Abstract 6: Heterozygous TGFβ1 Zinc-Finger Knockout Dahl S Rats Reveal Protection Against High-Salt Induced Renal Injury

We used a zinc-finger nuclease strategy to create heterozygous TGFβ1 knockout rats (TGFβ1 +/- ) on a Dahl SS/Jr genetic background with a 22 base-pair frame shift mutation between nucleotides 191-212 which introduced a premature stop codon at amino acid 34. Intercrossing TGFβ1 +/- rats did not produce homozygous knockout rats, indicating that the mutation is embryonic lethal. Wildtype (WT) littermates and TGFβ1 +/- rats were fed either a 0.4% (normal salt, NS) or 8% NaCl (high salt, HS) diet for 5 weeks. When fed a NS diet, WT and TGFβ1 +/- exhibit similar renal cortical TGFβ1 expression (1.00±0.12 vs 1.05±0.05, arbitrary units), urinary TGFβ1 excretion (3.9±1.2 vs 5.3±0.4, μg/day), proteinuria (43±5 vs 36±4, mg/day), and minimal glomerular injury and tubulointerstitial fibrosis (TIF). 5 weeks of HS increased renal cortical TGFβ1 protein expression to a greater extent in WT versus TGFβ1 +/- (1.89±0.14 vs 1.52±0.09, arbitrary units) and TGFβ1 levels in urine increased to a greater extent in WT (41±10 μg/day) versus TGFβ1 +/- rats (18±4 μg/day) fed a HS diet for 1 week. Systolic blood pressure (SBP), measured by tail-cuff, was similar in WT (161±6 mmHg) and TGFβ1 +/- (162±7 mmHg) fed a NS diet and increased to the same extent in both WT (235±2 mmHg) and TGFβ1 +/- (239±4 mmHg) fed a HS diet for 5 weeks. Urinary protein excretion increased to a greater extent in WT versus TGFβ1 +/- (463±28 vs 313±36 mg/day) fed a HS diet for 5 weeks. Glomerular injury and renal cortical interstititial fibrosis were markedly reduced in TGFβ1 +/- versus WT after 5 weeks on a HS diet. Similarly, TIF in the renal medulla was less in TGFβ1 +/- compared with WT. These findings suggest that loss of one copy of the TGFβ1 gene blunts the increase in renal TGFβ1 protein in Dahl S rats fed a HS diet and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis independent of changes in blood pressure.

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Heterozygous knockout of transforming growth factor-β1 protects Dahl S rats against high salt-induced renal injury

Physiological Genomics ◽

10.1152/physiolgenomics.00119.2012 ◽

2013 ◽

Vol 45 (3) ◽

pp. 110-118 ◽

Cited By ~ 14

Author(s):

Chun Cheng Andy Chen ◽

Aron M. Geurts ◽

Howard J. Jacob ◽

Fan Fan ◽

Richard J. Roman

Keyword(s):

Blood Pressure ◽

Growth Factor ◽

Systolic Blood Pressure ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Transforming Growth Factor Β1 ◽

High Salt ◽

Glomerular Injury ◽

Protein Excretion ◽

Tgf Β1

The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-β1 ( Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-β1+/− Dahl S). Intercrossing TGF-β1+/− rats did not produce any homozygous KO rats (66.4% +/−, 33.6% +/+), indicating that the mutation is embryonic lethal. Six-week-old wild-type (WT) littermates and TGF-β1+/− Dahl S rats were fed a 0.4% (low salt, LS) or 8% NaCl (high salt, HS) diet for 5 wk. Renal cortical expression of TGF-β1, urinary TGF-β1 excretion, proteinuria, glomerular injury and tubulointerstitial fibrosis, and systolic blood pressure were similar in WT and TGF-β1+/− Dahl S rats maintained on the LS diet. The expression and urinary excretion of TGF-β1 increased to a greater extent in WT than in TGF-β1+/−Dahl S rats fed an HS diet for 1 wk. Systolic blood pressure rose by the same extent to 235 ± 2 mmHg in WT and 239 ± 4 mmHg in TGF-β1+/− Dahl S rats fed a HS diet for 5 wk. However, urinary protein excretion was significantly lower in TGF-β1+/− Dahl S than in the WT animals. The degree of glomerular injury and renal cortical and outer medullary fibrosis was markedly less in TGF-β1+/− than in WT rats. These findings suggest that the loss of one copy of the TGF-β1 gene blunts the increase in renal TGF-β1 protein expression and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis in Dahl S rats fed an HS diet independently of changes in blood pressure.

