Renal Cortex Research Articles

Sex-Based Differences in Proximal Tubule Endocytic Capacity in Mice

Women are more protected from acute kidney injury, and chronic kidney disease (CKD) in women progresses more slowly to end-stage renal disease compared to men. CKD involves changes in the integrity of the glomerular barrier function with consequent proteinuria. The kidney proximal tubule (PT) normally reclaims albumin and other filtered proteins via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. PT health, and ultimately kidney function, is impacted by the uptake of excess albumin and by nephrotoxic drugs that enter cells in a megalin/cubilin dependent manner. Recent transcriptomic studies in mice have shown differences in the expression of megalin and cubilin between males and females and across the S1, S2, and S3 sub-segments that comprise the PT. Additionally, reduced reabsorption of water and salt by the PT has been demonstrated in female rodents. These differences in PT receptor expression and functionality likely contribute to sex-based differences in PT endocytic capacity. To determine if there are sex-based differences in the recovery of filtered proteins by the PT, urine and kidney tissue were collected from five male and five female 13-week-old C57BL/6 mice. Urinary albumin was quantified by ELISA. Kidneys were processed for Western blotting and immunofluorescence staining. We used imaging-based approaches to quantify the expression and distribution of albumin, megalin, and cubilin in SGLT2-positive S1 and SGLT2-negative, OAT1-positive S2 segments. Under normal conditions, S1 had greater intensity of subapical albumin staining in both males and females. However, females had greater intensity of albumin staining in S2 normalized to S1 compared to males. Female mice had lower levels of urinary albumin excretion when normalized to creatinine than males. Female mice had greater expression of total megalin (1.5x) and cubilin (1.4x) and decreased expression of NHE3 (0.7x) in the kidney cortex. Male and female mice had similar levels of megalin expression and surface localization in S1, however female mice had greater expression and surface localization of megalin in S2. Males had greater colocalization of megalin with lysosomal marker, LAMP1. In both males and females, the colocalization of megalin with LAMP1 decreased from S1 to S2. Together, these data suggest that, at baseline in female mice, S2 has greater endocytic capacity and contributes more to the uptake of normally filtered albumin than in males. This increased capacity could explain the reduced urinary excretion of albumin observed in rodent injury models and in women with CKD. Current studies are focused on incorporating these sex-based differences into a multiscale mathematical model of protein uptake along the length of the PT. National Institutes of Health: T32 DK007052, F31 DK121394, R01 DK125049, R01 DK118726, R01 DK083785, S10 OD021627, S10 OD028596, U54 DK137329, ASN Foundation for Kidney Research Pre-Doctoral Fellowship Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Male Offspring Exposed to Maternal Hyperandrogenemia Have Exaggerated Pressor Response to Angiotensin II That Is Partially Attenuated by Etanercept

Background: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy and major cause of hyperandrogenemia in women. Our group has previously shown that male offspring (F1) exposed to maternal hyperandrogenemia are born smaller (indicative of intra-uterine growth restriction) and have exaggerated pressor response to angiotensin (Ang) II as adults. Tumor necrosis factor-α (TNF-⍺) has been shown to play a role in Ang II-mediated hypertension in different models. Increased pressor response to Ang II could signal increased risk of cardiovascular diseases later in life. Therefore, the present study was designed to test the hypothesis that exposure to maternal hyperandrogenemia programs intrarenal inflammation that plays a role in the exaggerated pressor response to Ang II in male F1s. Methods: At 4 weeks (wks) of age, female Sprague Dawley (SD) rats were fed a purified diet (10% kcal from fat) and implanted (s.c.) with either 5α-dihydrotestosterone pellets (7.5 mg/90 d; hyperandrogenemic females (HAFs)) or placebo pellets (PBO), that were replaced throughout life. At 9-12 wks of age, HAFs and PBOs were paired with SD males and allowed to get pregnant, deliver and lactate their offspring (F1-HAF and F1-PBO) until weaning (3 wks of age). Urine was collected from male F1s (n = 6-10/grp, 4-5 months (mos) of age, 1 pup/litter) which were then euthanized. Kidney cortices were used for the assay of pro-inflammatory cytokines using Bioplex. Another batch of male F1s (n = 4-7/grp, 8-10 mos of age, 1 pup/litter) were implanted with radiotelemetry transmitters. After recovery, male F1s were treated with either etanercept (a TNF-⍺ antagonist; ETA; 1.2 mg/kg, twice a week s.c. injection; ETA-F1-HAF and ETA-F1-PBO grps) or vehicle (0.9% saline; VEH; VEH-F1-HAF and VEH-F1-PBO grps) for 2 wks. Mean arterial pressure (MAP) was then measured for 6 days (Pre-enalapril), followed by treatment with enalapril (25 mg/kg/d for 9 d in drinking water) then Enalapril + Ang II (150 ng/kg/min for 6 d, s.c. minipump). Results: Male F1-HAF had significantly higher renal cortical interleukin (IL)-1α, IL-1β, IL-7 and TNF-α (203.8 ± 14 vs 141.7 ± 10, 331.4 ± 27 vs 233.7 ± 13, 524.7 ± 41 vs 379.1 ± 46, and 579.8 ± 47 vs 423.7 ± 47 pg/g tissue, respectively; p<0.05 for all), proteinuria (14.1 ± 2.4 vs 8.6 ± 0.9 mg/24 h, p<0.05), urinary kidney injury molecule-1 (1058.0 ± 123 vs 557.0 ± 122 pg/24 h, p<0.05) compared to male F1-PBO. VEH-F1-HAF had exaggerated pressor response to Ang II compared to VEH-F1-PBO starting at day 3 until day 6 of Ang II (154 ± 6 vs 128 ± 6 mmHg, p<0.05) despite similar MAP pre-enalapril (116 ± 3 vs 118 ± 2 mmHg, p=NS) and after enalapril (97 ± 3 vs 96 ± 1 mmHg, p=NS). ETA-F1-HAF had significantly lower MAP compared to VEH-F1-HAF on days 3 (119 ± 10 vs 138 ± 3 mmHg, p<0.05), 4 (131 ± 9 vs 149 ± 5 mmHg, p<0.05) and 5 (142 ± 6 vs 154 ± 5 mmHg, p<0.05) of Ang II treatment. Yet, on day 6 of Ang II treatment, MAP was similar between VEH-F1-HAF and ETA-F1-HAF (154 ± 6 vs 146 ± 5 mmHg, p=NS) and significantly higher in both groups compared to ETA-F1-PBO (133 ± 12 mmHg) and VEH-F1-PBO (128 ± 6 mmHg) [p < 0.05]. Conclusion: Exaggerated pressor response to Ang II in male F1s exposed to maternal hyperandrogenemia could be partly mediated by the pro-inflammatory cytokine TNF-α. Future studies will determine the role of different immune cells in this response and the underlying epigenetic changes in male F1s exposed to maternal hyperandrogenemia. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM121334 (JFR, NMS), R01HL135089 (JFR), R01AG075963 (JFR). The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This work was also supported by the American Heart Association Career Development Award Number 938320 (NMS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bilateral renal dysplasia in a ferret (Mustela putorius furo)

