PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation
- Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, China
Figures
Figure 1 with 1 supplement
PSTPIP2 expression was decreased in the aristolochic acid I (AAI)-induced acute aristolochic acid nephropathy model in vivo and injured mouse renal tubular epithelial cells (mRTECs) in vitro.
(A) Representative H&E-stained images of kidney sections. Kidneys were isolated on day 0, 1, 3, and 5 after AAI treatment (n = 6 per group). Scale bar, 50 μm. (B) Western blotting analysis of …
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Figure 1—source data 1
Data represented by each point in Figure 1B, C, E, and F.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig1-data1-v2.zip
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Figure 1—source data 2
Uncropped western blots for Figure 1B, E, and F.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig1-data2-v2.zip
Figure 1—figure supplement 1
Aristolochic acid I (AAI)-induced acute aristolochic acid nephropathy (AAN) in vivo model and mouse renal tubular epithelial cell (mRTEC) injury in vitro model.
(A) Establishment of AAI-induced acute AAN mouse model (n = 6 per group). (B) Serum creatinine (Cr) assay at various times after AAI treatment (n = 6). (C) Blood urea nitrogen (BUN) assay at various …
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Figure 1—figure supplement 1—source data 1
Data represented by each point in Figure 1—figure supplement 1B–G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Uncropped western blots for Figure 1—figure supplement 1E–G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig1-figsupp1-data2-v2.zip
Figure 2 with 1 supplement
Pstpip2-cKI attenuates aristolochic acid I (AAI)-induced kidney damage.
(A) Scheme illustrating the genetic approach for generating Pstpip2 conditional knock-in (cKI) mice. (B) Genotypes of Pstpip2-cKI mice were confirmed using PCR. The floxed allele showed a …
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Figure 2—source data 1
Data represented by each point in Figure 2D, E, G, H, I, and J.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig2-data1-v2.zip
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Figure 2—source data 2
Uncropped western blots and gel electrophoresis for Figure 2B, D, and I.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig2-data2-v2.zip
Figure 2—figure supplement 1
Pstpip2-cKI reduced aristolochic acid I (AAI)-induced kidney apoptosis.
(A) TUNEL staining of kidney sections after AAI treatment for 3 d and quantification (n = 3). Scale bar, 50 μm. (B) Caspase-3 activity in kidney tissues quantified after AAI treatment for 3 d (n = …
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Figure 2—figure supplement 1—source data 1
Data represented by each point in Figure 2—figure supplement 1A–H.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Uncropped western blots for Figure 2—figure supplement 1C and G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig2-figsupp1-data2-v2.zip
Figure 3
AAV9-mediated Pstpip2 reduction re-induces renal injury and kidney apoptosis in Pstpip2-cKI mice.
(A) Scheme illustrating the rescue experiment in Pstpip2-cKI mice. (B) Fluorescence-labeled AAV9–PSTPIP2 was localized in the kidney. (C) Real-time PCR analysis of PSTPIP2 in kidney tissues (n = 6 …
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Figure 3—source data 1
Data represented by each point in Figure 3C, E –G, I–N.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig3-data1-v2.zip
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Figure 3—source data 2
Uncropped western blots for Figure 3E, I, and M.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig3-data2-v2.zip
Figure 4 with 2 supplements
Changes in renal Pstpip2 expression affect neutrophil infiltration and inflammatory factor production in mice with aristolochic acid I (AAI)-induced acute renal injury.
(A) Representative images of immunohistochemistry (IHC) staining with Ly6G+. Scale bar, 50 μm. Quantification of the percentage of Ly6G+ neutrophil infiltration. n = 6. (B, C) mRNA level of renal …
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Figure 4—source data 1
Data represented by each point in Figure 4A–O.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig4-data1-v2.zip
Figure 4—figure supplement 1
Mice were pretreated with anti-mouse Ly6G or IgG control antibody followed by aristolochic acid I (AAI) treatment.