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Abstract MP44: Proteolytic Activation Of ENaC Mediates Fructose-Induced Salt-Sensitive Hypertension

Hypertension ◽

10.1161/hyp.76.suppl_1.mp44 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Rashi Shukla ◽

Nancy Hong ◽

Ryan Henderson ◽

Agustin Gonzalez-Vicente ◽

Ulrich Hopfer ◽

...

Keyword(s):

Blood Pressure ◽

Systolic Blood Pressure ◽

High Salt ◽

Na Channel ◽

Glomerular Injury ◽

Proteolytic Activation ◽

Protein Excretion ◽

Dietary Fructose ◽

Before And After ◽

Salt Sensitive Hypertension

In nephrotic syndrome, glomerular injury leads to filtration of plasma proteases. Once in the urine, these enzymes cleave and activate the epithelial Na channel (ENaC) causing inappropriate Na retention and hypertension. Dietary fructose causes inappropriate Na retention and salt-sensitive hypertension but whether proteolytic activation of ENaC is involved, and the source of the proteases, is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt-sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high-salt diet without fructose (high salt) or one containing 4% NaCl plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 68±7% (p < 0.01). Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose) high salt/glucose) on blood pressure before and after oral amiloride. After 7 days the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but had no significant effect on the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose-induced salt-sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.

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Vasoactive intestinal peptide infusion reverses existing renal interstitial fibrosis via a blood pressure independent mechanism in the rat

European Journal of Pharmacology ◽

10.1016/j.ejphar.2020.172979 ◽

2020 ◽

Vol 873 ◽

pp. 172979

Author(s):

Karen A. Duggan ◽

George Hodge ◽

Juchuan Chen ◽

Sofie Trajanovska ◽

Tegan Hunter

Keyword(s):

Blood Pressure ◽

Vasoactive Intestinal Peptide ◽

Interstitial Fibrosis ◽

Renal Interstitial Fibrosis ◽

Independent Mechanism ◽

Pressure Independent

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Preclinical Study of DNA-Recognized Peptide Pyrrole-Imidazole Polyamide Targeting Human TGF-β1 Promoter for Progressive Renal Diseases in Common Marmoset

10.20944/preprints201908.0090.v1 ◽

2019 ◽

Author(s):

Masari Otsuki ◽

Noboru Fukuda ◽

Takashi Inoue ◽

Takayuki Mineshige ◽

Tomoyasu Otsuki ◽

...

Keyword(s):

Target Genes ◽

Interstitial Fibrosis ◽

Renal Medulla ◽

Common Marmoset ◽

Preclinical Study ◽

Renal Diseases ◽

Glomerular Injury ◽

Tubulointerstitial Injury ◽

Injury Score ◽

Dependent Inhibition

Pyrrole-imidazole (PI) polyamides are novel gene silencers that strongly bind the promoter region of target genes in a sequence-specific manner to inhibit gene transcription. We developed a PI polyamide targeting human TGF-b1 (hTGF-b1). To develop PI polyamide targeting hTGF-b1 (Polyamide) as a practical medicine for progressive renal diseases, we examined the effects of Polyamide in two common marmoset models of nephropathy.We performed lead optimization of PI polyamides targeting hTGF-b1 by the dose-dependent inhibition of the PMA-stimulated expression of TGF-b1 mRNA in marmoset fibroblasts. Marmosets were housed with a 0.05% NaCl and magnesium diet and treated with cyclosporine A (CsA; 37.5 mg/kg/day, 8 weeks) to establish chronic nephropathy. Marmosets with nephropathy were treated with Polyamide (1 mg/kg/week, 4 weeks). We also established a unilateral urethral obstruction(UUO) model and examined the effects of Polyamide (1 mg/kg/week, 4 times) in marmosets.Histologically, the renal medulla from CsA-treated marmosets showed cast formation and interstitial fibrosis in the renal medulla. Immunohistochemistry showed strong staining of Polyamide in the renal medulla from CsA-treated marmosets.Polyamidetreatment (1 mg/kg/week, 4 times) reduced hTGF-b1 staining and urinary protein excretion in CsA-treated marmosets. Polyamide reduced the glomerular injury score (GIS) and tubulointerstitial injury score (TIS) in UUO kidneys from marmosets. Polyamide significantly suppressed the hTGF-b1 and Snail mRNA expressionin UUO kidneys from marmosets.PI polyamide effectively improved CsA- and UUO-associated nephropathy, indicating its potential application in the prevention of renal fibrosis in progressive renal diseases.