AbstractRenal dysplasia is a histological diagnosis characterised by disorganised architecture of the renal cortex and medulla with disrupted nephron differentiation and primitive ducts surrounded by collarettes of the mesenchymal tissue. This condition has been reported once previously in ferrets as a component of multiple congenital abnormalities of the urogenital tract. A 1.5‐year‐old, male, neutered ferret was presented after being found minimally responsive under a fallen shelf in its enclosure. The ferret was obtunded, with mild intermittent muscle tremors and severe dehydration. Survey radiographs, including thoracic and abdominal ventrodorsal and right and left lateral views, were performed to assess for general trauma. The right renal silhouette was normal in size, and the left was moderately reduced. The capsular margins of both renal silhouettes were moderately undulating and mildly indistinct with perirenal retroperitoneal striations. No signs of trauma or other abnormalities were identified. Whole blood biochemical analysis demonstrated elevated creatinine, elevated blood urea nitrogen, hypernatraemia, hypercalcaemia, hyperphosphataemia and hyperproteinemia characterised by hyperalbuminemia and haemoconcentration. The hypernatraemia and mentation failed to improve with intravenous fluid therapy, and the ferret was euthanased. Bilateral renal dysplasia was confirmed via gross postmortem examination and histopathology.

Volume microscopic analysis of membrane contact sites in mouse kidney renal proximal tubule epithelial cells

Subcellular organelles communicate with one another and with the plasma membrane (PM). A wide variety of membrane contact sites (MCS) tether subcellular structures to one another and mediate inter-organelle communication and exchange. The nature, distribution and function of membrane contact sites within the segment-specific architecture of renal epithelial cells has only just begun to be elucidated. We have used advanced imaging techniques, including Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and machine learning-based segmentation, to create a comprehensive, high resolution, 3D and quantifiable volume microscopic atlas of the ultrastructure of proximal tubule epithelial cells of the mouse kidney. We applied this data set to identify and quantify MCS between the endoplasmic reticulum and mitochondria and the PM in these cells. FIB-SEM was performed on a 50 μm by 50 μm by 44 μm block of tissue prepared from the renal cortex of a perfusion fixed 12-week-old male mouse. OsO4-stained tissue was embedded in Durcupan and a 3D X-ray tomogram of the block was used to select a volume of interest that included extended lengths of multiple proximal tubule (PT). The volume was imaged by a customized enhanced FIB-SEM system. The X-Y scanning resolution was set to 4-nm, and 4-nm FIB milling was used to expose surfaces for successive imaging. Generation of the complete image stack required ~11,000 cycles of scanning/FIB ablation over ~4 weeks of uninterrupted imaging. Segmentation of the resultant image set was performed with Zeiss Arivis Pro technology. The boundary box for reconstruction had the dimensions of 29.2 μm x 24.8 μm x 8 μm with a volume of 5793.280 μm3 comprising of 2000 consecutive images at an isotropic voxel size of 4 nm. Deep learning segmentation teased apart 10 cells with distinct boundaries in the PT structure. The volume of the cells in our clipped volume ranged from 8.79 μm3 to 1438.53 μm3 with a median of 45.20 μm3. In one representative proximal tubule cell the volume of mitochondria makes up 84.91 μm3 (31.62%) of the cell volume and the volume of Endoplasmic Reticulum (ER) makes up 23.92 μm3 (8.91%) of the cell volume. The 3D reconstruction and quantitative analysis revealed that there is one dominant ER object accounting for most of the ER volume in the whole cell: i.e., the largest ER object takes up 22.37 μm3 or 93.5% of the ER volume in the cell. A post deep-learning processing algorithm used 3D object modifications to identify 11,109 3D MCS between mitochondria and ER in the 10 cells. The Mito-ER MCS ranged from 512 nm3 to 354,304 nm3 with a median volume of 1792 nm3. Visually we can see Mito-ER and Mito-PM MCS extending across a region of up to ~45 nm between the two organelles. Taken together, these data show that the basal lateral surfaces of PT cell PMs are generously invested with fenestrated smooth ER, as are mitochondria and peroxisomes. Extensive regions of direct contact between mitochondria and PM are also observed. The biochemical identities of these contacts and their physiological functions are being actively pursued. NIDDK DK072612 NIDDK DK120534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of ERK and NF-KB pathway in neurally mediated enhanced expression and traffcking of SGLT2 in congestive heart failure

