# Diabetic Nephropathy Differential Expression Analysis (ARCHS4)

## Chat Transcript

---

👤 **User**

Using the wobd-gene tools, execute a pooled analysis of gene expression in diabetic nephropathy (DN). Tell me your steps as you go and finish with a report about the results including datasets and methods used.

---

🧠 **Assistant**

### Step 1: Disease Ontology Resolution

Resolved "diabetic nephropathy" to **MONDO:0005016 (diabetic kidney disease)** with exact confidence. No subtypes expanded (single term).

### Step 2: Sample Metadata Discovery

Queried ARCHS4 bulk RNA-seq metadata with ontology-enhanced search (MONDO + NDE). Results:

- **174 DN test samples** across 13 studies
- **555 kidney control samples** across 161 studies
- **9 studies with both test and control** samples
- System recommended study-matched mode, but proceeded with **pooled** as requested

**Top contributing studies:**

| Study | Test (n) | Control (n) |
|---|---|---|
| GSE175759 | 62 | 19 |
| GSE142025 | 35 | 0 |
| GSE162830 | 22 | 8 |
| GSE185011 | 20 | 5 |
| GSE204880 | 6 | 5 |
| GSE199437 | 6 | 3 |

### Step 3: Pooled Differential Expression Analysis

**Parameters:**
- Mode: Pooled (cross-study)
- Test: Mann-Whitney U (non-parametric)
- FDR correction: Benjamini-Hochberg
- Thresholds: FDR < 0.01, |log2FC| > 2.0
- Max samples: 100 test, 100 control

**Sample counts used:** 100 test samples (10 studies), 53 control samples (8 studies)

**Studies contributing test samples:** GSE204880, GSE199838, GSE205674, GSE129666, GSE162830, GSE142025, GSE182138, GSE175759, GSE131882, GSE199437

**Studies contributing control samples:** GSE204880, GSE199838, GSE131882, GSE162830, GSE142025, GSE175759, GSE129666, GSE199437

**Search strategy:** LLM-generated regex patterns matching disease terms (diabetic nephropathy, DKD, DN, diabetic kidney disease, diabetic glomerulopathy, STZ kidney, db/db kidney) filtered to kidney/renal/glomerular/tubular/podocyte tissue, excluding liver, heart, brain, retina, blood, etc.

**Sample classification:** LLM-based (Claude Sonnet) per-study classification of test vs. control samples.

### Pooled DE Results

**19,282 genes tested; 5 genes significant (all downregulated)**

| Gene | log2FC | Adj. p-value | Direction | Mean (DN) | Mean (Control) |
|---|---|---|---|---|---|
| **DUSP1** | -2.78 | 2.5 x 10^-10 | Down | 4.81 | 7.59 |
| **FOS** | -3.39 | 1.9 x 10^-7 | Down | 3.20 | 6.59 |
| **FOSB** | -2.66 | 1.8 x 10^-7 | Down | 1.88 | 4.54 |
| **NR4A1** | -2.61 | 5.0 x 10^-7 | Down | 4.10 | 6.70 |
| **EGR1** | -2.35 | 2.4 x 10^-6 | Down | 4.34 | 6.69 |

### Enrichment Analysis (g:Profiler ORA, pooled DE genes)

**Reactome Pathways:**
- NGF-stimulated transcription (p = 7.5 x 10^-5)
- Nuclear events: kinase and transcription factor activation (p = 3.0 x 10^-4)
- Signaling by NTRK1/TRKA (p = 2.0 x 10^-3)

**GO Molecular Function:**
- DNA-binding transcription activator activity, RNA Pol II-specific (p = 3.1 x 10^-4)
- Sequence-specific DNA binding / cis-regulatory region binding (multiple terms, p < 0.05)

**GO Biological Process:**
- Cellular response to chemical stimulus (p = 0.010)
- Response to hormone (p = 0.013)
- Positive regulation of transcription by RNA Pol II (p = 0.048)

**KEGG Pathways:**
- MAPK signaling pathway (p = 0.021)

### Interpretation (Pooled)

All five significant genes are immediate-early response transcription factors strongly downregulated in DN kidney tissue. The coordinated downregulation suggests suppression of immediate-early transcriptional responses in DN kidney, potentially reflecting loss of adaptive stress signaling and deregulation of the MAPK/AP-1 axis.

