1.Imbalance between Endothelin-1 and eNOS Expression Associates with Tubular Injury in Mice with 5/6 Subtotal Nephrectomy
Muhammad Mansyur Romi ; Nur Arfian ; A.A.N Nata Baskara ; Dwi Cahyani Ratna Sari
Malaysian Journal of Medicine and Health Sciences 2020;16(Supp 3,June):82-85
Introduction: Chronic Kidney Diseases (CKD) leads to kidney fibrosis which characterized by tubular injury and atrophy with interstitial fibrosis. Endothelin-1 (ET-1) and endothelial Nitrite Oxide Synthase (eNOS) are known to play
role in CKD and kidney fibrosis, although their correlation with tubulo-interstitial injury have not been understood
clearly. Methods: 5/6 Subtotal Nephrectomy (SN) was performed in male Swiss Background mice to induce CKD.
Sham operation (SO, n=5) procedure was performed on mice as control. The mice were sacrificed in day 7 (1N, n=5)
and day 28 (4N, n=5) after operation. We measured creatinine serum to assess renal function. Tubular injury score
was quantified based on Periodic-Acid Schiff (PAS) staining. Prepro-ET-1 and eNOS were quantified using RT-PCR.
Results: SN_1N and SN_4N groups had significant higher of serum creatinine and tubular injury from SO group. Densitometry analysis of RT-PCR revealed up-regulation of prepro-ET-1 mRNA expression in SN_1N and SN_4N (p<0.05
vs SO). Meanwhile, we found a significant increase of eNOS expression in SN_1N, and then it reduced significantly
in SN_4N. We found significant parallel correlation between ET-1 and tubular injury expression (r: 0.768;p<0,05),
meanwhile there were insignificant inverse correlation between eNOS and tubular injury (r: -0.354;p>0.05). Conclusion: eNOS might play role as a counterbalance in the up regulation of ET-1 in acute condition after SN. However, it
failed in chronic condition. These lead to deterioration of renal function and tubular injury. An imbalance between
ET-1 and eNOS expression in chronic CKD model might play role in profound renal damage.
2.Ethanol Extract of Centella asiatica (Gotu Kola) Attenuates Tubular Injury Through Inhibition of Inflammatory Cytokines and Enhancement of Anti-Fibrotic Factor in Mice with 5/6 Subtotal Nephrectomy
Nur Arfian ; Wiwit Ananda Wahyu Setyaningsih ; Nungki Anggorowati ; Muhammad Mansyur Romi ; Dwi Cahyani Ratna Sari
Malaysian Journal of Medical Sciences 2019;26(5):53-63
Background: Chronic kidney disease (CKD) leads to inflammation, fibrosis and
destruction of the renal architecture. Centella asiatica (CeA) is an herbaceous plant with antiinflammatory
effects. We aimed to elucidate the effect of CeA on inflammation, fibrosis, vascular
remodelling and antifibrotic substances in a 5/6 subtotal nephrectomy (SN) model in mice.
Methods: Mice were divided into three groups: sham operation (SO, n = 6), 5/6 SN for
seven days (SN7, n = 7) and SN7 with oral CeA treatment (SN7-CeA, n = 7). At day 7, mice were
euthanised, kidneys were harvested and stained with periodic-acid Schiff (for tubular injury
and glomerulosclerosis) and sirius red (for fibrosis and vascular remodeling) staining. mRNA
expression of prepro-endothelin-1, nephrin, E-cadherin, bone morphogenic protein-7 (BMP-7),
toll-like receptor 4 (TLR4), tumour necrosis factor-α (TNFα) and hepatocyte growth factor (HGF)
were quantified using reverse transcriptase-PCR.
Results: SN group demonstrated significant higher interstitial fibrosis, vascular
remodeling, tubular injury and glomerulosclerosis (P < 0.01) compared to SO group. Meanwhile,
in SN7-CeA demonstrated attenuation of vascular remodeling as shown by significant higher lumen
area with lower Wall/Lumen area ratio compared to SN7. RT-PCR analysis showed up-regulation
of nephrin, BMP-7 and E-cadherin mRNA expression (P < 0.05) and down-regulation of ppET-1 in
SN7-CeA group compared to SN7 group (P < 0.05).
Conclusion: CeA may ameliorate renal injury in the SN model in mice.