(A) Schematic of intraperitoneal (IP) injection of anti-mouse Ly6G neutralizing antibody or IgG control antibody; purified anti-mouse Ly6G (10 µg/g mouse), and IgG control antibody (10 µg/g mouse) …
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Figure 4—figure supplement 1—source data 1
Data represented by each point in Figure 4—figure supplement 1B–D.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
In vivo neutrophil depletion attenuates aristolochic acid I (AAI)-induced kidney damage in mice.
(A, B) Serum creatinine (Cr) and blood urea nitrogen (BUN) assay of mice pretreated with anti-mouse Ly6G or IgG control antibody, followed by AAI treatment (n = 6 per group). (C) Representative H&E-s…
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Figure 4—figure supplement 2—source data 1
Data represented by each point in Figure 4—figure supplement 2A, B, and D–G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig4-figsupp2-data1-v2.zip
Figure 5 with 4 supplements
Pstpip2 conditional knock-in in the kidney significantly decreased neutrophil extracellular trap (NET) formation.
(A) Serum nucleosome level after aristolochic acid I (AAI) treatment for 3 d (n = 5). (B, C) Serum myeloperoxidase (MPO) and dsDNA level after AAI treatment for 3 d (n = 6). (D) Protein level of …
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Figure 5—source data 1
Data represented by each point in Figure 5A–D and F–I.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-data1-v2.zip
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Figure 5—source data 2
Uncropped western blots for Figure 5D and I.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-data2-v2.zip
Figure 5—figure supplement 1
AAV9-mediated Pstpip2 restoration re-induces neutrophil extracellular trap (NET) formation in Pstpip2-cKI mice.
(A) Serum nucleosome level (n = 6 per group). (B) Serum myeloperoxidase (MPO) level (n = 6 per group). (C) Serum dsDNA level (n = 6 per group). (D) Protein level of citrullinated histone 3 (Cit-H3) …
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Figure 5—figure supplement 1—source data 1
Data represented by each point in Figure 5—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Uncropped western blots for Figure 5—figure supplement 1D.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp1-data2-v2.zip
Figure 5—figure supplement 2
Neutrophil extracellular trap (NET) formation inhibitor, GSK484, or DNase I inhibited formation of NETs in aristolochic acid I (AAI)-treated mice.
(A) Schematic of GSK484 or DNase I injection in mice experiments. DNase I (10 U/100 μL, 0.9% NaCl) or GSK484 (4 mg/kg) was administered daily for 3 d before AAI treatment. (B, C) Serum nucleosome …
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Figure 5—figure supplement 2—source data 1
Data represented by each point in Figure 5—figure supplement 2B–E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
Uncropped western blots for Figure 5—figure supplement 2E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp2-data2-v2.zip
Figure 5—figure supplement 3
Inhibition of neutrophil extracellular traps (NETs) formation attenuates aristolochic acid I (AAI)-induced kidney damage and apoptosis.
(A, B) Serum creatinine (Cr) and blood urea nitrogen (BUN) assay performed on AAI-treated mice co-treated with GSK484 or DNase I (n = 6 per group). (C) H&E staining of kidney sections of AAI-treated …
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Figure 5—figure supplement 3—source data 1
Data represented by each point in Figure 5—figure supplement 3A, B, and D-G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp3-data1-v2.zip
Figure 5—figure supplement 4
Conditional medium (CM) derived from primary neutrophils of aristolochic acid nephropathy (AAN) mice co-treated with neutrophil extracellular traps (NETs) inhibitor prevents apoptosis of mouse renal tubular epithelial cells (mRTECs).
(A) Primary renal neutrophils were isolated from aristolochic acid I (AAI)- and DNase I-treated mice. (B) The level of neutrophil elastase (NE) in the primary renal neutrophils culture supernatant …
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Figure 5—figure supplement 4—source data 1
Data represented by each point in Figure 5—figure supplement 4B, C, E, and H–J.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp4-data1-v2.zip
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Figure 5—figure supplement 4—source data 2
Uncropped western blots for Figure 5—figure supplement 4E and J.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig5-figsupp4-data2-v2.zip
Figure 6 with 1 supplement
Expression of interleukin-19 (IL-19) was associated with kidney injury and regulated by PSTPIP2.