Congestive heart failure (CHF) is characterized by the activation of neurohumoral systems concomitant with avid fluid retention. Recently we showed enhanced expression of sodium-glucose cotransporter 2 (SGLT2) in rats with CHF. However, the exact molecular mechanism linking enhanced sympathetic tone and enhanced expression and translocation of SGLT2 in CHF is not clear. The objective of this study was to investigate the role of enhanced sympathetic tone on the expression and traffcking of SGLT2 via activation of protein kinase A (PKA), extracellular signal-regulated kinases (ERK), and nuclear factor kappa B (NF-KB) in the renal cortex of CHF rats. Male Sprague Dawley rats were subjected to either the left coronary artery ligation for the CHF group or Sham surgery. Four weeks after surgery, expression of protein levels (PKA, ERK and NF-KB) were assessed by Western blotting, whereas the location of SGLT2 expression was determined by immunohistological and immunofluorescent staining in the cortical region of kidneys from Sham and CHF rats. Effect of exogenous norepinephrine (NE) (10μM for 24hrs) on expression and traffcking of SGLT2, phosphorylation of ERK and NF-KB in HK2 cells were examined in an in vitro model. Expression of SGLT2 was significantly enhanced in the renal cortex of rats with CHF versus Sham (0.83±0.06 vs. 0.58±0.02). Also, the translocation of SGLT2 was significantly enhanced towards the membrane in CHF rats (0.92±0.11 vs. 0.33±0.07). Phosphorylation of PKA (0.05±0.01 vs. 0.01±0.01), ERK (0.32±0.02 vs. 0.23±0.02) and NF-KB (0.71±0.14 vs. 0.08±0.06) were significantly enhanced in CHF compared to Sham. NE treatment significantly increased expression of SGLT2 (0.26±0.03 vs. 0.15±0.02), traffcking of SGLT2 (0.45 ± 0.09 vs. 0.15 ± 0.03) towards membrane, phosphorylation of ERK (0.38±0.05 vs. 0.14±0.02) and NF-KB (0.61±0.08 vs. 0.17±0.07) in HK2 cells. Thus, NE treatment enhanced the phosphorylation of ERK, NF-kB as well as traffcking of SGLT2. Immunohistological and immunofluorescent staining demonstrated increased expression of SGLT2 in the kidneys of rats with CHF. Similar results were observed in immunofluorescent staining of human kidney cells treated with NE. In conclusion, these findings indicate that NE activates ERK and NF-KB pathways which leads to enhanced traffcking and overexpression of SGLT2 in CHF. These results provide a molecular link between enhanced sympathetic tone, ERK and NF-KB pathway and the traffcking of SGLT2 to the luminal membrane of renal proximal tubular cells and consequent increase in sodium retention in rats with CHF. This study was funded by NIH R01-DK-129311. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pathophysiology of Carnitine Palmitoyltransferase 2 (CPT2) Deficiency in the Dahl Salt-sensitive R at

Carnitine palmitoyltransferase 2 (CPT2) is an essential enzyme of the carnitine shuttle, which facilitates fatty acid -oxidation in mitochondria. This study aims to characterize the various effects of CPT2 deficiency on the Dahl salt-sensitive (SS) rat metabolome and its concomitant influence on renal homeostasis. CRISPR/Cas9 was used to insert a single base pair into the SS Cpt2 gene, causing a frameshift mutation found to be lethal in homozygotes (SSCpt2−/−). Western blotting and qPCR analysis of renal cortex tissue confirmed a reduced expression of CPT2 in heterozygous (SSCpt2+/-) rats compared to wild-type (WT) littermates; therefore, male and female heterozygotes were used for all experiments. Lipid metabolomics of renal cortex samples collected from SSCpt2+/- animals at 8 weeks old on a baseline 0.4% NaCl diet revealed a significant upregulation in inflammatory lipids such as 11-HETE (p<0.01) and 12-HHT (p<0.05) when compared to WT littermates. Urine analysis from the SSCpt2+/- group demonstrated a severe reduction in acetoacetic acid production (p<0.001), indicating ketogenesis is likely impacted by the loss of CPT2 in -oxidation. Additionally, TCA cycle metabolites fumaric acid (p<0.05) and oxaloacetic acid (p<0.01) are significantly downregulated in urine from SSCpt2+/- animals. Radio telemeters were implanted into the rats at 8 weeks of age to continuously record mean arterial pressure (MAP) and monitor the development of SS hypertension over the course of several dietary treatment protocols. Three weeks of either a 4% NaCl high-salt (HS) diet or a HS + ketogenic (HSK) diet with equivalent caloric content (4:1 caloric ratio, fat: carbohydrate) were administered to the animals. No difference in MAP elevation occurred between groups after the HS challenge alone, but SSCpt2+/- animals on the HSK diet had a significantly attenuated development of hypertension (p=0.03). Further metabolic analysis after the HSK diet illustrated that both groups had similar increases in long-chain fatty acids, liver enzymes, and cholesterol levels with a decrease in short-chain fatty acids. These experiments show that the partial loss of CPT2 in the Dahl SS rat leads to dysregulation across multiple metabolic pathways. Moreover, a ketogenic diet appears to protect heterozygous animals from the development of salt-sensitive hypertension. Funding: NIH R35 HL135749 and VA I01 BX004024. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Don’t lose your transporters! Optimizing renal transporter recovery after kidney freezing