3.Upregulation of Megalin, Cubilin, NGAL mRNA expression in kidney may represent tubular injury and apoptosis in chronic condition of rat diabetic model
Muhammad Mansyur Romi ; Nungki Anggorowati ; Dara Syifa Maulida ; Muhammad Wishka Al Hafiidh Suskalanggeng ; Wiwit Ananda Wahyu Setyaningsih ; Dwi Cahyani Ratna ; Nur Arfian
The Medical Journal of Malaysia 2021;76(1):87-92
Introduction: Diabetes mellitus (DM) leads to microvascular
injury development and produces diabetes nephropathy
(DN) with proteinuria, tubular injury, apoptosis and
autophagy with upregulation of Bax, BASP and mTORC-1.
Megalin, Cubilin and Neutrophil Gelatinase Associated
Lipocalin (NGAL) play role in acute pathological condition of
kidney injury, however its expression in chronic and slowly
progressive kidney injury such as DN has not been
elucidated yet. This study focuses upregulation of Megalin,
Cubilin and NGAL in association with tubular injury and
apoptosis in DN condition.
Materials and methods: Diabetic condition was performed
with intraperitoneal injection of Streptozotocin 60 mg/kg
body weight (BW) in Sprague Dawley rats (2 months old,
n=24), and were kept for 1, 2, and 4 months (DM1, DM2, and
DM4, respectively). Control group was injected with NaCl
0.9%. Serum glucose level and proteinuria score were
assessed, furthermore tubular injury score was quantified
based on Periodic-Acid Schiff (PAS) staining. Reverse
Transcriptase-PCR (RT-PCR) was carried out for NGAL,
Megalin, Cubilin, m-TOR, Bax, and BASP-1 mRNA
expression. Data were analyzed using SPSS 22 software.
Results: DM led to kidney injury in this model with
significant higher glucose level, proteinuria and tubular
injury, especially in DM4 group which represented chronic
phase of DN and CKD. These findings associated with
upregulation of Megalin,Cubilin and NGAL mRNA
expression in DM groups, especially in DM4 group. DM4
group also revealed higher expression of Bax, BASP and
mTOR mRNA expression which demonstrated apoptosis.
Conclusion: Megalin, Cubilin and NGAL upregulation may
represent tubular injury and apoptosis as progression of
DN.
4.Ethanol Extract of Yacon Leaves (Smallanthus sonchifolius) Attenuates Fibroblast and Myofibroblast Expansion in Association With Downregulation of TGF β1 and Snail mRNA Expression in 5/6-Subtotal Nephrectomy Model in Mice
Ermawati ; Nur Arfian ; Setyo Purwono ; Muhammad Mansyur Romi ; Dwi Cahyani Ratna Sari
Malaysian Journal of Medicine and Health Sciences 2022;18(No.3):85-91
Introduction: Chronic kidney disease (CKD) is characterized by fibroblast activation, myofibroblast formation, and
up-regulation of transforming growth factor-β1 (TGF-β1) that may activate Snail in fibroblast to myofibroblast transition. Ethanol extract of Yacon leaves is known to have a renoprotective effect on diabetic nephropathy but its effect
in the CKD model is unknown. This experimental study aimed to elucidate the effect of ethanol extract from Yacon
leaves in attenuating renal failure in a CKD mice model. Methods: Male Swiss-Webster mice (3 months, 30–40
grams, n=25) underwent 5/6 subtotal nephrectomy (SN) to induce CKD. The mice were divided into five groups: SN,
SN mice with oral treatment of Yacon leaves ethanol extract with doses 0.735 μg/kg (SN+YK1), 1.47 μg/kg (SN+YK2),
and 2.94 μg/kg (SN+YK3), and a Sham operation (SO) group with aquadest 0.1% supplementation. Mice were euthanized on day 14 after the operation and kidneys were harvested. Paraffin sections were used for histological analysis.
Immunostaining was done for quantifying fibroblasts and myofibroblasts. We performed RT-PCR to measure TGF-β1
and Snail mRNA expressions. Results: The SN group had significantly higher fibroblast number, myofibroblast fraction area, TGF-β1 and Snail mRNA expressions compared to the SO. The fibroblasts number (p<0.001) and myofibroblast fraction areas (p<0.001) were significantly lower in Yacon treated-groups compared to the SN group. RT-PCR
analysis showed lower mRNA expressions of TGF-β1 and Snail, but no significant differences were found among the
various Yacon treated-groups. Conclusion: Ethanol extracts of Yacon leaves improved kidney damage in male mice
with 5/6 subtotal nephrectomy model.