(A) PSTPIP2 was overexpressed in mouse renal tubular epithelial cells (mRTECs) by the plasmid pEGFP-C1/PSTPIP2; the cells were subsequently treated with aristolochic acid I (AAI, 40 μM) for 20 hr. (B…
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Figure 6—source data 1
Data represented by each point in Figure 6B–D and F–L.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig6-data1-v2.zip
Figure 6—figure supplement 1
Expression of interleukin-19 (IL-19) in mouse kidneys and mouse tubular epithelial cells (mRTECs) in aristolochic acid I (AAI)-induced aristolochic acid nephropathy (AAN).
(A) Immunofluorescence (IF) staining of IL-19 (red) and E-cadherin (green) in kidney tissues of AAI-induced AAN mice. E-cadherin was used as an epithelial cell marker (mRTECs, n = 6). Scale bar, 50 …
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Figure 6—figure supplement 1—source data 1
Data represented by each point in Figure 6—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig6-figsupp1-data1-v2.zip
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Figure 6—figure supplement 1—source data 2
Uncropped western blots for Figure 6—figure supplement 1E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig6-figsupp1-data2-v2.zip
Figure 7
In vivo administration of rIL-19 in mice exacerbated the progression of aristolochic acid nephropathy.
(A) Representative H&E staining images of kidney sections from vehicle and rIL-19-treated mice after being administered with aristolochic acid I (AAI) (n = 6 per group). Scale bar, 50 μm. (B, C) …
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Figure 7—source data 1
Data represented by each point in Figure 7B–I.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig7-data1-v2.zip
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Figure 7—source data 2
Uncropped western blots for Figure 7F.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig7-data2-v2.zip
Figure 8
Administration of rIL-19 in mice promotes neutrophil infiltration, inflammatory factor production, and formation of neutrophil extracellular traps in aristolochic acid nephropathy.
(A, B) mRNA level of renal tissue monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) assessed by real-time PCR (n = 6 per group). (C) Representative images of …
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Figure 8—source data 1
Data represented by each point in Figure 8A, B, and E–H.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig8-data1-v2.zip
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Figure 8—source data 2
Uncropped western blots for Figure 8E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig8-data2-v2.zip
Figure 9 with 1 supplement
Interleukin-19 (IL-19) induces neutrophil extracellular trap (NET) formation in vitro through IL-20Rβ receptor signaling.
(A) Schematic of Transwell migration assay for neutrophils. (B) Neutrophils were isolated from mouse bone marrow and loaded into the upper Transwell chamber. Serum-free RPMI-1640 medium (500 μL) …
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Figure 9—source data 1
Data represented by each point in Figure 9D–G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig9-data1-v2.zip
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Figure 9—source data 2
Uncropped western blots for Figure 9G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig9-data2-v2.zip
Figure 9—figure supplement 1
Conditional medium (CM) derived from mouse bone marrow-derived neutrophils co-treated with interleukin-19 (IL-19) promotes apoptosis of injured mouse renal tubular epithelial cells (mRTECs).
(A) Mouse bone marrow-derived neutrophil/mRTEC-conditioned medium culture model. (B) Activity of caspase-3 quantified after treatment with different CMs (n = 3). (C) Protein levels of cleaved …
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Figure 9—figure supplement 1—source data 1
Data represented by each point in Figure 9—figure supplement 1B, C, and E.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig9-figsupp1-data1-v2.zip
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Figure 9—figure supplement 1—source data 2
Uncropped western blots for Figure 9—figure supplement 1C.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig9-figsupp1-data2-v2.zip
Figure 10
PSTPIP2 reduces interleukin-19 (IL-19) transcription by inhibiting NF-κB signaling.
