HCO3- expression (p < 0.01). These results suggest that, similar to extracellular Ang II, intracellular Ang II may also play an important role in Ang II receptor-mediated proximal tubule NHE3, Na+/HCO3-, and Sglt2 expression by activation of AT1a/MAPK/ERK1/2/NF-kB signaling pathways.

Chronic Kidney Disease Induced by Cadmium and Diabetes: A Quantitative Case-Control Study.

Kidney disease associated with chronic cadmium (Cd) exposure is primarily due to proximal tubule cell damage. This results in a sustained decline in glomerular filtration rate (GFR) and tubular proteinuria. Similarly, diabetic kidney disease (DKD) is marked by albuminuria and a declining GFR and both may eventually lead to kidney failure. The progression to kidney disease in diabetics exposed to Cd has rarely been reported. Herein, we assessed Cd exposure and the severity of tubular proteinuria and albuminuria in 88 diabetics and 88 controls, matched by age, gender and locality. The overall mean blood and Cd excretion normalized to creatinine clearance (Ccr) as ECd/Ccr were 0.59 µg/L and 0.0084 µg/L filtrate (0.96 µg/g creatinine), respectively. Tubular dysfunction, assessed by β2-microglobulin excretion rate normalized to Ccr(Eβ2M/Ccr) was associated with both diabetes and Cd exposure. Doubling of Cd body burden, hypertension and a reduced estimated GFR (eGFR) increased the risks for a severe tubular dysfunction by 1.3-fold, 2.6-fold, and 84-fold, respectively. Albuminuria did not show a significant association with ECd/Ccr, but hypertension and eGFR did. Hypertension and a reduced eGFR were associated with a 3-fold and 4-fold increases in risk of albuminuria. These findings suggest that even low levels of Cd exposure exacerbate progression of kidney disease in diabetics.

PCK1 Protects against Mitoribosomal Defects in Diabetic Nephropathy in Mouse Models.

Renal gluconeogenesis plays an important role in the pathogenesis of diabetic nephropathy (DN). Proximal tubular phosphoenolpyruvate carboxykinase1 (PEPCK1) is the rate-limiting enzyme in gluconeogenesis. However, the functions of PEPCK1 have not been elucidated. We describe the novel role of PEPCK1 as a mitoribosomal protector using Pck1 transgenic (TG) mice and knockout mice. Pck1 blocks excessive glycolysis by suppressing the upregulation of excess HK2 (the rate-limiting enzyme of glycolysis). Notably, Pck1 overexpression retains mitoribosomal function and suppresses renal fibrosis. The renal and mitoribosomal protective roles of Pck1 may provide important clues for understanding DN pathogenesis and provide novel therapeutic targets. Phosphoenolpyruvate carboxykinase (PEPCK) is part of the gluconeogenesis pathway, which maintains fasting glucose levels and affects renal physiology. PEPCK consists of two isoforms-PEPCK1 and PEPCK2-that the Pck1 and Pck2 genes encode. Gluconeogenesis increases in diabetic nephropathy (DN), escalating fasting and postprandial glucose levels. Sodium-glucose cotransporter-2 inhibitors increase hepatic and renal gluconeogenesis. We used genetically modified mice to investigate whether renal gluconeogenesis and Pck1 activity are renoprotective in DN. We investigated the expression of Pck1 in the proximal tubule (PTs) of streptozotocin (STZ)-treated diabetic mice. We studied the phenotypic changes in PT-specific transgenic (TG) mice and PT-specific Pck1 conditional knockout (CKO) mice. The expression of Pck1 in PTs was downregulated in STZ-treated diabetic mice when they exhibited albuminuria. TG mice overexpressing Pck1 had improved albuminuria, concomitant with the mitigation of PT cell apoptosis and deposition of peritubular type IV collagen. Moreover, CKO mice exhibited PT cell apoptosis and type IV collagen deposition, findings also observed in STZ-treated mice. Renal fibrotic changes in CKO mice were associated with increasing defects in mitochondrial ribosomes (mitoribosomes). The TG mice were protected against STZ-induced mitoribosomal defects. PCK1 preserves mitoribosomal function and may play a novel protective role in DN.

Role of Coactivator Associated Arginine Methyltransferase 1 (CARM1) in the Regulation of the Biological Function of 1,25-Dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, activates the nuclear vitamin D receptor (VDR) to mediate the transcription of target genes involved in calcium homeostasis as well as in non-classical 1,25(OH)2D3 actions. In this study, CARM1, an arginine methyltransferase, was found to mediate coactivator synergy in the presence of GRIP1 (a primary coactivator) and to cooperate with G9a, a lysine methyltransferase, in 1,25(OH)2D3 induced transcription of Cyp24a1 (the gene involved in the metabolic inactivation of 1,25(OH)2D3). In mouse proximal renal tubule (MPCT) cells and in mouse kidney, chromatin immunoprecipitation analysis demonstrated that dimethylation of histone H3 at arginine 17, which is mediated by CARM1, occurs at Cyp24a1 vitamin D response elements in a 1,25(OH)2D3 dependent manner. Treatment with TBBD, an inhibitor of CARM1, repressed 1,25(OH)2D3 induced Cyp24a1 expression in MPCT cells, further suggesting that CARM1 is a significant coactivator of 1,25(OH)2D3 induction of renal Cyp24a1 expression. CARM1 was found to act as a repressor of second messenger-mediated induction of the transcription of CYP27B1 (involved in the synthesis of 1,25(OH)2D3), supporting the role of CARM1 as a dual function coregulator. Our findings indicate a key role for CARM1 in the regulation of the biological function of 1,25(OH)2D3.

Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis.

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.