Renal ion transporters are critical to maintain fluid and electrolyte homeostasis and are primary targets for (patho)physiology studies and drug discovery. Transporter abundance and covalent modifications are determined by immunoblots. Our standard protocol is quick kidney removal and homogenization in buffer containing 0.2 mM PMSF, 0.9 ug/ml aprotinin, and Sigma P0044 phosphatase inhibitors followed by isolation of a low speed supernatant (the homogenate). Collaborations with other labs requires shipping frozen kidneys. We tested whether flash freezing (FZ) Sprague Dawley (SD) kidneys impacts ion transporter recovery compared to quick removal without freezing (QR) defined as 100% recovery; assessed by immunoblot with specific antibodies. FZ and ~ 3 d storage before homogenization in inhibitors significantly reduced recoveries of proximal tubule (PT) NHE3 and megalin to 20% and Cldn2 to 40%. Medullary NHE3 and NKCC2 remained at 100%, DCT/CD proteins were reduced ~ 10-60% after FZ vs. QR. Renal perfusion pre-FZ further reduced recovery of all transporters vs. non-perfused kidneys. To test whether Fz activates proteases not inhibited in our standard buffer, Roche protease inhibitor (minitabs) were added to homogenization buffer. Adding minitabs to FZ kidneys increased NHE3 recovery to 85% and Cldn2 recovery to 100% of QR, NKCC2 and ENaC-beta to 85% and NCC to100%. Lastly, we tested, in male and female (M,F) SD rats, whether minitab addition to QR renal cortex homogenates followed by Fz prevents proteolysis vs. minitab addition to QR homog without Fz. In F, minitab addition with Fz preserved 100% recovery of transporters: NHE3, Cldn2, AQP1, megalin, NKCC2, NCC, ENaC-beta, Cldn-7 and AQP2. In M, minitab addition with Fz preserved 100% recovery of only AQP1, villin, Cldn7 and AQP2; all other M transporters exhibited ~40% less recovery. In conclusion, flash freezing kidney tissue differentially activates proteolysis of ion transporters - up to 80% in cortex and not at all in medulla - despite inclusion of standard inhibitors in homogenizing buffers. Strong sex differences are evident: addition of minitabs prevents proteolysis of F not M cortical ion transporters. Single cell ‘omics approaches may be useful to identify tubule and sex specific proteases. NIH: DK083785, DK134695. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physical and functional interaction between NHE3 and SGLT2 in the renal proximal tubule of male and female rats

Sodium-glucose cotransporter type 2 (SGLT2) and sodium/hydrogen exchanger-3 (NHE3) serve as the primary renal proximal tubule transporters responsible for the apical uptake of filtered glucose and NaHCO3/NaCl, respectively. Our previous studies demonstrated that chronic treatment with the selective SGLT2 inhibitor empagliflozin reduces NHE3 activity in the proximal tubule. Furthermore, we observed that the non-specific SGLT1/SGLT2 inhibitor, phlorizin, acutely inhibits NHE3 in the absence of luminal glucose, suggesting that the impact of SGLT inhibition on NHE3 may not be attributed to high tubular glucose concentrations. Additionally, our investigations revealed that SGLT2, but not SGLT1, colocalizes with NHE3 in the renal proximal tubule. This study aimed to gain insights into the functional and physical association of NHE3 and SGLT2 in the renal proximal tubule. In vivo microperfusion was conducted in male and female Wistar rats using glucose-free perfusates containing vehicle or empagliflozin (1 μM). Empagliflozin treatment significantly decreased the flow rate of bicarbonate reabsorption (JHCO3−) in both male (2.109±0.091 vs. 1.119±0.096 nmol/cm2∙s) and female rats (1.750±0.037 vs. 1.128±0.062 nmol/cm2∙s), indicating that SGLT2 inhibitors inhibit NHE3 activity through glucose-independent mechanisms. By employing nondenaturant detergents, we utilized blue native polyacrylamide gel electrophoresis (BN-PAGE) to preserve native protein-protein interactions. Double staining with antibodies against specific partner candidates revealed that bands attributed to NHE3, MAP17, PDZK1, and SGLT2 migrate similarly at ~480 kDa in samples from rat renal cortex, as supported by independent experiments. Rigorous controls were implemented to address concerns related to autofluorescence, antibody cross-reactivity, and signal specificity, confirming the validity of our results. Furthermore, complexes involving SGLT2 and NHE3 at an apparent ~480 kDa molecular weight were observed in male and female rats. Unlike SGLT2, SGLT1 bands in BN-PAGE did not overlap with the NHE3 signal, which is consistent with our earlier immunofluorescence experiments. Collectively, our data suggest that NHE3 and SGLT2 are functionally and physically associated with the renal proximal tubule. Future experiments will be undertaken to further characterize the physical association between NHE3 and SGLT2 and to determine whether the inhibition of NHE3 by SGLT2 inhibitors is mediated through the disruption of the NHE3-PDZK1-MAP17-SGLT2 multiprotein complex. Funding: São Paulo Research Foundation (FAPESP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Juxtaglomerular (JG) cell-specific deletion of the NADPH oxidase accessory protein, NOXA1, decreases pro-renin and proteinuria in vivo in a mouse model of diabetes

