MicroRNA-29b Suppresses TGF-β-Induced Epithelial-Mesenchymal Transition in Renal Interstitium of Spontaneously Hypertensive Rats

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MicroRNA-29b Suppresses TGF-β-Induced Epithelial-Mesenchymal Transition in Renal Interstitium of Spontaneously Hypertensive Rats

Hypertension represents a major cardiovascular disorder with profound effects on renal structure and function. The kidney, a critical regulator of fluid balance and blood pressure, becomes susceptible to hypertensive injury through mechanisms involving glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria. Among molecular regulators implicated in chronic kidney disease, microRNAs (miRNAs) have emerged as key players in modulating pathological processes. miR-29b, a member of the miR-29 family, has previously demonstrated renoprotective properties by attenuating angiotensin II-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. However, its functional role in hypertensive nephropathy in vivo remained unclear. This study investigated the therapeutic potential of miR-29b in a spontaneously hypertensive rat (SHR) model, elucidating its effects on renal function, extracellular matrix (ECM) accumulation, EMT, and TGF-β signaling.

Experimental Design and Animal Model

Fifteen-week-old male SHRs and normotensive Wistar-Kyoto (WKY) rats were utilized to model hypertensive renal injury. Rats were divided into five groups (n=5 per group):

  1. WKY Group: Normotensive rats treated with physiological saline.
  2. SHR Group: Hypertensive rats treated with saline.
  3. SHR+C Group: SHRs receiving nonspecific control lentiviral vectors.
  4. SHR+M Group: SHRs administered lentiviral vectors expressing miR-29b mimics.
  5. SHR+I Group: SHRs treated with lentiviral vectors inhibiting miR-29b.

Lentiviruses (lv-miR-29b for overexpression, lv-miR-29b-In for inhibition, and lv-miRC as control) were delivered via intrarenal parenchymal injection. After four weeks, renal tissues and blood samples were collected for molecular, histological, and functional analyses. Parameters assessed included serum creatinine (SCr), blood urea nitrogen (BUN), urinary protein, collagen I deposition, α-smooth muscle actin (α-SMA, an EMT marker), and TGF-β expression.

miR-29b Ameliorates Hypertensive Renal Dysfunction

At baseline, SHRs exhibited significantly elevated SCr (1.2-fold increase) and BUN (1.3-fold increase) compared to WKY rats, indicating impaired renal function. These markers further increased by 26% (SCr) and 31% (BUN) in untreated SHRs over four weeks. Urinary protein levels in SHRs were 2.4-fold higher than in WKY controls, with a 35% increase within the four-week observation period, confirming progressive renal injury.

miR-29b overexpression (SHR+M group) reversed these trends. SCr and BUN levels decreased by 18% and 22%, respectively, compared to the SHR+C group. Urinary protein content declined by 6.75% in miR-29b-treated SHRs. Conversely, miR-29b inhibition (SHR+I group) exacerbated renal dysfunction, with SCr, BUN, and urinary protein rising by 28%, 33%, and 15%, respectively, relative to the SHR+C group. Notably, renal function parameters in the SHR+M group approximated those of WKY rats, demonstrating the therapeutic efficacy of miR-29b.

Suppression of ECM Accumulation

Hypertension-induced renal fibrosis was evaluated through collagen I expression and Sirius red staining. SHR kidneys displayed 4.2-fold higher collagen I mRNA levels and 3.8-fold greater protein expression compared to WKY rats. miR-29b overexpression reduced collagen I mRNA by 54% and protein by 61% versus the SHR+C group, while miR-29b inhibition increased these levels by 75% (mRNA) and 82% (protein). Immunohistochemical analysis corroborated these findings, showing dense collagen I deposition in SHR interstitial regions, which diminished markedly in the SHR+M group. Sirius red staining quantified interstitial fibrosis at 3.29% in SHR+M rats versus 9.12% in SHR+C and 14.8% in SHR+I groups, highlighting miR-29b’s anti-fibrotic effects.

Inhibition of EMT via α-SMA Downregulation

EMT, a hallmark of renal fibrosis, was assessed through α-SMA expression. SHR kidneys exhibited 3.1-fold higher α-SMA mRNA and 2.9-fold elevated protein levels compared to WKY controls. miR-29b overexpression suppressed α-SMA expression by 48% (mRNA) and 53% (protein), whereas inhibition increased levels by 71% and 67%, respectively. Immunostaining revealed abundant α-SMA-positive myofibroblasts in SHR tubulointerstitial regions, which decreased by 44% in the SHR+M group. These results indicate miR-29b’s capacity to attenuate EMT-driven fibrosis.

Modulation of TGF-β Signaling

TGF-β, a central mediator of fibrosis, was significantly upregulated in SHR kidneys (2.7-fold mRNA increase; 3.1-fold protein increase vs. WKY). miR-29b overexpression reduced TGF-β mRNA and protein by 62% and 66%, respectively, while inhibition caused 85% and 89% increases. Immunostaining confirmed TGF-β suppression in the SHR+M group, with signal intensity reduced to one-third of controls. This inverse correlation between miR-29b and TGF-β underscores a regulatory mechanism where miR-29b blunts TGF-β-driven fibrotic pathways.

Mechanistic Implications

The study establishes miR-29b as a critical inhibitor of hypertensive renal fibrosis via dual mechanisms:

  1. ECM Regulation: By directly targeting collagen I, miR-29b mitigates ECM accumulation, preventing peritubular capillary compression and tubular damage.
  2. EMT Suppression: Through downregulation of α-SMA and TGF-β, miR-29b impedes myofibroblast activation and epithelial cell transition, preserving renal architecture.

The observed TGF-β suppression aligns with prior studies showing miR-29b’s transcriptional inhibition of TGF-β in human trabecular meshwork cells. This crosstalk between miR-29b and TGF-β provides a mechanistic foundation for its anti-fibrotic effects.

Therapeutic Significance

The restoration of near-normal renal function in SHRs following miR-29b overexpression highlights its clinical potential. By targeting upstream regulators like TGF-β, miR-29b offers a multifaceted approach to counteract hypertension-induced nephropathy. Lentiviral delivery, though requiring further optimization for human use, presents a viable strategy for localized miRNA modulation in renal tissues.

Conclusion

This study delineates miR-29b’s role as a potent renoprotective agent in hypertensive renal injury. Through suppression of TGF-β, collagen I, and α-SMA, miR-29b attenuates ECM remodeling, EMT, and fibrosis, preserving renal function. These findings advocate for miR-29b-based therapies as a promising avenue for treating hypertensive nephropathy and related fibrotic disorders.

doi.org/10.1097/CM9.0000000000001922

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