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Interactions between 11β-hydroxysteroid dehydrogenase and COX-2 in kidney

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00786.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. R1767-R1773 ◽

Cited By ~ 27

Author(s):

Bing Yao ◽

Raymond C. Harris ◽

Ming-Zhi Zhang

Keyword(s):

Blood Pressure ◽

Renal Medulla ◽

Hydroxysteroid Dehydrogenase ◽

High Salt ◽

Adult Rats ◽

Water Excretion ◽

Suckling Rats ◽

Apparent Mineralocorticoid Excess ◽

Cox 2 ◽

Salt Sensitive Hypertension

The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11β-hydroxysteroid dehydrogenase (11βHSD2). In this disorder, cortisol is not inactivated by 11βHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11βHSD2 inhibition and examined its possible role in the development of hypertension. 11βHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11βHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11βHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11βHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11βHSD2 deficiency and that 11βHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.

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Sodium bicarbonate loading limits tubular cast formation independent of glomerular injury and proteinuria in Dahl salt-sensitive rats

Clinical Science ◽

10.1042/cs20171630 ◽

2018 ◽

Vol 132 (11) ◽

pp. 1179-1197 ◽

Cited By ~ 6

Author(s):

Sarah C. Ray ◽

Bansari Patel ◽

Debra L. Irsik ◽

Jingping Sun ◽

Hiram Ocasio ◽

...

Keyword(s):

Blood Pressure ◽

Sodium Bicarbonate ◽

Renal Injury ◽

High Salt ◽

Proton Channel ◽

Pathological Feature ◽

Glomerular Injury ◽

High Salt Diet ◽

Salt Diet ◽

Inflammatory Status

Sodium bicarbonate (NaHCO3) slows the decline in kidney function in patients with chronic kidney disease (CKD), yet the mechanisms mediating this effect remain unclear. The Dahl salt-sensitive (SS) rat develops hypertension and progressive renal injury when fed a high salt diet; however, the effect of alkali loading on kidney injury has never been investigated in this model. We hypothesized that NaHCO3 protects from the development of renal injury in Dahl salt-sensitive rats via luminal alkalization which limits the formation of tubular casts, which are a prominent pathological feature in this model. To examine this hypothesis, we determined blood pressure and renal injury responses in Dahl SS rats drinking vehicle (0.1 M NaCl) or NaHCO3 (0.1 M) solutions as well as in Dahl SS rats lacking the voltage-gated proton channel (Hv1). We found that oral NaHCO3 reduced tubular NH4+ production, tubular cast formation, and interstitial fibrosis in rats fed a high salt diet for 2 weeks. This effect was independent of changes in blood pressure, glomerular injury, or proteinuria and did not associate with changes in renal inflammatory status. We found that null mutation of Hv1 also limited cast formation in Dahl SS rats independent of proteinuria or glomerular injury. As Hv1 is localized to the luminal membrane of TAL, our data suggest that alkalization of the luminal fluid within this segment limits cast formation in this model. Reduced cast formation, secondary to luminal alkalization within TAL segments may mediate some of the protective effects of alkali loading observed in CKD patients.

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Qingxuan Jiangya Decoction Mitigates Renal Interstitial Fibrosis in Spontaneously Hypertensive Rats by Regulating Transforming Growth Factor-β1/Smad Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/1576328 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Wangyu Liu ◽

Shan Lin ◽

Qiaoyan Cai ◽

Ling Zhang ◽

Aling Shen ◽

...