A large fraction of patients with diabetes develops diabetic nephropathy. Plasma pro-renin levels are elevated in diabetic patients and thought to contribute to hypertension, renal damage and inflammation. Studying the mechanisms by which diabetes reprograms JG cells to increase pro-renin release could provide new targets for kidney disease. We previously found that JG cells from diabetic mice had higher reactive oxygen species production (superoxide and H2O2), pro-renin expression and pro-renin and renin release. We hypothesized that increased ROS production by specific NADPH oxidases (NOXs) in JG cells is responsible for higher pro-renin release and contributes to glomerular damage in diabetes. Western Blots showed that the NADPH oxidase isoform NOX1 and NOXA1 are upregulated in JG cells from diabetic mice (C57=100%; Akita=147±10%; n=4; p<0.05), whereas NOX2 or NOX4 were not different. Confocal microscopy showed that NOX1 and NOXA1 are present in JG cells and co-localize with renin-containing granules (n=3). Silencing NOX1 in primary cultures of JG cells from diabetic mice decreased renin release to C57 control levels; (n=6; p<0.01) whereas silencing NOX4 had no effect (n=6). To study the role of NOXA1 and NOX1 in JG cells in vivo, we used Cas9 mediated silencing in kidneys of control mice. Adenoviruses expressing Cas9 under control of the Ren1 promoter (AV-Ren1-Cas9) and AAV-gRNA-NOXA1 were loaded into 28-day minipumps and delivered into the renal subcapsular space in C57 mice. After 5 weeks, we obtained control (non-transduced) and Ren-Cas9-NOXA1-edited kidneys and isolated JG cells. NOXA1 expression was reduced by 60±22% in isolated JG cells and pro-renin expression was decreased by 46±19% compared to non-transduced kidneys (n=3). We then specifically deleted NOXA1 from JG cells in diabetic mice by subcapsular delivery of AV-Ren1-Cre in the kidney cortex of transgenic NOXA1 floxed diabetic Akita mice. JG cell-specific deletion of NOXA1 in diabetic mice decreased urinary ROS (8-Isoprostane pg/24hrs: Akita=4091±307; NOXA1 JG/KO=3086 ±137; n=4; p<0.05), and glomerular damage as indicated by decrease proteinuria (24h Ualbumin Akita= 98.6± 9.4; NOXA1 JG/KO= 57.8± 16.4 μg/24h; n=4; p<0.05). We conclude that enhanced NOX1/NOXA1 induced ROS in JG cells drive enhanced pro-renin release from JG cells which contribute to glomerular damage in diabetes. Thus, NOXA1/NOX1 in JG cells are a potential new target for diabetic nephropathy. DK105300-RO3 Henry Ford Hospital Institutional Funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mammalian target of rapamycin mediates expression and activity of ADAM 17 in diabetic kidney disease

Purpose: Previous data have illustrated a role for the matrix metalloprotease A Disintegrin And Metalloprotease 17 (ADAM17), known to cleave growth factors and cytokines, in renal cell injury in diabetes. The goal of this study was to identify upstream regulators of ADAM17 in the cascade of events contributing to extracellular matrix accumulation in diabetic nephropathy. Rationale: Diabetic kidney disease is a serious complication faced by type 1 and type 2 diabetic patients alike. Albuminuria and extracellular matrix accumulation are prominent features of the disease and this accumulation of extracellular matrix is a contributing factor to renal fibrosis and decline in renal function. The mechanisms involved in the pathogenesis of diabetic kidney disease have not been completely identified. Methodology: Age and weight-matched Sprague Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). Type 1 diabetes was induced through tail vein injection of streptozotocin. After rats were determined to be diabetic by blood glucose measurement, rapamycin treatments were administered intraperitoneally for two months. Both kidneys were removed and frozen in liquid nitrogen for microscopy and experimental analyses or formalin fixed for morphometric imaging at the experimental endpoint. Kidney cortex homogenates were used for western blot analyses and enzymatic activity assays. Findings: Using the mTOR complex 1 inhibitor rapamycin, it was determined that increased ADAM17 enzymatic activity and ADAM17 protein expression is dependent on mTORC1 in streptozotocin-induced type 1 diabetic rats. Inhibition of mTORC1 with rapamycin abrogated the increase in collagen IV α 2 protein expression observed in diabetic rat cortex. Additionally, this study is the first to provide evidence that mTOR complex 1 activates ADAM17 contributing to extracellular matrix accumulation in diabetic nephropathy. Studies are continuing looking at the molecular mechanisms involved in mTOR activation of ADAM17 resulting in downstream effects in cultured human proximal tubular epithelial cells. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ceramides as Mediators of Mitochondrial Dysfunction Driving Kidney Injury

