This document discusses a study that assessed how nicotine exacerbates diabetic nephropathy through upregulation of the Grem 1 gene expression. The study used various methods like TUNEL staining, real-time PCR, western blotting, and RNA sequencing to analyze gene expression and protein levels in mouse kidney tissues and cells. The results found that nicotine reduces nephrin expression in diabetic rat kidneys and increases Grem 1 expression when combined with high glucose in podocytes. The conclusions state that Grem 1 plays an important role in nicotine-exacerbated diabetic nephropathy and may be a potential therapeutic target for chronic smokers with this condition.

1. Gelena Marsiglia Romero
third semester
PONTIFICAL BOLIVARIAN UNIVERSITY AUGUST 14- 2023

2. is a highly addictive stimulant found in tobacco and
vaping devices. The bloodstream quickly absorbs this
substance.
Nicotine is just as addictive as heroin or cocaine,
making it extremely difficult to stop using.
The physical effects of consuming nicotine include
rapid heart rate, high blood pressure, shortness of
breath, and an increased tendency to contract colds
and complaints.
Nicotine users have a higher risk of lung disease, heart
disease, and stroke. Brain development can be
affected by nicotine, even in late adolescence.
Nicotine withdrawal symptoms include anxiety, anger,
restlessness, and insomnia.
NICOTINE

3. diabetic
nephropathy
In the study, DNA sequencing was performed to analyze damage
to gene expression caused by nicotine-exacerbated DN.
It was found that Grem 1 is a gene that promotes kidney damage
in response to nicotine in the diabetic milieu.
This gene encodes a member of the BMP (bone morphogenic
protein) antagonist family. Like BMPs, BMP antagonists contain
cystine knots and typically form homo- and heterodimers.
Is a common microvascular complication of diabetes (20–40%) .
it affects the kidneys' ability to do their normal job of removing
waste and excess fluid from the body. Over the years, the
condition slowly damages the delicate filtering system of the
kidneys. Early treatment could prevent or delay the progression
of the disease and reduce the chance of complications.

4. General objective
Assess nicotine exacerbates diabetic nephropathy
through upregulation of grem 1 expression

5. Methods
1
Apoptosis determination
In mouse tissues, apoptotic cells were
analyzed by TUNEL staining with Click-iT™, In
in vitro studies, we determined cell apoptosis
using Hoechst33342 staining.
Cells with condensed and fragmented nuclei
They were identified as apoptotic cells. the
health cores cells appeared blue (TUNEL-
negative), and nuclei of apoptotic cells
stained brown (TUNEL positive)
2
Real‑time PCR
Total RNA was isolated from mouse kidney
samples using TRIzol reagent. Five micrograms
of total RNA were reverse transcribed using the
first-strand synthesis system.
was done in a Sequence Detection System
Using the Comparative CT method, a
determined GAPDH internal control and
standardized relative levels of mRNA.

6. 3 Western blotting analysis
The acquired images were analyzed
using the NIH imaging program in the
public domain. primary antibodies
used were rabbit anti-Grem1 and
mouse anti-actin.
For protein expression quantification,
the films were scanned with a Canon
Scan scanner.
4 RNA sequencing and data analysis
In the subsequent procedure, mRNA
molecules were fed with total RNA puri
using Oligo(dT)-linked magnetic molecules.
Clean readings were obtained of the raw
data after removing the adapter, ploy-N
and low quality readings. Fuzzy C-means
clustering was used to identify mice
gene cluster that responds to ND
exacerbated by nicotine.
Methods

7. Nicotine reduces the expression of nephrin
in the kidneys of diabetic rats.
Immunofluorescence staining and q-PCR
were performed to detect
Nephrin manifestations.
Immunofluorescence staining was
performed to detect nephrin protein
expression in the kidneys of Con and Nic,
Mouse DM and Nic + DM.
In this result, the test control
and the gene have been
compared.
Representative gels were shown,
protein bands were scanned
Results

8. I need a test control which in this case is
actin. We observe how in one test the
expression increases and in the other it
decreases.
A Western blot was performed to investigate
expression is displayed
Western blot was performed to
Investigate the expression of Grem1, nephrin and
cleaved caspase 3 in high glucose and nicotine co-
treated podocytes. B knocking down
Grem1, a Western blot was performed to investigate
the expression of Grem1, nephrin, and cleaved
caspase 3 in co-treated podocytes.
nicotine and high glucose.
Results

9. Discussion

10. Conclusions
Grem1 plays a vital role in nicotine-
exacerbated DN. Grem1 may be a potential
therapeutic target for
chronic smokers with DN
Administration of nicotine alone did not cause
apparent renal injury, but significantly
increased hyperglycemia-induced
albuminuria, BUN, plasma creatinine, and
renal tissue mRNA expression of KIM-1 and
NGAL.
.