5.Uric Acid Induces Inflammation, Hepatocyte Apoptosis and Deterioration of Liver Function
Dwi Cahyani Ratna Sari ; Rina Nofrienis ; Muhammad Mansyur Romi ; Untung Tranggono ; Eryna Ayu Nugra Desita ; Nur Arfian
Malaysian Journal of Medicine and Health Sciences 2020;16(Supp 3,June):49-55
Introduction: Uric acid is a common cause of liver tissue damage due to its hepatotoxic effect. This study is aimed to
investigate: (1) the effect of uric acid on liver damage which can be seen from the serum levels of SGOT and SGPT,
(2) the inflammatory response demonstrated by TLR-4 and MCP-1 mRNA expression, and (3) the proportion of hepatocytes apoptosis in mice. Methods: A total of 25 adult male Swiss-Webster mice were divided into five groups:
one control group and four uric acid groups (AU7, AU14, AU21 and AU28). The uric acid groups were administered
with 125 mg/kgBW uric acid for 7, 14, 21, and 28 days. Following the treatment, mice were terminated and the liver
was harvested. Blood sample was taken from retro-orbital vein to assess serum uric acid, SGOT, and SGPT levels.
RT-PCR was performed to examine the mRNA expressions of TLR-4 and MCP-1. TUNEL staining was used to assess
the proportion of apoptotic hepatocytes. Results: Induction of uric acid caused hyperuricemia, increased expression
of TLR-4 and MCP-1 mRNA significantly (p<0.05) which indicated an inflammatory reaction. The levels of SGOT
and SGPT were elevated significantly (p<0.05), as well as the number of hepatocyte apoptosis (p<0.05). Conclusion: Hyperuricemia affected the inflammatory response by increasing the mRNA expression of TLR-4 and MCP-1.
An increased number of apoptotic hepatocytes was likely caused by the ongoing inflammatory reaction during the
induction of uric acid.
6.Upregulation of ppET-1/ETBR/eNOS mRNA Expression After Calcitriol Treatment in Chronic Kidney Diseases Model in Rats
Anak Agung Ngurah Nata Baskara ; Nur Arfian ; Wiwit Ananda Wahyu Setyaningsih ; Muhammad Mansyur Romi ; Dwi Cahyani Ratna Sari
Malaysian Journal of Medicine and Health Sciences 2022;18(No.5):23-29
Introduction: Myofibroblast formation in the interstitial area is the hallmark of chronic kidney disease (CKD). Endothelin signalling has been known to play role in physiology and pathophysiology in the kidney. Vitamin D has a
reno-protective effect through inhibiting inflammation and fibrosis. However, the interaction between vitamin D and
endothelin signalling in the CKD model has not been elucidated yet. Therefore, we aimed to check the difference
impact of endothelin (ET) receptor in CKD. Methods: Sprague Dawley rats (3-months-old, 150-250grams) underwent
5/6 subtotal nephrectomy (SN) to induce CKD. Then, it was divided into 4 groups (each contains 6 rats): sham operation (SO), 5/6 subtotal nephrectomy (SN), calcitriol groups (0.01µg/100grBW/day (SN-D1), and 0.05µg/100grBW/day
(SN-D2). Calcitriol was administered for 14 days after the surgery. The Sham Operation (SO) group was injected with
NaCl. At the specified date, the rats were sacrificed and the kidneys were harvested. Fibrosis was quantified based
on Sirius Red staining. Immunostaining was done for localizing fibroblast (PDGFRβ). The mRNA expressions of prepro-ET-1, endothelin receptor A (ETAR), endothelin receptor B (ETBR), and endothelial nitrite oxide synthase (eNOS)
were quantified using reverse-transcriptase PCR (RT-PCR). Results: The CKD promotes an elevation of prepro-ET-1,
ETBR, and eNOS, and reduction of ETAR (p<0.05) mRNA expression compared to the SO group. Administration of
calcitriol (SN-D1 and SN-D2) showed the vice versa effects. However, only SN-D2 group consistently showed statistically significant differences whenever compared to either SO or SN groups. Conclusion: Calcitriol might attenuate
interstitial fibrosis in CKD model via ET-1/eNOS signalling.