Objective. Perturbations in proximal tubule mitochondrial function and lipid metabolism potentiate acute kidney injury and failed repair. Sphingolipids such as ceramides are recognized as lipotoxic drivers of metabolic dysfunction and cell death. We hypothesized that ceramides drive mitochondrial dysfunction and tubular insult and that lowering ceramides would prevent acute kidney injury in a mouse model of bilateral ischemia reperfusion injury (IRI). Methods. We examined acute effects of ceramide treatment on primary mouse proximal tubule epithelial cell (PTEC) mitochondrial oxygen consumption and mitochondrial supercomplex assembly by Seahorse respirometry and blue native PAGE, respectively. In vivo, we profiled accumulation of ceramides via HPLC-MS/MS in kidney cortex tissue of mice challenged with warm bilateral IRI. We lowered whole-body ceramides via Degs1 inducible global knockout ( Degs1iGKO) in male C57Bl/6J mice prior to challenge with IRI. Clinical, transcriptional, and histopathological renal outcomes were compared to sham-operated and Degs1fl/fl controls 24 hours after kidney reperfusion. Results. Treatment of PTECs with 5-50uM C2-ceramide resulted in acute and dose-dependent decreases in mitochondrial ATP production rates (15-65% decrease from baseline, respectively, p<0.05), as well as disruption of electron transport chain supercomplex structures (i.e., III2+IV and I+III2+IV). In mice, IRI led to a 25% increase in total cortex ceramides (sham: 995±110 vs IRI: 1239±185 pmol lipid/mg protein, p<0.05). Lipid species within the de novo ceramide synthesis pathway were elevated as early as 5 minutes after IRI (e.g., 34% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 5 minutes: 3.20±0.81 pmol lipid/mg protein, p<0.05) and continued to accumulate throughout the progression of the AKI up to 24 hours after reperfusion (e.g., 122% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 24 hours: 5.29±1.71 pmol lipid/mg protein, p<0.001). Cortex ceramides, particularly deleterious long-chain (e.g., Cer(d18:1/16:0)) species, correlated positively with kidney injury biomarkers (plasma creatinine, Pearson r=0.57; BUN, r=0.74; and cortex expression of Lcn2, r=0.60 , and Havcr1, r=0.62 , p<0.05). Degs1 global knockout decreased cortex ceramide levels by 56% (fl/fl: 995±110 vs KO: 435±174 pmol lipid/mg protein, p<0.001). Degs1iGKO animals had improved cortex histopathology, blood urea nitrogen (fl/fl: 81.02±2.13 vs KO: 49.82±6.34 mg/dL, p <0.001), plasma creatinine (fl/fl: 1.28±0.4 vs KO: 0.32±0.11 mg/dL, p<0.001), and cortex Lcn2 expression (fl/fl: 36.36±21.50 vs KO: 5.31±4.61, p<0.01) following IRI. Conclusions. Renal ceramides are implicated as drivers of kidney injury and mitochondrial dysfunction. Global Degs1 knockout prevented AKI induced by ischemia reperfusion injury. Thus, ceramide-lowering interventions may be an effective strategy to prevent AKI. This work was supported by funding from the National Institutes of Health [R01KD130296 (SAS, WLH), R01DK131609 (SAS), U01CA272529 (SAS), R44DK116450 (SAS), R01DK112826 (WLH), T32DK091317 (RJN), F31DK134088 (RJN)]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl salt-sensitive rats

The essential role of inwardly rectifying K+ Channel Kir5.1 (encoded by the Kcnj16 gene) in renal salt handling and blood pressure control has been demonstrated in both human and animal models. However, the underlying mechanisms are still not fully understood. Here, we aimed to integrate transcriptomics and metabolomics to profile the comprehensive changes of genes and metabolites under the deletion of Kcnj16 in the Dahl salt-sensitive (SS) rat background to identify novel mechanisms. For the transcriptomics profiling, we performed RNA-sequencing on kidney cortex tissue of Kcnj16 knockout (KO) and wild-type (WT) SS rats (N=5 per group). For the metabolomics profiling, we performed untargeted metabolomics of kidney cortex tissue, plasma, and urine of KO (N=6) and WT (N=5) rats. Transcriptomics analysis revealed over 6,000 differentially expressed genes in KO rats compared to WT rats, with renin Ren (the gene encoding renin) being the most upregulated gene. Consistent with the phenotype observed in the KO rats, ingenuity pathway analysis (IPA) based on the transcriptomic profile predicted reduced blood pressure and kidney damage and increased ion transport. Canonical pathway analysis suggested activation of metabolic-related pathways [e.g., xenobiotic metabolism, tricarboxylic acid (TCA) cycle, phenylalanine degradation, etc.] and suppression of immune responses-related pathways [e.g., pathogen-induced cytokine storm signaling pathway, macrophage classical activation signaling pathway, nuclear factor of activated T cells (NFAT) pathway, etc.] in KO rats. Untargeted metabolomic analysis suggested distinct metabolic profiles of plasma, urine, and kidneys between WT and KO rats, with 219, 790, and 31 differentially expressed metabolites identified in plasma, urine, and kidney in KO rats compared to WT rats. Canonical pathway analysis identified spermine and spermidine degradation pathways in both plasma and urine and taurine biosynthesis pathways in both urine and kidney. Integration analysis based on transcriptomic and metabolomic profiles suggested an altered TCA cycle, amino acid, and reactive oxygen species metabolism. In conclusion, besides significantly altered ion transport, our transcriptomics and metabolomics data suggest significant suppressed immune responses-related and altered metabolic-related pathways in SS rats lacking Kir5.1, which provides new insights into the underlying mechanisms of the regulation of blood pressure and renal function by Kir5.1. This work is supported by National Institutes of Health Grants R35 HL135749 and R01 DK135644 (to A.S.), Department of Veteran Affairs grant I01 BX004024 (to A.S.), the American Physiological Society Postdoctoral Fellowship (to B.X.) and the American Society of Nephrology Dimitrios G. Oreopoulos Research Fellowship Award (to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of Renal Cortical PPAR-alpha and Downstream Targets by Sodium Glucose Cotransporter, Type 2 Inhibition (SGLT2i) in High-Fat Fed Mice