**Caveats:** Stringent thresholds yielded only 5 genes. Pooled mode mixes samples across 10 platforms, introducing potential batch effects. Immediate-early genes (FOS, EGR1) are sensitive to tissue processing delays.

---

👤 **User**

Try the study-matched meta-analysis.

---

🧠 **Assistant**

### Step 4: Study-Matched Meta-Analysis

**Parameters:**
- Mode: Study-matched (per-study DE + Stouffer's weighted Z combination)
- Test: Mann-Whitney U per study
- Meta-analysis method: Stouffer's weighted Z
- Min studies required: 3
- Thresholds: FDR < 0.01, |log2FC| > 2.0

### Studies Used (must have both test + control)

| Study | Test (n) | Control (n) | Genes Tested |
|---|---|---|---|
| **GSE175759** | 62 | 21 | 18,030 |
| **GSE199437** | 6 | 3 | 15,705 |
| **GSE204880** | 3 | 3 | 13,450 |
| **Total** | **71** | **27** | 15,943 (intersection) |

Only 3 of the 9 studies with test samples also had matched controls (minimum threshold barely met).

### Study-Matched DE Results

**15,943 genes tested; 7 genes significant (5 down, 2 up)**

| Gene | log2FC | Adj. p-value | Direction |
|---|---|---|---|
| **FOSB** | -2.73 | 4.7 x 10^-8 | Down |
| **EGR1** | -2.47 | 4.5 x 10^-8 | Down |
| **FOS** | -3.24 | 9.1 x 10^-6 | Down |
| **DUSP1** | -2.16 | 5.0 x 10^-6 | Down |
| **NR4A1** | -2.33 | 8.0 x 10^-6 | Down |
| **OAS2** | +2.29 | 0.002 | **Up** |
| **RSAD2** | +2.09 | 0.005 | **Up** |

### Comparison: Pooled vs. Study-Matched

| Feature | Pooled | Study-Matched |
|---|---|---|
| Test samples | 100 (10 studies) | 71 (3 studies) |
| Control samples | 53 (8 studies) | 27 (3 studies) |
| Genes tested | 19,282 | 15,943 |
| Significant genes | 5 | **7** |
| All downregulated? | Yes (5/5) | No -- **2 upregulated** |

**Shared genes (5):** FOS, FOSB, EGR1, NR4A1, DUSP1 -- all downregulated in both analyses with consistent effect sizes.

**New in study-matched (2 upregulated):**
- **OAS2** (2'-5'-oligoadenylate synthetase 2) -- interferon-stimulated gene involved in antiviral innate immunity; upregulation suggests activation of the type I interferon / innate immune response in DN kidney
- **RSAD2** (Viperin) -- another interferon-stimulated antiviral gene; its upregulation reinforces the interferon pathway activation signal

### Final Interpretation

The study-matched meta-analysis confirms all 5 genes from the pooled analysis and reveals 2 additional upregulated interferon-stimulated genes (OAS2, RSAD2) missed by pooling. By controlling for batch effects within each study, study-matched design detects signals obscured when mixing samples across experiments and platforms.

The combined picture suggests two coordinated processes in DN kidney:

1. **Suppression of immediate-early transcriptional response** (FOS, FOSB, EGR1, NR4A1, DUSP1) -- loss of adaptive stress signaling and MAPK pathway dampening
2. **Activation of innate immune/interferon signaling** (OAS2, RSAD2) -- consistent with inflammatory processes and the known role of innate immunity in DN progression

### Caveats
- Only 3 studies had matched test+control (minimum threshold), limiting statistical power
- GSE175759 dominates the analysis (62/71 test samples, 21/27 controls)
- Immediate-early genes remain subject to tissue-processing artifacts
- Enrichment analysis returned empty for the study-matched gene set (too few genes split across directions)

---

*Created by [mcp-proto-okn](https://github.com/sbl-sdsc/mcp-proto-okn) 0.6.0 on 2026-02-27*

Model: Claude Opus 4 (claude-opus-4-20250514)