Keyword(s):

Blood Pressure ◽

Signaling Pathway ◽

Spontaneously Hypertensive Rats ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Hypertensive Rats ◽

Renal Interstitial Fibrosis ◽

Spontaneously Hypertensive ◽

Smad Signaling ◽

Smad Signaling Pathway

Qingxuan Jiangya Decoction (QXJYD) is a traditional Chinese medicine commonly used in the clinical treatment of hypertension. Earlier studies had shown that QXJYD could inhibit the elevation of blood pressure in spontaneously hypertensive rats (SHRs) and prevent remodeling of arterial vessels. This study examines the therapeutic efficacy of QXJYD against elevated blood pressure using the SHR model, as well as the mechanisms behind its antihypertensive activity and protection against renal fibrosis. The results showed that QXJYD significantly attenuated the increase in blood pressure in SHRs and mitigated the development of renal interstitial fibrosis. In addition, QXJYD also robustly decreased the excess accumulation of extracellular matrix and attenuated the elevated expression of MMPs. The antihypertensive effects and renal protection of QXJYD were determined to be strongly associated with inhibition of TGF-β1/Smad signaling pathway.

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The Effect of a High Salt Diet and Gender on Blood Pressure, Urinary Protein Excretion and Renal Pathology in Shr Rats

Clinical and Experimental Hypertension Part A Theory and Practice ◽

10.3109/10641969109042072 ◽

1991 ◽

Vol 13 (5) ◽

pp. 687-697 ◽

Cited By ~ 3

Author(s):

David A. Blizard ◽

Wanda N. Peterson ◽

Samy S. Iskandar ◽

Z. K. Shihabi ◽

Nelson Adams

Keyword(s):

Blood Pressure ◽

Urinary Protein Excretion ◽

Urinary Protein ◽

High Salt ◽

Renal Pathology ◽

Shr Rats ◽

High Salt Diet ◽

Salt Diet ◽

Protein Excretion ◽

And Gender

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Preclinical Study of DNA-Recognized Peptide Compound Pyrrole-Imidazole Polyamide Targeting Human TGF-β1 Promoter for Progressive Renal Diseases in the Common Marmoset

Molecules ◽

10.3390/molecules24173178 ◽

2019 ◽

Vol 24 (17) ◽

pp. 3178 ◽

Cited By ~ 3

Author(s):

Otsuki ◽

Fukuda ◽

Inoue ◽

Mineshige ◽

Otsuki ◽

...

Keyword(s):

Target Genes ◽

Interstitial Fibrosis ◽

Renal Medulla ◽

Common Marmoset ◽

Renal Diseases ◽

Dependent Manner ◽

Glomerular Injury ◽

Tubulointerstitial Injury ◽

Injury Score ◽

Tgf Β1

Pyrrole-imidazole (PI) polyamides are novel gene silencers that strongly bind the promoter region of target genes in a sequence-specific manner to inhibit gene transcription. We created a PI polyamide targeting human TGF-β1 (hTGF-β1). To develop this PI polyamide targeting hTGF-β1 (Polyamide) as a practical medicine for treating progressive renal diseases, we examined the effects of Polyamide in two common marmoset models of nephropathy. We performed lead optimization of PI polyamides that targeted hTGF-β1 by inhibiting in a dose-dependent manner the expression of TGF-β1 mRNA stimulated by PMA in marmoset fibroblasts. Marmosets were housed and fed with a 0.05% NaCl and magnesium diet and treated with cyclosporine A (CsA; 37.5 mg/kg/day, eight weeks) to establish chronic nephropathy. We treated the marmosets with nephropathy with Polyamide (1 mg/kg/week, four weeks). We also established a unilateral urethral obstruction (UUO) model to examine the effects of Polyamide (1 mg/kg/week, four times) in marmosets. Histologically, the renal medulla from CsA-treated marmosets showed cast formation and interstitial fibrosis in the renal medulla. Immunohistochemistry showed strong staining of Polyamide in the renal medulla from CsA-treated marmosets. Polyamide treatment (1 mg/kg/week, four times) reduced hTGF-β1 staining and urinary protein excretion in CsA-treated marmosets. In UUO kidneys from marmosets, Polyamide reduced the glomerular injury score and tubulointerstitial injury score. Polyamide significantly suppressed hTGF-β1 and snail mRNA expression in UUO kidneys from the marmosets. Polyamide effectively improved CsA- and UUO-associated nephropathy, indicating its potential application in the prevention of renal fibrosis in progressive renal diseases.

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