SGLT2i has been shown to be protective of renal function and health in type 2 diabetes by unclear means. Recently we reported that empagliflozin (EMPA) treatment of mice on a high-fat diet (HFD) led to marked changes in the renal cortical lipidome, including significant upregulation of many carnitine species (11 species > 35% increased, all p < 0.05). Carnitines are critical in the oxidation of long-chain fatty acids by increasing their uptake into the mitochondria. The mechanism underlying this upregulation was unclear, but the peroxisome proliferator activated receptor, subtype alpha (PPARα) has been demonstrated to upregulate carnitines in other tissues. To the test the impact of EMPA (E) or control (C) treatment on renal cortical PPARα and downstream targets, male (M) and female (F) C57Bl6 mice were treated with HFD (60% by weight) ± EMPA (100 mg/kg·diet) for 12 weeks. EMPA caused a reduction in final body weight (20%) only in females. No differences were observed in final kidney weights (n = 16-18/group). Glomerular filtration rate (GFR) measured by FITC-sinistrin clearance showed a trend (p = 0.052 two-way ANOVA) for an increase by EMPA (mean ± sem, ml/min, n = 5-9): MC- 246 ± 13; ME- 291 ± 5; FC- 252 ± 13; and FE- 275 ± 36. Western blotting of cortex homogenates revealed a modest, insignificant rise in PPARα, itself with EMPA, and a higher overall abundance in females (p = 0.024 for sex, mean band density, % MC): MC-100 ± 14; ME- 108 ±11; FC- 123± 12, FE- 151± 18. Fibroblast growth factor 21 (FGF21) is a reported target of PPARα, and has been shown to be increased in liver in response to treatment with a PPARα agonist. However, FGF21 is also increased compensatorily in metabolic stress conditions, e.g., obesity. We found EMPA treatment substantially reduced circulating FGF21 levels in M, while F levels were low regardless of treatment (ng/ml): MC-3.28 ± 1.32; ME- 0.07 ± 0.027; FC- 0.19 ± 0.08, FE- 0.17 ± 0.08 (p = 0.031, 0.044, and 0.033, for treatment, sex, and interaction, respectively). FGF21 levels in liver, site of most production (measured in males only) was likewise reduced by EMPA (84%, p = 0.029, Mann-Whitney test). In contrast, no significant differences were found for renal cortical FGF21 levels. Previously, we showed PPARα whole-body knockout mice had higher renal expression of Na+K+ATPase, which was associated with higher blood pressure. Western blotting of EMPA-treated mice showed the α-1 subunit of Na+K+ATPase, a surrogate marker of proximal tubule transport activity, was significantly increased in the kidney cortex of EMPA-treated mice (band density % MC): MC-100 ± 26; ME- 145 ± 25; FC- 99 ± 22, FE- 175 ± 21 (p = 0.54, 0.016, and 0.52, for treatment, sex, and interaction, respectively). This was not associated with higher blood pressure in EMPA-treated mice, as measured by tail-cuff. Taken together, these findings do not suggest an overall increase in activity of PPARα in kidney or systemically (in liver) with EMPA. The upregulation of carnitine species in kidney may have resulted from an alternative mechanism. Additional studies are warranted to gauge the role of PPARα activation, emanating from a relatively fasted state, with SGLT2 inhibition, in the kidney and in regard to whole body metabolism. Supported by GHUCCTS (Howard and Georgetown, UL1TR001409) pilot award to CE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

CD226 promotes renal fibrosis by regulating macrophage activation and migration.

It has been found that CD226 plays an important role in regulating macrophage function, but its expression and function in macrophages during renal fibrogenesis have not been studied. Our data demonstrated that CD226 expression in macrophages was obviously upregulated in the unilateral ureteral obstruction model, while CD226 deficiency attenuated collagen deposition in renal interstitium along with fewer M1 within renal cortex and renal medulla and a lower level of proinflammatory factors compared to that of control littermates. Further studies demonstrated that Cd226-/- bone marrow-derived macrophages transferring could significantly reduce the tubular injury, collagen deposition, and proinflammatory cytokine secretion compared with that of Cd226+/+ bone marrow-derived macrophages transferring in the unilateral ureteral obstruction model. Mechanistic investigations revealed that CD226 promoted proinflammatory M1 macrophage accumulation in the kidney via suppressing KLF4 expression in macrophages. Therefore, our results uncovered a pathogenic role of CD226 during the development of chronic kidney disease by promoting monocyte infiltration from peripheral blood into the kidney and enhancing macrophage activation toward the inflammatory phenotype by suppressing KLF4 expression.

Resting-state MRI reveals spontaneous physiological fluctuations in the kidney and tracks diabetic nephropathy in rats.