(A) Co-immunoprecipitation (Co-IP) of PSTPIP2 and NF-κB p65 in mouse renal tubular epithelial cell (mRTEC) using anti-NF-κB p65 and anti-PSTPIP2 antibodies. (B) Western blotting results showed that …
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Figure 10—source data 1
Data represented by each point in Figure 10B, C, F, and G.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig10-data1-v2.zip
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Figure 10—source data 2
Uncropped western blots for Figure 10A, B, C, and F.
- https://cdn.elifesciences.org/articles/89740/elife-89740-fig10-data2-v2.zip
Figure 11
PSTPIP2 alleviates aristolochic acid I-induced acute kidney injury and renal tubular epithelial cell apoptosis by suppressing interleukin-19-mediated neutrophil extracellular trap formation.
Author response image 1
Tables
Table 1
Primer sequences for the quantitative real-time PCR analysis of mouse renal tubular epithelial cells (mRTECs) and mouse tissues.
| Terms(mouse) | Forward primer (5'–3') | Reverse primer (5'–3') |
|---|---|---|
| IL1α | TCTATGATGCAAGCTATGGCTCA | CGGCTCTCCTTGAAGGTGA |
| IL1β | GAAATGCCACCTTTTGACAGTG | TGGATGCTCTCATCAGGACAG |
| IL3 | GGGATACCCACCGTTTAACCA | AGGTTTACTCTCCGAAAGCTCTT |
| IL4 | GGTCTCAACCCCCAGCTAGT | GCCGATGATCTCTCTCAAGTGAT |
| IL5 | GCAATGAGACGATGAGGCTTC | GCCCCTGAAAGATTTCTCCAATG |
| IL6 | CTGCAAGAGACTTCCATCCAG | AGTGGTATAGACAGGTCTGTTGG |
| IL7 | TTCCTCCACTGATCCTTGTTCT | AGCAGCTTCCTTTGTATCATCAC |
| IL9 | ATGTTGGTGACATACATCCTTGC | TGACGGTGGATCATCCTTCAG |
| IL10 | CTTACTGACTGGCATGAGGATCA | GCAGCTCTAGGAGCATGTGG |
| IL11 | GCGCTGTTCTCCTAACCCG | GAGTCCAGACTGTGATCTCCG |
| IL12α | CAATCACGCTACCTCCTCTTTT | CAGCAGTGCAGGAATAATGTTTC |
| IL13 | TGAGCAACATCACACAAGACC | GGCCTTGCGGTTACAGAGG |
| IL14 | TCCTGAGTACATACTGTGTGGAC | GCTGCATAGGTTCGGGACTTC |
| IL15 | CATCCATCTCGTGCTACTTGTG | GCCTCTGTTTTAGGGAGACCT |
| IL16 | AAGAGCCGGAAATCCACGAAA | GTGCGAGGTCTGGGATATTGC |
| IL17A | TCAGCGTGTCCAAACACTGAG | CGCCAAGGGAGTTAAAGACTT |
| IL17F | TGCTACTGTTGATGTTGGGAC | CAGAAATGCCCTGGTTTTGGT |
| IL18 | GTGAACCCCAGACCAGACTG | CCTGGAACACGTTTCTGAAAGA |
| IL19 | CTCCTGGGCATGACGTTGATT | GCATGGCTCTCTTGATCTCGT |
| IL20 | GTCTTGCCTTTGGACTGTTCT | AGGTTTGCAGTAATCACACAGC |
| IL21 | GGACCCTTGTCTGTCTGGTAG | TGTGGAGCTGATAGAAGTTCAGG |
| IL22 | ATGAGTTTTTCCCTTATGGGGAC | GCTGGAAGTTGGACACCTCAA |
| IL23 | CAGCAGCTCTCTCGGAATCTC | TGGATACGGGGCACATTATTTTT |
| IL24 | GAGCCTGCCCAACTTTTTGTG | TGTGTTGAAGAAAGGGCCAGT |
| IL25 | ACAGGGACTTGAATCGGGTC | TGGTAAAGTGGGACGGAGTTG |
| IL27 | CTGTTGCTGCTACCCTTGCTT | CTCCTGGCAATCGAGATTCAG |
| IL28B | GTTCAAGTCTCTGTCCCCAAAA | GTGGGAACTGCACCTCATGT |
| IL31 | TCAGCAGACGAATCAATACAGC | TCGCTCAACACTTTGACTTTCT |
| IL33 | ATTTCCCCGGCAAAGTTCAG | AACGGAGTCTCATGCAGTAGA |
| IL34 | TTGCTGTAAACAAAGCCCCAT | CCGAGACAAAGGGTACACATTT |
| IL40 | ACTGGAAGTTTATCCCCAAAGC | CGGAGTCATGCACAACCTTTTT |
| Kim-1 | TAAACCAGAGATTCCCACAC | GATCTTGTTGAAATAGTCGTG |
| MCP-1 | GCTTGAGGTGGTTGTGGAAAA | CTCACCTGCTGCTACTCATTC |
| β-Actin | GATTACTGCTCTGGCTCCTAGC | GACTCATCGTACTCCTGCTTG |
| TNF-α | CCCTCACACTCAGATCATCTTCT | GCTACGACGTGGGCTACAG |
Table 2
Clinical features of the patients with kidney diseases.