The kidneys maintain fluid-electrolyte balance and excrete waste in the presence of constant fluctuations in plasma volume and systemic blood pressure. The kidneys perform these functions to control capillary perfusion and glomerular filtration by modulating the mechanisms of autoregulation. An effect of these modulations are spontaneous, natural fluctuations in glomerular perfusion. Numerous other mechanisms can lead to fluctuations in perfusion and flow. The ability to monitor these spontaneous physiological fluctuations in vivo could facilitate the early detection of kidney disease. The goal of this work was to investigate the use of resting-state magnetic resonance imaging (rsMRI) to detect spontaneous physiological fluctuations in the kidney. We performed rsMRI of rat kidneys in vivo over 10 min, applying motion correction to resolve time series in each voxel. We observed spatially variable, spontaneous fluctuations in rsMRI signal between 0 and 0.3 Hz, in frequency bands associated with autoregulatory mechanisms. We further applied rsMRI to investigate changes in these fluctuations in a rat model of diabetic nephropathy. Spectral analysis was performed on time series of rsMRI signals in the kidney cortex and medulla. The power from spectra in specific frequency bands from the cortex correlated with severity of glomerular pathology caused by diabetic nephropathy. Finally, we investigated the feasibility of using rsMRI of the human kidney in two participants, observing the presence of similar, spatially variable fluctuations. This approach may enable a range of preclinical and clinical investigations of kidney function and facilitate the development of new therapies to improve outcomes in patients with kidney disease.NEW & NOTEWORTHY This work demonstrates the development and use of resting-state MRI to detect low-frequency, spontaneous physiological fluctuations in the kidney consistent with previously observed fluctuations in perfusion and potentially due to autoregulatory function. These fluctuations are detectable in rat and human kidneys, and the power of these fluctuations is affected by diabetic nephropathy in rats.

Three-dimensional morphometry of kidney in New Zealand rabbit using unbiased design-based stereology.

The rabbit is widely used as a laboratory animal in experimental models of kidney diseases. This species is also important from a veterinary perspective as a companion animal. Stereology has been accepted as an accurate approach to kidney morphometry. The objective of the present project was to provide normal quantitative stereological parameters for adult rabbit kidneys. The left kidneys of five adult male New Zealand rabbits were used. Isotropic sections were obtained using the orientation method. Total kidney volume was calculated by the Cavalieri principle. The volume fraction of the renal structures was estimated using the point counting system. The lengths of the proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) were calculated using counting frames. The total glomerular number was accounted for using the physical/fractionator technique. The mean glomerular volume was obtained by dividing the total volume of glomeruli by their total number. The total volume of rabbit kidneys calculated was 10.39 ± 1.98 cm3. The fractional volume of the kidney cortex and medulla accounted for 57.79 ± 0.65% and 42.2 ± 0.65%, respectively. The total glomerular volume was 2.18 ± 0.32% of the whole kidney. The total number of glomeruli in the rabbit kidney was estimated as 204.68 ± 12 × 103. The mean glomerular volume measured 1.07 ± 0.12 × 106 μm3. The total length of PCT and DCT was 2.96 ± 0.29 km and 1.38 ± 0.24 km, respectively. These findings can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals by comparing with available data on humans and other species. RESEARCH HIGHLIGHTS: Three-dimensional morphometry of adult rabbit kidney structures was analyzed using quantitative stereology. Total volume of kidney, fractional volume of cortex and medulla, length of renal tubules and number of nephrons were estimated. These three-dimensional morphometrical data can be used as a reference in experimental nephrology research and may help to expand the knowledge of nephrology in mammals.

Renal Lesions with Low-level Enhancement on Contrast-enhanced CT Promotes Early Detection of Drug-induced Kidney Injury in Patients Administered Anticancer Drugs.

Some patients with cancer-administered anti-cancer drugs may develop renal lesions with low-level enhancement on follow-up abdominal computed tomography (CT). To explore the clinical significance of renal lesions with low-level enhancement on CT after exposure to anti-cancer drugs. Medical records of patients with cancer who developed renal lesions on CT after exposure to anti-cancer drugs were retrospectively reviewed. Renal lesions were scored according to the extent of involvement, CT attenuation values of lesions and normal parenchyma were measured on precontrast CT and three phases of contrast-enhanced CT, and changes in serum creatinine (SCr) from one week before exposure to drugs to one week before and after the appearance of renal lesions were recorded. This study included 54 patients (86 lesions). Lesions were slightly lower density on pre-contrast CT, and less enhancing than normal renal parenchyma, especially in the delayed phase. Lesions were wedge-shaped, and involved the renal pyramid and associated renal cortex, as well as, were single or multiple, and occurred in the unilateral or bilateral kidneys. There were patchy and cord-like shadows of increased density in adjacent perirenal adipose tissue. During follow-up, lesions disappeared in 15 patients and persisted in 39 patients without significant progression. There were significant differences in renal lesions and normal renal parenchyma CT attenuation values in each phase of contrast-enhanced CT. Change in SCr level was significantly positively correlated with lesion score. Renal lesions with low-level enhancement on CT suggest early drug-induced kidney injury. These findings will inform clinical decision-making.

Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

UroAngel: a single-kidney function prediction system based on computed tomography urography using deep learning.

Accurate estimation of the glomerular filtration rate (GFR) is clinically crucial for determining the status of obstruction, developing treatment strategies, and predicting prognosis in obstructive nephropathy (ON). We aimed to develop a deep learning-based system, named UroAngel, for non-invasive and convenient prediction of single-kidney function level. We retrospectively collected computed tomography urography (CTU) images and emission computed tomography diagnostic reports of 520 ON patients. A 3D U-Net model was used to segment the renal parenchyma, and a logistic regression multi-classification model was used to predict renal function level. We compared the predictive performance of UroAngel with the Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations, and two expert radiologists in an additional 40 ON patients to validate clinical effectiveness. UroAngel based on 3D U-Net convolutional neural network could segment the renal cortex accurately, with a Dice similarity coefficient of 0.861. Using the segmented renal cortex to predict renal function stage had high performance with an accuracy of 0.918, outperforming MDRD and CKD-EPI and two radiologists. We proposed an automated 3D U-Net-based analysis system for direct prediction of single-kidney function stage from CTU images. UroAngel could accurately predict single-kidney function in ON patients, providing a novel, reliable, convenient, and non-invasive method.