| No. | Pathological diagnosis | Sex | Age (years) | Serum creatinine (μmol/L) | Serum IL-19 (ng/mL) |
|---|---|---|---|---|---|
| 1 | Kidney transplant | Female | 19 | 198.4278 | 274.4815 |
| 2 | Kidney transplant | Female | 33 | 144.0580 | 297.7699 |
| 3 | Hydronephrosis with ureteral stones | Male | 48 | 216.9878 | 125.0563 |
| 4 | Hydronephrosis with nephrolithiasis | Male | 47 | 158.1964 | 199.1192 |
| 5 | Renal allograft dysfunction | Male | 27 | 450.1882 | 308.2646 |
| 6 | Allograft dysfunction, renal allograft | Male | 36 | 444.1835 | 161.4381 |
| 7 | Renal anemia, 5 CKD stage | Male | 58 | 1225.2303 | 340.7484 |
| 8 | CKD stage 3, type 2 diabetes | Female | 76 | 129.5376 | 199.1192 |
| 9 | Nephrotic syndrome with minimal change nephropathy | Female | 21 | 160.7620 | 231.6030 |
| 10 | Renal end-stage disease | Male | 35 | 1250.3954 | 391.4230 |
| 11 | Nephrotic syndrome | Male | 56 | 124.0242 | 240.0487 |
| 12 | Polycystic kidney disease | Female | 57 | 210.0129 | 147.1452 |
| 13 | Renal end-stage disease | Female | 38 | 1229.0289 | 468.0847 |
| 14 | Chronic renal failure | Male | 57 | 807.2540 | 218.6095 |
| 15 | Hydronephrosis with nephroureterolithiasis, type I diabetes | Female | 53 | 83.8521 | 226.4056 |
| 16 | Diabetes | Male | 66 | 82.1029 | 229.0043 |
| 17 | Nephrolithiasis with hydronephrosis | Male | 34 | 139.9357 | 208.2147 |
| 18 | CKD stage 4 | Male | 55 | 134.3601 | 201.7179 |
| 19 | Membranous nephropathy | Male | 40 | 145.3473 | 253.6919 |
| 20 | Nephrotic syndrome | Male | 46 | 76.5273 | 262.7874 |
| 21 | Hydronephrosis | Female | 52 | 92.5981 | 135.4511 |
| 22 | Renal insufficiency | Male | 82 | 754.3408 | 225.1062 |
| 23 | SLE, lupus nephritis | Male | 32 | 456.8682 | 391.4230 |
| 24 | Hydronephrosis with nephrolithiasis | Female | 68 | 128.7846 | 266.6854 |
| 25 | Diabetes | Male | 43 | 107.3569 | 283.5770 |
| 26 | Membranous nephropathy | Male | 53 | 174.3729 | 442.0977 |
| 27 | Nephrotic syndrome | Male | 46 | 186.9453 | 188.7244 |
